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CLONING AND CHARACTERIZATION OF OUTER MEMBRANE
PROTEIN(S) OF Pasteurella multocida SEROTYPE B:2 (P52)
Thesis
Submitted to the
Govind Ballabh Pant University of Agriculture and Technology,
PANTNAGAR-263 145 (Udham Singh Nagar), Uttarakhand, INDIA
By
Archana Yadav
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
Doctor of Philosophy
(MICROBIOLOGY)
April, 2008
Acknowledgement
I feel great pleasure to express my regards, deep sense of gratitude and
indebtedness towards my advisor, Dr. Anita Sharma Assistant Professor,
Department of Microbiology and Chairman of my advisory committee for her help
and encouragement in the preparation of the manuscript.
I express deepest gratitude and heartiest thanks to Dr, V. D. P. Rao,
Professor, Veterinary Microbiology and Registrar of the University for providing
me facilities for conducting the research.
I would like to take this opportunity to express my profound sense of
gratitude to Dr. Mumtesh Kumar Saxena, Assistant Professor, Animal
Biotechnology Centre and member of my advisory committee for his invaluable and
critical suggestions. It was due to his planning, constructive criticism,
encouragement and support through out research that made possible for me to
complete this study.
I would like to express my profound sense of gratitude to the members of my
advisory committee Dr. Reeta Goel, Professor and Head Department of
Microbiology, and Dr. Dinesh Yadav, Associate Professor, Molecular Biology and
Genetic Engineering, for their generous support and guidance during the course of
my study.
Thanks are due to Dean C.B.S.H. and Dean P.G.S. for providing all the
necessary facilities during the course of investigation.
I take this opportunity in expressing my heartfelt thanks to Dr, Sameer
Srivastava, Assistant Professor for his gregarious nature and continuous motivation.
Thanks are due to Dr. Manvika Sahgal, J.R.O. Microbiology, Dr. Soma
Marla Assoc Professor and Vinay Singh of Bioinformatics centre of GBPUAT
Pantnagar for their sincere help.
I am highly thankful to Mr. A. B. Sati, Mr. L. M. Padaliya, Mr. Pathak , Mr.
Mahesh, Ramchandra and Shriram for providing necessary help whenever it was
needed during full period of my study.
Wordly thanks cannot express my respect towards my seniors Moni mam,
Gunjan mam and Bablu sir for their co-operation and help during the course of
study. Thank you Dr. Shantanu for sharing the quest for knowledge in all aspects.
My friends Hemlata, Manu, Sunita, Shraddha and Amit deserve a special thanks.
Words fail to express the depth of my feelings for my loving parents who
displayed affection love, care, silent support and constant encouragement and ever
open arms, which has rought to me this stage. I owe my ever feelings to my motherin-law, brother-in-law, brother (Sanjay bhaiya), sisters (Madhu Di, and Shashi Di )
for their ever encouraging words, extreme confidence, true love and faith they
shower upon me. I place my compliments to my dear sister Neelam who support me
in tough phases during the study.
No words of gratitude will be able to express my feelings to words my
beloved husband Dr. V. K. Singh (strength of my life), for their numerous sacrifices
in all aspects during these four years period and tremendous understanding which
were moral boosting to me during my study.
ABOVE ALL THANK YOU GOD
Pantnagar
April, 2008
(Archana Yadav)
Authoress
Dr. Anita Sharma
Department of Microbiology
College of Basic Sciences & Humanities
G. B. Pant Univ. of Agric. & Tech.,
Pantnagar - 263 145,
Distt.– Udham Singh Nagar,
Uttarakhand, INDIA
Assistant Professor
CERTIFICATE
This is to certify that the thesis entitled “CLONING
CHARACTERIZATION OF OUTER MEMBRANE PROTEIN(S) OF
multocida
SEROTYPE
AND
Pasteurella
B:2 (P52)” submitted in partial fulfilment of
the requirements for the degree of DOCTOR
OF
PHILOSOPHY with major
in MICROBIOLOGY and minor in MOLECULAR BIOLOGY
AND
GENETIC
ENGINEERING of the College of Post-Graduate Studies, Govind Ballabh
Pant University of Agriculture and Technology, Pantnagar, is a record
of bona-fide research carried out by ARCHANA YADAV, Id. No. 31741
under my supervision and no part of the thesis has been submitted
for any other degree or diploma.
The assistance and help received during the course of this
investigation have been acknowledged.
(Anita Sharma)
Chairman
Advisory Committee
C E R T I F I C A T E
We, the undersigned, members of the Advisory Committee of
ARCHANA YADAV, Id. No. 31741 a candidate for the degree of
DOCTOR
OF
PHILOSOPHY with major in MICROBIOLOGY and minor in
MOLECULAR BIOLOGY
AND
entitled
AND
“CLONING
PROTEIN(S) OF
GENETIC ENGINEERING, agree that the thesis
CHARACTERIZATION
Pasteurella multocida
OF
SEROTYPE
OUTER
MEMBRANE
B:2 (P52)” may be
submitted in partial fulfilment of the requirements for the degree.
(Anita Sharma)
Chairman
Advisory Committee
(V.D.P. Rao)
Member
(Dinesh Yadav)
Member
(Reeta Goel)
Member
(Mumtesh Saxena)
Member
CONTENTS
S. N.
Chapters
1.
Introduction
2.
Review of Literature
3.
Materials and Methods
4.
Results
5.
Discussion
6.
Summary
References
Appendix
Annexure
Vita
Abstract
Pages
Chapter 1
Introduction
Haemorrhagic septicaemia (HS) is an acute pasteurellosis, caused
by particular serotypes of Pasteurella multocida and manifested by an
acute and highly fatal septicemia mainly in cattle and water buffaloes;
the latter are thought to be more susceptible.
HS has a wide distribution particularly in tropical countries like
Africa and Asia. In Asia, HS epidemics occur as an alarming and
devastating disease problems in cattle and buffaloes. Disease outbreaks
mostly occur during mansoon season when high temperature prevails
with high humidity.
This disease is caused by Pasteurella multocida, a Gram-negative
coccobacillus residing as a commensal organism in the upper
respiratory tract of the animals. Asian serotype B:2 and the African
serotype E:2 (Carter and Heddleston system), corresponding to 6:B and
6:E (Namioka-carter system) are mainly responsible for the disease. In
wild animals, serotype B:2,5 is predominantly present. The association
of other serotypes, namely A:1, A:3 with a HS-like condition in cattle
and buffaloes in India has also been recorded (OIE, 2005).
On the basis of distribution of the disease, three distinct
categories of different countries have been identified by FAO-WHO-OIE.
India comes under the category A, where the disease is endemic and is
of great importance.
HS is a disease of utmost economic importance particularly in
Asia and to a lesser extent in Africa. In Asia, susceptible animal
population consists of 432 million cattle and 146 million buffaloes,
constituting 30 and 95% of the world’s cattle and buffalo population
respectively. The high population of buffalo in Asia, their high
susceptibility and fatality to HS and high fatality show the significance
of the economic losses due to this disease. In other Asian countries like
Sri Lanka around 15% buffaloes and 8% cattle in the HS endemic areas
died of HS annually in 1970. Other countries in South Asia also ranked
HS as the most economically important infectious disease. In Pakistan,
the annual economic losses have been estimated at 1.89 billion rupees
due to this disease (De Alwis, 2002). Economic losses due to HS are
not only confined to animal industry but rice production is also affected
on account of its high prevalence among draught animals used in rice
fields.
The organism causing HS does not survive outside the animal
body to any significant degree. Moist conditions prolong the survival of
the organism. Thus the disease tends to spread more during the wet
season. The onset of the monsoon in Asian countries also set into
motion other activities such as rice cultivation which bring about
movements of animals, work stress in work animals, etc. all of which
favour the precipitation of outbreaks of this disease.
HS is a primary bacterial disease and could be effectively treated
by the wide range of antibiotics currently available. However, treatment
is constrained by a host of practical considerations. Animals can be
cured only if treated in the earlier stages of the disease. Usually
chemotherapy is done by either streptomycin or oxytetracycline
administered by intramuscular route at fairly high dosage. Penicillin
and
ampicillin
are
also
widely
used.
Antibiotic
resistance
for
streptomycin and sulfonamides has been reported in P. multocida
(Kedrak and Borkowska-Opacka, 2001).
Tragically, treatment for HS is of limited value due to the acute
nature of the disease and vaccination is the only effective method of
controlling the disease. Numerous whole cell and subunit vaccines were
previously developed and tried against this disease with varying degree
of success. Hence there is a paramount urgency of identification of
potential immunogens of P. multocida which can lead to preparation of
more effective vaccines and implementation of new vaccination
strategies.
Effective vaccines against HS are formalin killed bacterins or
dense bacterins with adjuvants. Adjuvants enhance the level and
prolong the duration of immunity. The most effective bacterin is oiladjuvant-one dose provides protection for 9-12 months. The alumprecipitated-type bacterin is given at 6- months intervals. These
vaccines provide only short term immunity (Chandrasekaran et al.,
1994) and require annual administration for maximum effectiveness
(De Alwis 1992). The oil- adjuvanted vaccines have disadvantage of
high viscosity which makes them unpopular among field users.
A live intranasal vaccine prepared from a B: 3,4 serotype of deer
origin is being used with reported success in southeast Asia. Live
vaccines stimulate protection against a wide range of P. multocida
serotypes and for longer periods, but at present live vaccines are
undefined and may revert to virulence leading to death in vaccinated
population. For live strains to be used as vaccines, the mode of
attenuation should be well defined and constructed in such a way that
the possibility of reversion to virulence is minimized (Tabatabaei et al.,
2007).
Earlier P. multocida was considered to be non pathogenic for
human being but recent reports indicate that it may cause lung cancer
in human (Goussard et al., 2006). So, for mass production of killed
vaccine handling of such dreaded organism is not advisable.
Previous studies on vaccine development against P. multocida
using
experimental
animals
demonstrated
that
the
protective
component(s) resided primarily in the outer membrane of the organism.
The outer membrane proteins (OMPs) of P.multocida play
significant
role in host-pathogen interactions and are important determinants of
immunoprotection hence can serve as vaccine candidate against
haemorrhagic septicaemia (Basagoudanavar et al., 2006).
To overcome from these problems several efforts have been made
to develop safe and efficient vaccine. Aromatic mutant vaccine
(Homchampa et al., 1992, 1997) and r-DNA vaccine have been tried
and some promising results have been reported in lab animals (Lee et
al., 2007).
Recombinant vaccine is a novel vaccine technology that has been
applied to stimulate protective immunity against many infectious
agents. Recombinant vaccines do not harm the host, since it is a single
protein, not the organism itself. In r-DNA vaccine preparation the most
important criteria is to target an immunopotent gene. OMP(s) have been
targeted as subunit vaccine showing promising results (Ruffolo and
Adler 1996; Luo et al., 1997; Lee et al., 2007). Present study was
taken up to study the immunopotential of OMP(s) of Pasteurella and to
clone the gene encoding outer membrane protein(s) of P. multocida P52
(serotype B:2) in E. coli with the following objectives:
1. To purify outer membrane proteins of P. multocida P52 strain
2. To identify major immunodominant OMPs.
3. To clone and sequence the gene(s) encoding 87 and 34 kDa outer
membrane protein(s) of P. multocida P52.
Review of
Literature
Chapter 2
Review of Literature
2.1
HISTORY AND DISTRIBUTION
Haemorrhagic septicaemia is one of the most economically
important diseases of cattle and buffaloes. It is caused by two specific
serotypes
of
a
gram
negative
organism,
Pasteurella
multocida.
‘Pasteurellosis’ is one of the oldest diseases described in literature. The
first systematic study of an outbreak of septicaemic pasteurellosis in
deer, cattle, and swine was carried out by Bollinger (1878) in Germany
and the causative agent was isolated by Kitt (1885). During the same
era, the so-called ‘Golden age of Bacteriology’ the microorganisms
causing fowl cholera (Pasteur, 1880), and rabbit septicaemia (Gaffky,
1881) were also discovered. The disease in buffalo was described as
‘Barbone’ by Oreste and Armanni (1886). A German pathologist
Hueppe (1886) proposed the name ‘Haemorrhagic septicaemia’ for the
disease. In 1901 Ligniers used generic name ‘Pasteurella’. Presently
accepted name Pasteurella multocida (Multocida : fatal for many) was
suggested by Rosenbusch and Merchant (1956).
Pasteurella multocida, a Gram negative, non motile, facultative,
coccobacillary organism is incriminated for a number of animal
diseases (Confer et al., 1991) and has been classified into five
serogroups (A, B, D, E, and F) based on capsule antigens. They are
further
classified
into
16
serotypes
(1-16)
based
primarily
on
lipopolysaccharide antigens using the Heddleston scheme (Carter,
1955; Heddleston et al., 1972). Two haemorrhagic septicaemia
serotypes of the organism designated as B:2 and E:2 (Carter Heddleston
system) were found equivalent to Nomioka 6:B and 6:E serotypes.
Infections caused by P. multocida include fowl cholera of poultry
(Rhoades and Rimler, 1989), progressive atrophic rhinitis of pigs
(Chanter and Rutter, 1989), pneumonia of cattle, sheep and pigs
(Chanter
and
Rutter,
1989;
Frank,
1989), and haemorrhagic
septicaemia of cattle and water buffaloes in certain enzootic areas of
Asia and Africa (Carter and De Alwis, 1989). This pathogen is also
associated with atrophic rhinitis (Krametter et al., 2004) and
septicaemia of sheep (Watson and Davies, 2002). In addition, it is also
responsible for infections in deer (Aalbaek et al., 1999), rhinitis
(‘snuffles’) and pneumonia in rabbits (Manning et al., 1989). Although
relatively uncommon, human infections have also been observed in a
range of sites, commonly following cat or dog bites (Holm and Tarnvik,
2000).
Asian serotype B: 2 and African serotype E:2 are considered
primarily as the causative agent of classical HS of cattle and buffaloes
(Dawkins et al., 1991). Kumar (1996) have reported association of A:
1, A: 3, F: 3 and F: 3, 4 with HS-like and/or similar diseases in India.
Association of these organisms with classical fatal HS still needs to be
confirmed, though these serotypes may play a role in causing other
clinical
manifestations
like
pneumonic
Pasteurellosis
leading
to
animal’s death. The most predominant serotype reported in Asian
region is B:2. In African countries the predominant serotype is E: 2. In
countries like Egypt and Sudan, both B:2 and E:2 serotypes have been
reported by Mustafa and Shigidi., (1979). Voigts et al. (1997)
reported HS due to B: 2 serotype of P. multocida in Namibia. In wild
animals, serotype B:2,5 is predominantly present (OIE, 2005).
2.2
THE DISEASE: HAEMORRHAGIC SEPTICAEMIA (HS)
Haemorrhagic septicaemia (HS) of cattle and buffaloes occurs as
catastrophic epidemics with high morbidity and mortality in South East
Asia, South Africa, Australia, Europe and India (DeAlwis, 1999).
This disease has been characterized having three phases
(DeAlwis, 1996). During the first phase, there is elevation of
temperature, second phase is characterized by respiratory distress
followed by third phase or terminal phase of recumbency. In natural
conditions, generally the disease is characterized by high fever and
depression which is followed by sudden death of the animal. Edema of
head and neck and bleeding from natural orifices may be observed
(Shewen and Conlon, 1994). Haemorrhages and edema of serous and
mucous surface and in lymph node, spleen, lung and other visceral
organs are the predominant post-mortem findings of this disease
(Carter and DeAlwis, 1989). The extent of lesions has been shown to
depend on duration of the clinical disease, in per acute case, where
death occurred within 24-36 h, no more than a few scattered petechial
hemorrhages could be observed in some experimental studies (DeAlwis
et al., 1978).
Adherence of the organism to respiratory tract is considered to be
an important factor for pathogenesis. Some surface and secreted factors
of P. multocida have been identified for adhesion, but no single factor
could be associated with the virulence of organism. It has been shown
that highly virulent type B strains produce hyaluronidase enzyme which
contributes significancy in outcome of infection. Bivalent cation like
iron has an important role in this respect. Due to excessive
hemorrhage, Fe++ level increases favoring bacterial adherence and their
growth in the animal (Babiuk and Campos, 1993).
The septicaemia in HS is essentially a terminal symptom that’s
why blood samples taken from the sick animals before death may not
always contain P. multocida (DeAlwis, 1989). Organisms are not
consistently present in the nasal secretions of sick animals (DeAlwis,
1989) which indicates that bacterimea is followed quickly by death of
the animal.
Humoral type immunity is predominantly reported in HS. As the
organism does not grow facultatively or intracellularly, so the role of
cellular immunity is limited. The oil adjuvant and bacterin vaccines
elicit immune response of IgG for longer duration and IgM for short
duration (Dawkins et al., 1991). Verma and Jaiswal (1997) reported
the appearance of cellular and humoral immunity as early as 7 days of
post inoculation which persist upto one year in cattle.
2.3
DISEASE INCIDENCES AND ECONOMIC IMPORTANCE
2.3.1 International status
HS is a disease of utmost economic importance particularly in
Asia and to a lesser extent in Africa. In Asia, the susceptible animal
population consists of 432 million cattle and 146 million buffaloes,
constituting 30 and 95% of the world’s cattle and buffalo population
respectively. The high population of buffalo in Asia, their high
susceptibility to HS and high fatality show the significance of the
economic losses due to this disease. In Sri Lanka, in1970, around 15%
buffaloes and 8% cattle in the HS endemic areas died of HS annually.
Other countries in South Asia also ranked HS as the most economically
important infectious disease. In Pakistan the annual economic losses
have been estimated of 1.89 billion rupees (De Alwis, 2002).
2.3.2 Indian status
HS is endemic in most parts of India and seasonal outbreaks are
quite common. Andhra Pradesh ranks first in the total number of HS
attacks reported in India in the past five years followed by Gujarat and
Karnataka (Rajasekhar, 2005). Dutta et al., (1990) have reported that
the overall relative risk due to HS in India during the period of 19741986 was highest in Manipur and lowest in Dadra and Nagar Haveli. In
Assam, Karnataka and Maharastra an increase in the relative risk was
observed during the period of 1977 to 1979 but this trend decreased
during subsequent years. The authors have also identified Andhra
Pradesh, Himachal Pradesh, Manipur, Meghalaya, Rajasthan and
Arunachal Pradesh as high risk states, Karnataka and Maharastra as
medium risk areas and Sikkim, Dadra and Nagar Haveli, Goa,
Pondicherry and Chandigarh as disease free zones for 1983-1986. It
accounts for loss of more than 10 million rupees per annum in India
(Singh et al., 1996).
2.4 ANTIGENS OF PASTEURELLA MULTOCIDA
In general, bacterial strains that possess capsules are more
virulent than their acapsular variants (Snipes et al., 1987; Tsuji and
Matsumoto, 1989). Role of capsule in the pathogenesis of P. multocida
has been clearly demonstrated by Boyce and Adler, (2000). A
genetically defined acapsular serotype B mutant was more susceptible
to murine macrophages than to its wild-type parent.
Lipopolysaccharide also plays a critical role in pathogenesis of
disease. It stimulates humoral immunity and is considered to be
protective antigen. Intravenous inoculation of lipopolysaccharide from
serotype
B:
2
could
reproduce
clinical
signs
of
haemorrhagic
septicaemia in buffalo (Horadagoda et al., 2002). A complete
lipopolysaccharide structure was required for replication in vivo and
causing disease (Harper et al., 2004).
Although the role of hyaluronidase in pathogenesis has not been
determined, but it is present in B: 2 serotype of P. multocida that causes
bovine haemorrhagic septicaemia. A study of 176 strains of P. multocida
representing different serotypes has observed hyaluronidase activity
confined to serotype B, but more specifically to B: 2. It was suggested
that hyaluronidase activity could be used to presumptively identify B:2
strains (Rimler and Rhoades, 1994).
Colonisation of host tissues by Gram- negative bacteria is
facilitated by various adhesins, one of which is type 4 fimbriae (pili).
These structures have been associated with pathogenesis in several
bacterial species and have also been shown to mediate colonisation of
epithelial surfaces. Type 4 fimbriae were identified and characterised in
P. multocida strains A, B and D (Ruffolo et al., 1997), it’s gene ptfA
has been sequenced from a number of strains indicating variations in
the ptfA sequences at serotype level (Doughty et al., 2000).
2.4.1 Iron regulated and iron acquisition proteins
Iron is an essential growth factor for all bacteria. The low
concentration of free iron on the mucous membranes and tissues is one of
the first line of defence against bacterial infection. The acquisition of iron
is possibly the major determinant for a pathogen to maintain itself inside
an animal host. Bacteria present several mechanisms to take up this
element. Organisms like E.coli or Salmonella Typhimurium, produce
siderophores which are secreted outside cells and are able to remove iron
from the host environment or from the host iron-binding molecules
(Ratledge and Dover, 2000). Other bacteria, like Haemophillus influenza
and Neisseria meningitides, present outer membrane proteins able to
interact with iron binding host molecules, such as haemin, haemoglobin,
transferin or lactoferin (Ratledge and Dover, 2000). In both the cases,
transport of iron across the outer membrane is an active process requiring
a functional TonB system. Similarly P. multocida has developed multiple
mechanisms for iron uptake. Sequence analysis of P. multocida PM70
revealed that a relatively large proportion of the genome (over 2.5%)
encodes 53 proteins with similarity to proteins involved in iron uptake or
acquisition (May et al., 2001). P. multocida expresses several outer
membrane proteins ranging from 35 to 109 kDa molecular weight when
grown under iron restricted conditions, which mediate iron acquisition in
vivo. (Veken et al., 1994; Zhao et al., 1995; Ikeda and Hirsh, 1988).
Comparison of P. multocida grown in iron-rich, iron-depleted media
or in vivo has demonstrated that many high molecular weight outer
membrane proteins are regulated by iron levels and have
been called
iron- regulated outer membrane proteins (IROMPs) (Snips et al., 1988;
Choi-Kim et al., 1991). P. multocida grown under iron limited conditions
also induces a stronger protective response in mice compared with the
same strain grown under iron-replete conditions (Kennett et al., 1993),
and it is thought that IROMPs may play significant role in cross-protective
immunity (Glisson et al., 1993; Ruffolo et al., 1998).
IROMPs have several attributes to be used as vaccine candidates.
They are surface exposed molecules expressed in vivo during infection
and elicit protective and bactericidal antibodies in laboratory animals.
2.5
MULTIPLE DRUG RESISTANCE
Various antibiotics like sulfonamides, tetracyclines, penicillin and
chloramphenicol are effective against HS if administered early. Because
of the rapid course of the disease and the difficulty to access the
animals,
antimicrobial
therapy
remains
impracticable.
Although
resistance to multiple antibiotics has been reported for some strains of
P. multocida but it is not described for HS serotypes. The complete
nucleotide sequence of a naturally occurring 5.36 kb streptomycin and
sulphonamide resistance plasmid, designated pIG1, isolated from typed
D P. multocida was determined by Wright et al., 1997.
Verma et al. (2004) revealed that the majority of P. multocida
isolates were sensitive to enrofloxacin, gentamycin and chloramphenicol
and
resistant
to
sulphadimidine,
oxytetracycline,
streptomycin,
amoxicillin and tetracycline. Shivachandra et al. (2004) found that
avain strains were most sensitive to chloramphenicol (73.98%), followed
by enrofloxacin (71.54%), lincomycin (64.23%) norfloxacin (61.79%) and
doxycycline-HCl (56.91%).
Arora et al. (2005) showed that P. multocida isolates from
different animal species were sensitive to enrofloxacin, pefloxacin,
gentamicin and chloramphenicol and resistant to cotrimoxazole,
erythromycin and streptomycin.
2.6
BIOLOGICALS IN CURRENT USE
Haemorrhagic septicaemia if treated well in time, responds to
antibacterial agents. But due to acute nature of the disease and short
duration of clinical symptoms, it becomes difficult to treat the animal
well in time. So timely vaccination is the only practical approach to
control the disease. Various vaccine types have been developed against
this disease, among which broth bacterin, oil adjuvant vaccine, double
emulsion vaccine and a live vaccine
are quite common (Verma and
Jaiswal, 1998).
2.6.1 Bacterins
Broth bacterins provide only one and a half to two months
immunity and may induce toxic shock due to the endotoxin present in
the bacterin (Carter and De Alwis, 1989).
2.6.2 Alum percipitated vaccine
Iyer et al. (1955) developed alum precipitated vaccine, which
provided immunity for a period of six months. Despite the fact that APV
is known to provide immunity for short duration but it is still the most
common vaccine in use, as it is the easiest vaccine to inject. It
constitutes
nearly
80
%
of
haemorrhagic
septicaemia
vaccine
production in south-east and south Asian countries (Myint and Jones,
2007).
2.6.3 Adjuvant vaccine (OAV)
Bains and Jones (1955) have described for the first time the oil
adjuvant vaccine (OAV) using formalin killed whole organism which is
now being used in our country as a prophylactic measure. OAV
provided stronger immunity lasting atleast for one year but due to its
high viscosity it is not convenient to administer which makes it
unpopular among field users (De Alwis, 1992).
Shah et al. (1997)
prepared an improved oil adjuvant vaccine using Mineral oil and Mercol
52 as adjuvant together with Span 85 and Tween 85 as emulsifiers
which when tested in cattle showed good protection upto 250 days upon
challenge with 109 viable bacteria sub-cutaneously. Atthi et al. (2001)
studied
the
onset
and
duration
of
immunity
to
haemorrhagic
septicaemia oil adjuvant (water in oil) vaccine in cattle containing P.
multocida B: 2,5 bacterin. The vaccine was reported to be protective in
cattle even after 24 months of post vaccination. Burns et al. (2003)
evaluated the effect of heat on oil-emulsion P. multocida bacterin.
Commercial bacterin when heated to 410C for 5 hr reduced local tissue
reaction without any deleterious effect on its immunity as measured by
ELISA and other challenge studies. Indonesia and Srilanka have been
successfully using lower levels of lanoline, an emulsifying agent, to
reduce viscosity. In Thailand, OAV with lower viscosity has been
developed and routinely used (FAO, 2005).
2.6.4 Multiple Emulsion Vaccine (MEV)
To overcome from the problem of high viscosity of OAV, double
emulsion vaccine (Yadav and Ahooja, 1983) has been developed.
Chandrasekaran et al. (1991) showed that double emulsion vaccine
was as effective as the oil adjuvant vaccine and demonstrated immunity
for 52 weeks of post vaccination. Verma (1995) reported that MEV
against HS was able to protect calves upto one year. Verma and
Jaiswal (1997) vaccinated calves with multiple emulsion HS vaccine
and observed that both humoral and cell mediated immune response
contribute to protect vaccinated animals.
2.6.5 Live vaccines
A live vaccine made with P. multocida serotype B:3, 4 isolated
from a fallow deer protected cattle against a serotype B:2 challenge and
conferred immunity against HS for one year in cattle vaccinated
subcutaneously (Myint et al., 1987). A local reaction in the form of
lump at the site of inoculation (S/C and I/D) was observed in
vaccinated animals (Khar et al., 1992). Safety, efficacy and crossprotectivity of a live intranasal HS vaccine were tested in young cattle
and buffaloes in Myanmar (Myint et al., 2005). Seven months after
vaccination, three out of three buffaloes were protected and twelve
months after vaccination, three of four buffaloes were protected against
a subcutaneous challenge with serotype B:2. The serum of vaccinated
cattle cross-protected mice against infection with P. multocida serotypes
E:2, F:3, 4 and A:3, 4.
2.7
OUTER MEMBRANE PROTEINS
2.7.1 Structure and function
The outer membrane of gram-negative bacteria contains a
number of components: phospholipid layer, outer membrane proteins
(OMP), and lipolpoysaccharides (LPS). It contains a number of proteins
including major outer membrane porins and other proteins. It protects
gram-negative bacteria against a harsh environment. At the same time,
the embedded proteins fulfill a number of tasks that are crucial to the
bacterial cell, such as solute and protein translocation, as well as signal
transduction.
Henderson et al. (1996) mentioned that approximately 50% of
the dry matters of outer membranes of gram-negative bacteria consisted
of more than twenty immunochemically different fragments. Some of
the
major
outer
membrane
proteins
called
porins,
are
highly
immunogenic and expose epitopes on the bacterial surfaces. They are
conserved in the bacterial species because they show high homology in
primary amino acid sequence and secondary structures and are
antigenically related (Jeanteur et al., 1991).
About 50% of the outer membrane mass consists of protein,
either in the form of integral membrane proteins or as lipoproteins that
are anchored to the membrane by means of N-terminally attached
lipids. More than a dozen, different outer membrane lipoproteins have
been identified in E.coli (Blattner et al., 1997). Exposure at the cell
surface has led to the exploitation of outer membrane proteins by
pathogenic agents such as bacteriophages and bacteriocins (Table 2.1).
During the last two decades we have been witnessing exciting
advances in the field of membrane proteins. The three-dimensional
structures of membrane proteins revealed the existence of two
structural motifs, α-helices and β-barrels in these proteins. β-barrel
membrane proteins (outer membrane proteins, OMPs) differ from the
all-β structural class of globular proteins due to the presence of a lipid
environment and different structural motifs compared with α-helical
membrane proteins.
Table 2.1: Structure and functional features of prototype outer membrane proteins from E.coli.
Protein family
Small β barrel
membrane
anchors
Small β barrel
membrane
anchors
Membrane integral
enzymes
General(non
specific)
porins
Substrate
specific porins
TonBdependent
receptors
Prototype protein
OmpA
OmpX
PldA(OMPLA)
OmpF
LamB
FhuA
Function
Physical linkage
between OM and
peptidoglycan
Neutralizing
host defence
mechanisms
Hydrolysis of
phospholiids
Diffusion pore
for ions and
other small
molecules
Maltose and
maltodextrin
uptake
Uptake of ironsiderophore
complexes;
Bacteriophages
K3, M1
K20
Bacteriocins
Colicin K,colicin
L
Colicin N
Oligomeric state
Monomer
Monomer
8
8
Number of
transmembrane β
strands
Domain structure
Two co-linear
Domains
One domain
Monomer/dimer
12
One domain
λ
Homotrimer
16
One domain
T1, T5
Colicin M
Homotrimer
18
One domain
Monomer
22
Two inter
connected
domains
The dielectric constant within a lipid bilayer is very low compared
with that of the aqueous environment. Membrane proteins thus expose
a hydrophobic surface to the lipid bilayer core, a property that
distinguishes them from water-soluble proteins. This also implies that a
maximum number of hydrogen bonds of the protein segment located in
the lipid bilayer are formed. Therefore, long before the first structure of
a membrane protein was determined, it was predicted that only regular
secondary structure elements (a-helices and b-sheet) could occur within
the lipid bilayer in order to saturate the entire main-chain hydrogen
bonding potential. All donor and acceptor groups could be saturated
either intrasegmentally, as in the case of α helices, or intersegmentally
by the formation of hydrogen bonds between adjacent β-strands
(Rosenbusch, 1988).
2.7.2 Influence of amino acid residues in sequence and structure of
OMPs
The analysis on the three dimensional structures of OMPs shows
the presence of Ser, Asn and Gln residues which play important role in
the stability and function of OMPs and also required in the formation of
β-barrel structures in the membrane, stability of binding pockets and
the function of OMPs (Gromiha et al., 2006).
OmpA is one of the major outer membrane proteins and plays a
structural role in the integrity of the bacterial surface. X-ray structural
analysis of OmpA revealed that eight antiparallel β-strands are
connected by three short periplasmic turns and the presence of four
relatively long surface exposed loops (Fig 2.1). A cluster of highly
conserved charged residues (Lys-12, Glu-52, Arg-96, Arg-138 and Glu140) was uncovered, which builds up a network of salt bridges and
hydrogen bonds and may explain extraordinarily high thermal stability
of OmpA.
Fig 2.1:
Structure of OmpA transmembrane domain
The general diffusion pores formed by porins allow the diffusion of
hydrophilic molecules (<600 Da) and show no particular substrate
specificity despite some selectivity for either cations or anions. Porins
form homotrimers in the outer membrane. In each monomer, 16 βstrands span the outer membrane. Unlike the other loops, the third
loop, L3, is not exposed at the cell surface but folds back into the
barrel, forming a constriction zone at half the height of the channel,
giving it an hourglass-like shape. Therefore, this loop contributes
significantly to the permeability properties, such as exclusion limit and
ion selectivity, of the pore. Interestingly, this loop contains a sequence
motif, PEFGG, that is highly conserved among enterobacterial porins
(Jeanteur et al., 1991). At the constriction site, a strong transverse
electrostatic field is caused by acidic residues in loop L3 and a cluster
of basic residues at the opposite barrel wall (Fig 2.2).
Fig 2.2: Constriction site of OmpF
2.8 OMP - PRIME CANDIDATE FOR VACCINE
The surface of Gram-negative bacteria is critical for interaction of
the bacterium with the host cell environment as it mediates nutrient
uptake, secretion of toxins and other products and is involved in
avoidance of the host immune system (Neiman et al., 2004).
Furthermore, it is the bacterial surface molecules that are the targets
for host immunity. Bacterial surface proteins have been shown to be
important for conferring protective immunity in a range of infection
models (Brown et al., 2001; Frazer et al., 2006). P. multocida PlpB
protein was identified as a cross-protective antigen (Tabatabai and
Zehr, 2004; Rimler, 2001) and this protein is located in the outer
membrane (Boyce et al., 2006). Outer membrane proteins also
promote adherence to host cell surfaces and are therefore likely to be
involved in P. multocida virulence (Boyle and Finlay 2003).
Bioinformatics analysis of the P. multocida genome predicted 129
proteins as secreted, located in the outer membrane, or lipoproteins
(Al-Hasani et al., 2007). They identified novel immunogens like PlpB,
Lpp, OmpA, Omp16, Omph, PM1614 (Outer membrane antigenic
lipoprotein) and Oma87 in P. multocida which are expressed during
natural infection in chicken with the organism.
Outer membrane proteins of P. multocida are believed to be
important protective antigens. Knight et al. (1990) isolated outer
membrane of various serogroups of P. multocida . Their electrophoretic
patterns were remarkably different from those of P. haemolytica. SDSPAGE and immunoblot analysis of P. multocida serotype B:2 revealed
that two polypeptides of 30 and 37 kDa were prominent. It was
postulated that the 30 and 37 kDa polypeptides were the major
polypeptides present in serogroup B:2.
Confer et al. (1996) quantified the serum antibody response of
outer membrane proteins (OMPs) of P. multocida A: 3 for cattle
vaccinated with the homologous serogroup. Antibody responses to
individual OMP were detected by western blot analysis and were
generated by densitometry. Antibody to 11 prominent OMPs of 100, 97,
90, 85, 74, 53, 46, 35, 32, 21, and 16 kDa were identified and
quantified.
Purified OMP from P. multocida serotype B:2 was used to prepare
vaccine against HS (Pati et al., 1996). Buffalo calves vaccinated with
OMP provided complete protection against challenge with virulent
organism. Srivastava et al., (1998) have grown P. multocida serotype
B: 2 under iron restricted condition to enhance the production of iron
regulated OMPs. No difference in conferring protection in mice and
rabbits was observed using vaccines prepared from P. multocida cells
grown under iron deficient and iron sufficient medium. But the Ab titres
were found to be significantly higher in case of vaccine consisting P.
multocida grown under iron-restricted condition .The author suggested
that vaccine prepared from P. multocida under iron restricted condition
might be more effective than the vaccine prepared from the organism
grown in normal medium. However, proteomic analysis of IROMPs
identified PM0805, that was upregulated and the other, OmpW, that
was down regulated under low-iron conditions (Boyce et al., 2006).
Srivastava et al., (1998) extracted OMP from P. multocida B:2
and studied its ability to immunize against P. multocida infection and
resist phagocytosis by murine peritoneal macrophages. Inoculation of
OMP in rabbits resulted in the production of agglutinating antibodies,
which passively protected mice against P. multocida challenge and
caused lysis of virulent P. multocida in vitro.
Confer et al. (2001) vaccinated rabbits intranasally on day 0, 7
and 14 with P. multocida A:3 outer membrane protein (OMP) expressing
iron regulated OMP (IROMP). Some vaccines included cholera toxin (CT)
as an adjuvant. OMP-CT vaccinated individuals developed enhanced
resistance with both mucosal and systemic antibody responses against
challenge exposure but intranasal counts were not significantly
reduced. Vaccination with IROMP-CT resulted in mucosal and systemic
antibodies to challenge exposure and significantly reduced nasal
bacterial counts.
Chawak et al. (2001) characterized the OMP extracted from the
P. multocida grown in iron sufficient and iron restricted media by SDSPAGE analysis and immunoblotting. Under iron sufficient condition
presence of nine proteins ranging from 17kDa to 87kDa and seven
immunogens with 17 and 25.7 as immunodominant proteins were
observed using immunoblotting. Under iron restricted medium an
additional protein of 97.8kDa was found to be immunogenic.
Pal et al. (2002) studied the heat modifiable characteristics of
OMP from vaccine strain to know their basic characteristics on event of
temperature rise. A major band of 32kDa and two minor bands of
approximately 38 and 28kDa were found to be heat modifiable. They
suggested that boiling at 1000C in the presence of β-mercaptoethanol
for 5 min is sufficient for characterization of OMP by SDS-PAGE.
Tomer et al. (2002) characterized the outer membrane proteins
of vaccine strain and observed about 20 polypeptide bands with
molecular weight ranging from 16 to 90 kDa. They found three
polypeptides of MW 31, 33 and 37 kDa as the major OMPs. Anshu et
al. (2005) revealed the presence of 11 protein fractions of HS vaccine
strain and found two major OMPs of 32 and 35 kDa in capsular type B
isolates. Arora et al. (2007) found a homogenous OMP profiles of 17
different P. multocida isolates of bovine origin comprising 23 different
polypeptides bands ranging in molecular weight from 13 to 94 kDa. On
the basis of stain intensity, 32 kDa protein appeared to be major
protein band followed by the presence of two bands of 25 kDa and 28
kDa. Apart from this other significant protein bands were of 13, 34,
44.5, 46, 80 and 84 kDa. The 32 kDa protein was found to be the
immunodominant along with 25 kDa protein band in all the isolates.
Thus 32 kDa protein band represented a type specific marker for the
Asian HS isolates, so it might be a potent candidate antigen for a
subunit HS vaccine and can be exploited in immunodiagnosis of HS.
OmpA, a β-barrel ion channel protein, has been reported to have
a direct role in bacterial adhesion. Homologs of this protein are
important adhesins in Escherichia coli, Haemophillus influenza and
other bacteria. Recombinant OmpA binds to bovine kidney cells and
interacts with host extracellular molecules like heparin and fibronectin
(Dabo et al., 2003).
The ability of P. multocida to bind with host extracellular matrix
protein has shown that the bacteria can adhere to fibronectin and
collagen type IX. Proteins identified as possible adhesins include OmpA,
Oma87, Pm1069 and iron related proteins, Tbp (Transferrin binding
protein) and the putative TonB receptor HgbA (Dabo et al., 2005).
The outer membrane protein A (OmpA) of P. multocida A:1 was
cloned and sequenced by Dabo et al. (2003). Mice vaccinated with
purified Omp28 (member of OmpA family) developed a significant
antibody titre compared to the control but did not protect the animal
from a homologous intraperitoneal bacterial challenge. Even though
Omp28 is surface exposed and antigenic but it did not stimulate
immunity (Gatto et al., 2002).
Earlier studies on the P. multocida outer membrane showed that
a 37kDa protein was among five identified as possible protective
immunogens
based
on
radioimmunoprecipitation
results
using
protective immune rabbit sera and on their location in the outer
membrane (Lu et al., 1988). Monoclonal antibodies raised against the
37kDa protein were able to passively protect mice against infection with
P. multocida with strong protection afforded against homologous
strains, and some limited protection against heterologous strains (Lu et
al., 1991).
A protein of similar molecular mass (39 kDa) was identified in the
P. multocida A:3 strain P1059; its expression was in relation to the
presence and amount of capsule present on the cell (Borrathybay et
al., 2003b; Ali et al., 2004a). P. multocida can adhere and invade
chicken embryo fibroblasts. Adherence of the organism was inhibited by
both monoclonal and polyclonal antibodies raised against the 39 kDa
protein (Borrathybay et al., 2003a; Al-haj Ali et al., 2004; Ali et al.,
2004 a, b). The actual identity of 39 kDa protein was reported, but
recently a 39 kDa protein which can stimulate cross-serotype protection
was also isolated from outer membrane protein extracts of the same A:3
strain, P1059 (Rimler, 2001). This protein was identified as PlpB
(Pasteurella
lipoprotein
B),
using
peptide
mass
fingerprinting
(Tabatabai and Zehr, 2004) and is predicted to be an ABC transport
protein required for the uptake of methionine into the cell (Merlin et
al., 2002).
Antibodies raised against major outer membrane proteins (OmpH)
of P. multocida is provided some protection against the disease.
Monoclonal antibodies specific for OmpH passively protected mice
against P. multocida challenge (Marandi and Mittal, 1997) and
vaccination with the native OmpH protein (not recombinant) elicited
protective immunity in birds against homologous challenge (Luo et al.,
1997). In addition, antibodies raised to an OmpH synthetic peptide,
Cyclic-L2, provided partial protection in chickens against homologous
challenge (Luo et al., 1999). OmpH had significant similarity in both
primary and secondary structure with those of other serotypes.
Antibodies
raised
against
recombinant
OmpH
provided
strong
protection so it can be an useful vaccine candidate antigen for P.
multocida.
The immunoprotective efficacy of P. multocida (6:B) outer
membrane proteins (OMPs) was examined by Basagoudanavar et al.
(2006) and it was found that OMPs are important determinants of
immunoprotection hence can serve as vaccine candidates against
haemorrhagic septicaemia.
2.9
CLONING OF OUTER MEMBRANE PROTEINS
Among the methods that have been developed for genetic
manipulation; one of the most challenging task is the expression of gene
into a heterologous system. With the advancement of recombinant DNA
technology, a number of E. coli expression systems have been designed
and proved to be efficient means for mass production of naturally
scarce protein.
Ruffolo and Adler (1996) have cloned and expressed an 87 kDa
outer membrane antigen Oma 87 from P. multocida serotype A: causing
fowl cholera. The sequence of this gene showed extensive similarity with
D15 protective surface Ag of H. influenza. The expressed protein was
localized predominantly in the membrane fraction. Antiserum raised
against recombinant protein protected the animals against homologous
challenge.
The gene encoding major OMP of P. multocida X-73 has been
identified and sequenced from a genomic library by Luo et al. (1997).
Expression of ompH gene was performed in E. coli system using
expression vector pQE30 and pQE32. Recombinant protein conferred
immunity to chicken against homologous challenge. The gene was
found to be distributed among 15 serotypes of P. multocida.
The gene omp16, encoding a 16 kDa outer membrane protein,
was amplified and cloned into a pBluescript SK (-) vector and sequenced
by Goswami et al., (2004). Presence of this gene was reported among
different serotypes of P. multocida and found to localize in a 6.0 kb Hind
II of the P. multocida genome.
Haemolysins or cytolysins are membrane-damaging agents which
have been described as bacterial virulence factors due to their ability to
lyse erythrocytes and other host cells, and therefore inducing a greater
inflammatory response (Ruffolo et al., 2000). P. multocida was found
to be haemolytic under anaerobic conditions. Gene ahpA is responsible
for haemolysis of bovine and equine erythrocytes. The ahpA gene of P.
multocida B:2 was cloned and sequenced by Rani et al. (2006). It was
an inner membrane protein with two strong hydrophobic regions at the
N and C terminals.
2.10 E. COLI EXPRESSION SYSTEM
Expression
of
a
functional
protein
depends
upon
correct
transcription of the gene, efficient translation and in many cases post
translational processing and targeting of the nascent polypeptide. Any
fault in these steps may result in non expression of a gene. E. coli is
used most commonly for expression of foreign genes. Large numbers of
vectors which are compatible to E. coli system are available for high
level expression of a desired gene. Availability of multiple cloning sites
in the newer generation expression vectors make the task easy for
cloning of gene in correct orientation and proper reading frame.
Expression vectors like pQE, pGEX, pMAL and pCAL etc are
available in which foreign genes are expressed as fusion proteins. E. coli
system for direct expression and secretion has been developed and
refined (Goeddel et al., 1990). Many proteins of biological interest are
produced in very limited quantities in natural condition, thereby
making it difficult to study those proteins having the property of
conferring protection. To make a subunit vaccine, large quantities of
antigens must be produced and purified. For this purpose bacterial
systems are quite convenient and express antigens at very high levels.
There are several bacterial expression systems that can be used, but E.
coli is the most popular (Makrides et al., 1996; William et al., 1995).
Bacterial systems are suitable for expressing vaccine antigens that do
not require any post translational modification. Indeed E. coli has been
used extensively for the expression of large number of genes at levels
sufficient for structural biochemical analysis and even product
development (Rosenberg et al., 1996). Several advantages of E. coli
have ensured that it will remain valuable organism for high level
production of recombinant protein (Olins and Lee, 1993). Higher
expression of foreign protein in E. coli is deleterious to the host,
resulting in decreased growth rate or even lysis of the cell. Thus it is
essential that a tightly regulated, inducible promoter system be used to
limit protein expression until the cells have grown to maximal density in
culture. Mostly foreign proteins expressed at high levels in E. coli are in
the form of insoluble inclusion bodies. This can preclude purification
unless a chaotropic agent first solubilizes it.
Some epitopes have strict conformational requirements that may
be affected by treatment with a chaotropic agent. Thus exposure to
such agent may affect the protective efficacy of the antigen. A vaccine
antigen solubilized using a chaotropic agent may permanently lose its
biological activity, but may still posses the epitope required to elicit a
protective immune response.
E. coli has been used to express antigens for enormous variety of
vaccines. Many of these bacterial expressed recombinant antigens conferred
protective immunity. These antigens include bacterial derived proteins,
genetically modified toxins and virus derived peptides as products.
2.11 NEW APPROACHES OF VACCINE DEVELOPMENT
Looking the present scenario of the available vaccines it is
difficult to control the disease. To overcome from limitations of present
available vaccines, several new approaches have been utilized.
2.11.1 Subunit vaccine
Vaccines made from well defined components of microorganisms
are called subunit vaccines. These vaccines can be based on peptides,
proteins or polysaccharides that have been shown to contain protective
epitopes. Many of the cell surface carbohydrates of pathogenic bacteria
like capsular polysaccharides are important antigenic determinants for
vaccine development.
2.11.2 Recombinant subunit vaccine
A subunit vaccine that is produced using recombinant techniques
is called a recombinant vaccine. These vaccines are created by utilizing
bacteria or yeast to produce large quantities of a single viral or bacterial
protein. This protein is then purified, injected into the patient, and the
patient's immune system makes antibodies against the disease agent's
protein, protecting the patient from natural disease (Stephen, 1998).
Recombinant DNA technology allows controlled production of
protein subunit vaccines in heterologous hosts. Such strategies have
several advantages. Recombinant strategies further offer the possibility
of delivery protein subunits with the help of live delivery systems,
bacterial or viral or even as antigen encoding genes, so-called nucleic
acid vaccines.
1.
Recombinant technology begins with the isolation of a gene of
interest. The gene is then inserted into a vector and cloned. A
vector is a piece of DNA that is capable of independent growth;
commonly used vectors are bacterial plasmids and viral phages.
The gene of interest (foreign DNA) is integrated into the plasmid
or phage, and this is referred to as recombinant DNA.
2.
Before introducing the vector containing foreign DNA into the
host cells to express the protein, it must be cloned. Cloning is
necessary to produce numerous copies of the DNA since the
initial supply is inadequate to insert into host cells.
3.
Once the vector is isolated in large quantities, it can be
introduced into the desired host cells such as mammalian, yeast,
or special bacterial cells. The host cells will then synthesize the
foreign protein from the recombinant DNA. When the cells are
grown in vast quantities, the foreign or recombinant protein can
be isolated and purified in large amounts.
Table 2.2: Recombinant subunit vaccines and examples of their
advantages (+) and drawbacks (-)
Recombinant vaccine
Advantages/drawbacks
Protein immunogens
+ No risk of pathogenicity since
pathogenic organism is not present
the
+Efficient production systems available
Live delivery system
-
Multiple doses required
+ May induce both humoral and cellular
responses
Bacterial
Viral
Nucleic acid vaccines
- Risk of reversion when using attenuated
pathogens as carriers
+ Surface display of antigens possible
+Mucosal administration possible
+Efficient induction of cellular responses
+ No risk of pathogenicity
+May induce both humoral and cellular
responses
-Variable immune responses
-Inefficient transfection
(Source: Stahl et al., 2000)
Recent advances in immunology and protein engineering have
allowed the design and production of recombinant subunit vaccines
(Liljeqvist and Stahl., 1999). The epitopes recognized by neutralizing
antibodies are usually found in just one or a few proteins present on
the surface of the pathogenic organism. Isolation of the genes encoding
such epitope-carrying protein immunogens and their expression in
heterologous hosts form the basis of recombinant-subunit-vaccine
development (Stahl et al., 2000).
OmpH is a major antigenic outer membrane protein from P.
multocida and has high immunogenicity in antibody production.
Although the short fragment of recombinant OmpH has lower protective
immunity while antibodies against full-length of recombinant OmpH
appear to be protective in mice. Therefore, recombinant OmpH might be
an useful vaccine candidate antigen (Lee et al., 2007).
Leptospiral putative outer membrane proteins (OMPs) were cloned
and expressed by Chang et al. (2007). Primary screening for
immunoprotective potential was performed in hamsters challenged with
an LD50 inoculum of low passage serovar Pomona. They found that
rLp1454, rLp1118, and rMceII showed protection individually and
synergistically against serovar Pomona infection and might be helpful in
development of multicomponent vaccine for leptospirosis.
Recombinant outer membrane proteins of V. parahaemolyticus
zj2003, including OmpW, OmpV, OmpU and OmpK,were found to be
immunogenic during in vivo infection (Mao et al., 2007). This was the
first report of successful vaccination against V. parahaemolyticus with
purified recombinant outer membrane proteins. Zhang et al. (2007)
concluded that a multicomponent OMP antigen i.e. the fusion protein rOmpk-GAPDH could induce a higher frequency of immune effectors
than a single OMP (r-Ompk or r-GAPDH). These results presented a
good suggestion for the subunit vaccine design based on the OMPs of
gram-negative pathogens.
2.11.2.1 Live delivery system
Beside
the
possibility
of
producing
recombinant
protein
immunogens in heterologous hosts, technologies to construct live viral
and bacterial vaccine delivery vectors carrying foreign immunogens
have been developed. Wang et al. (2007) evaluated the humoral and
cellular
immune
responses
of
recombinant
Mycobacterium bovis
Bacillus Calmette-Guérin strains expressing the antigen ESAT-6 from
Mycobacterium tuberculosis in BALB/c mice. In immunized mice, the
IgG antibody titres, IFN-gamma level and splenocyte proliferation index
of rBCG group were significantly higher than that of BCG group and
therefore
might
be
the
better
vaccine
against
Mycobacterium
tuberculosis.
Zhou et al. (2007) found that recombinant adenovirus containing
the major outer membrane protein gene of Chlamydophila psittaci might
be a candidate vaccine against avian chlamydiosis.
2.11.2.2 Nucleic acid vaccines
Nucleic acid vaccines constitute a new class of recombinant
subunit vaccine, consisting of, for example, plasmid DNA containing the
gene encoding the antigen of interest under the control of a strong
mammalian promoter. Besides this, DNA vaccines are exceedingly
potent in priming the immune response as evidenced by the generation
of very high immune responses upon booster immunization with a low
dose of a traditional vaccine expressing the same antigen (Feng et al.,
2001). The antigen encoding gene will be expressed by the vaccine upon
delivery of the plasmid DNA. DNA vaccines expressing three variola
major (VARV) antigens (A30, B7 and F8) and their recombinant protein
counterparts elicited high-titer, cross-reactive, VACV neutralizing
antibody responses in mice (Sakhatskyy et al., 2007).
2.11.3 Aromatic mutant
Other molecular approaches to vaccine development include the
creation of attenuated strain by mutation of specific targets. Such
attenuated strain would be used as live vaccines, which are usually
more effective than killed whole cell subunit vaccines because they have
the advantage of a natural route of entry into the host, which allows
targeting of immunostimulatory factors to the same sites of the immune
system that occur in the natural infection. Such mutants can be
created by allelic exchange and further attenuated in mouse models.
The aroA gene encodes 5-enolpyruvylshikimate-3-phosphate (EPSP)
synthase, which is involved in the conversion of shikimic acid to
chorismic acid, a common intermediate in the biosynthesis of aromatic
amino acids. Mutation in the aroA gene creates a dependency for growth
on aromatic compounds that are not available in the host, as this
pathway is not operative in mammalian cells. This means that aroA
mutants are capable of only limited replication before they are cleared
from the host. As described by Homchampa et al. (1992, 1997) and
Tabatabaei et al. (2002, 2007), attenuated aroA mutants of P.
multocida serotypes A and B:2 causing fowl cholera and HS respectively,
have been shown to provide protection against challenge in chickens
(Scott et al., 1999) and mouse (Tabatabaei et al., 2002, 2007),
respectively.
On the other hand, HS is only one of a wide range of diseases
caused by P. multocida. Live aroA mutant organisms may be of use as
vaccines for other pasteurelloses, such as rabbit snuffle, fowl cholera
and pneumonic form of bovine and ovine pasteurellosis.
The production of bacterial ghosts is a new approach in non living
vaccine technology and is based on the controlled expression of the
PhiX174 derived lysis gene E. Bacterial ghosts are empty cells devoid of
cytoplasmic and genomic material. Marchart et al. (2003) used
Pasteurella ghosts for immunization of rabbits and mice. They reported
that animals which received 1.15x 108 ghosts and a challenge dose of
upto 60 cfu (LD90) showed 100% protection.
Materials &
Methods
Chapter 3
Materials and Methods
3.1 BACTERIAL STRAINS
Pasteurella multocida serotype B:2 (vaccine strain P52), obtained
from Indian Veterinary Research Institute, Izatnagar, U.P. India
was
used in this study and maintained in blood agar medium. It was
routinely cultured in brain-heart infusion (BHI) broth.
Escherichia coli DH5 used in the cloning experiments was
purchased from Bangalore Genei and grown in Luria broth (LB).
All the cultures were stored at 40C in their respective agar media
in slants. Their glycerol stocks were maintained at -200C.
3.3 CHEMICALS
All the chemicals and solvents used in the study were purchased
from Himedia, Sisco Research Laboratory, Bangalore Genei and Sigma.
3.4 GLASSWARE AND PLASTICWARES
All the glassware used in the study were of Borosil. Microfuge tubes
and micropipette tips were purchased from Axygen, Tarsons etc.
3.5 EQUIPMENTS USED
Name
Make
Refrigerated centrifuge
Remi
Rotatory shaker
Remi
pH meter
Sartorious
3.6
Electrophoresis assembly
Bangalore Genei
Electrophoresis Power supply
Bangalore Genei
Electronic balance
Sartorious
Laminar bench
(MAC) Macro scientific works
Thermocycler
Biometra
Water bath
Biometra
REVIVAL AND CHARACTERIZATION OF P. multocida P52
STRAIN
The culture of P. multocida P52 was revived on Brain Heart
Infusion (BHI: Hi Media Ltd., India) broth and blood agar and the
identity of the culture was tested by Gram’s staining, growth on
McConkey (Hi Media, India) agar, oxidase and indole reaction.
3.7 PATHOGENICITY TEST OF THE ORGANISM
Approximately 0.5ml of 10-5 dilution of 18 hour old culture of P52
was injected intraperitoneally into three healthy mice. All of them died
within 36-38 hours of inoculation. Post mortem was conducted and the
organisms were reisolated in pure culture from heart blood and spleen
of the dead animals.
3.8 P. multocida (B:2) SPECIFIC PCR
Type specific P. multocida (B:2) PCR was performed to amplify the
unique gene sequences in P. multocida B:2 serotype by using KTSP61KTT 72 primers as per method described by Townsand et al., (1998).
Primer1. KTSP 61: 5’ATCCGCTAACACACTCTC 3’
Primer2. KTT 72: 5’AGGCTCGTTTGGATTATGAAG 3’
Reaction mixture for PCR
Total volume
25μl
Assay buffer (10X) with
1.5mM MgCl2
2.5 μl (1X)
dNTPs
2μl (200μmol)
Template DNA
5μl (40 ng)
Primers
2 μl + 2 μl (20pmol each)
Taq polymerase
1.0U
Total volume was maintained with sterilized ultra pure water.
Amplification was done using the following programme
Temperature
Time
Initial denaturation
94 oC
5 min
Denaturation
94 oC
1 min
Anealing
55 oC
1 min
Extension
72 oC
1 min
72 oC
10 min
30 cycles from step 2
Final extension
3.8.1 Electrophoresis And Determination Of PCR Product
Five μl of amplified product was mixed with 1 μl of 6X loading dye
and loaded onto 1.5% agarose gel using 1X TAE buffer. Sample was run
at 5volts/cm for one hour and gel was observed under ultraviolet light
to visualize the bands. The band size was determined by comparing
with standard molecular weight marker. Gel was photographed by gel
documentation system (AlphaImager 2200 Documentation and Analysis
System, Alpha Innotech Corporation, USA).
3.9
1.
ISOLATION OF BACTERIAL WHOLE CELL PROTEINS
Single colony of bacterial culture was inoculated in 1ml of broth
and grown overnight.
2.
One ml from overnight grown culture was inoculated in 1 litre of
broth and again grown for 12 hours.
3.
Culture was centrifuged at 10,000 rpm for 10 minutes and pellet
were washed thrice in PBS.
4.
Pellet were then suspended in 10mM HEPES buffer and sonicated
for 10 minutes in ice.
5.
Cell suspension was centrifuged for 30 minutes at 10,000 Xg.
6.
The clear supernatant was collected and filtered through .45μm
filter paper.
7.
The filtrate was used as sonicated antigen for raising hyperimmune
serum.
8.
Protein concentration was estimated by Lowry’s method (1951).
3.10 PURIFICATION OF OUTER MEMBRANE PROTEINS (Choi-Kim
et al., 1991)
1.
For the purification of OMPs sonicated antigen was centrifuged at
1,00,000 x g for 60 minutes at 4oC in an ultracentrifuge.
2.
The pellet obtained were resuspended in 2 ml of 2 % (w/v) sodium
lauryl sarcosine in 10 mM HEPES buffer (pH 7.4) and incubated at
room temperature for 1 hour.
3.
The mixture was centrifuged at 1,00,000 x g for 1 hour at 40C and
pellet were washed twice with distilled water.
4.
The pellet containing purified OMPs were dissolved in PBS.
5.
Obtained proteins (OMPs) were used for analysis of polypeptides
and immunoblotting.
3.11 SODIUM
DODECYL
SULPHATE-POLYACRYLAMIDE
ELECTROPHORESIS (SDS-PAGE)
GEL
Whole cell protein and outer membrane proteins of P. multocida
P52 were analyzed by SDS-PAGE using the method of Laemmli (1970).
The vertical slab gel electrophoresis apparatus (Atto, Japan) with glass
plates of 14 x 14 cm and spacer of 1.5 mm thickness was used for
performing SDS-PAGE by discontinous buffer system using 12%
resolving gel and 5% stacking gel.
3.11.1 Gel preparation
The vertical slab gel unit was assembled in casting mode with
1.5mm spacers. The resolving gel solution was prepared according to
the given composition. The solution was mixed well and poured into the
sandwich to a level of 4 cm from the top, then 3 ml of distilled water
was gently added along the uniform gel surface after polymerization.
The water layer was poured off.
Stacking gel was prepared according to the described composition
and overlayed on the resolving gel. After putting the comb into the
sandwich, the gel was allowed to polymerize.
3.11.2 Sample Preparation
Fifty μl of the sample was mixed with equal volume of 2X sample
buffer and boiled for 5 minutes in a water bath.
3.11.3 Loading and running the gel
The comb was slowly removed from the gel after polymerization. The
wells were filled with electrode buffer. The samples and markers protein
were underplayed in each well. The lower and upper chambers of the tank
were also filled with electrode buffer. The electrophoresis unit was
connected to the power pack and the gel was run initially at 80V and then
at 120V till the tracking dye reached to the bottom of the gel.
3.11.4 Staining and Destaining
The gel mould was carefully disassembled after completion of the
run and gel was stained with coomassie brilliant blue for two hours.
Later, the gel was destained in destaining solution with intermitant
shaking. Finally gel was rinsed in distilled water and scanned.
3.11.5 Determination of molecular weight of ploypeptides
After destaining of gel by coomassie brilliant blue R, the
molecular weight of polypeptides were determined by the molecular
weight analysis tool of the gel documentation system. The molecular
weight of protein was determined by standard protein molecular weight
markers (Fermantas).
3.12 RAISING OF HYPER IMMUNE SERUM
Hyper immune serum against sonicated antigen of P. multocida
P52 was raised in New Zealand white rabbit in three doses as described
in Table 3.1.
1.
For the initial dose antigen was emulsified with equal volume of
Fruend’s complete adjuvant and injected in rabbit. Booster doses of
antigen were given along with Freund’s incomplete adjuvant.
2.
The first booster dose was given on day 10 and repeated thrice at
10 days interval.
3.
After 7 days of last booster, rabbit was bled and serum was
separated and stored at -20 0C.
Table 3.1: Immunization Schedule
Sl.
Days of
Amount of Volume of
No immunization antigen (μg) antigen (μl)
Volume of
Adjuvant (μl)
Total
Mode of
injection
1000
Subcutaneous,
Intradermal,
Footpad
1
0
500
500
500(complete
fruend’s
adjuvant)
2
10
250
250
500(incomplete
fruend’s
adjuvant)
500
Subcutaneous,
Intradermal,
Footpad
3
20
250
250
500(incomplete
fruend’s
adjuvant)
500
Subcutaneous,
Intradermal,
Footpad
3.13 AGAR GEL PRECIPITATION TEST (AGPT)
Agar gel precipitation test was performed according to the method
of Ouchterlony (1949) to detect antibodies against P. multocida in
hyper immune sera raised in rabbits.
Agarose (1%) was prepared in PBS, pH 7.4 in boiling water bath.
The molten agarose was poured on Petri dish and allowed to solidify.
One central and four peripheral wells of 3mm diameter at a distance of
3mm were punched and sealed with molten agarose .The central well
was filled with sonicated antigen of P.multocida and peripheral wells
were charged with hyper immune sera raised against it.
3.14 IDENTIFICATION OF IMMUNOGENIC PROTEINS BY WESTERN
BLOTTING
Polypeptides separated on 12% SDS-PAGE using discontinuous
buffer system were transferred on nitrocellulose membrane by semi-dry
method
of
electroblotting
(Electrophoretic
transfer
unit,
Atto
Corporation, Japan) as per the method of Towbin et al. (1979) with
minor modifications. Five sheets of thick Whatman filter paper (Hi
Media, India) were soaked in transfer buffer and placed on centre of
graphite anode electrode plate. The distilled water soaked NCM
(Nitrocellulose membrane) was then placed on the top of filter papers.
The gel was placed on the membrane followed by stacking of five sheets
of filter papers soaked in transfer buffer. The assembled transfer stack
was covered with cathode plate and current of 0.8 mA/cm2 was applied
for 1 hour. To assess the quality of transfer, prestained marker
(Bangalore Genei, India) was used.
The electrophoretic blot was kept in blocking buffer overnight at 4 oC.
After washing with wash buffer four times for 5 minutes each, the
membrane was incubated at 37 oC for 1 hour with hyperimmune serum
against whole cell antigen diluted 1:50 in blocking buffer. After washing 4
times with washing buffer each for 5 minutes, the blot was again incubated
at 37 oC for 1 hour with anti- rabbit horseradish peroxidase conjugate at a
dilution of 1:2000 in blocking buffer. The blot as described above, was
transferred to freshly prepared 50 ml of substrate solution containing
diaminobenzidine tetrahydrochloride and 6 μl of 30% (v/v) hydrogen
peroxide for few minutes. The reaction was stopped by washing with
distilled water. After drying, the membrane was stored in a dark place.
3.15 CLONING OF OUTER MEMBRANE PROTEIN GENES
3.15.1 Extraction of Genomic DNA
Genomic DNA of Pasteurella multocida P52 was isolated by C-TAB
method (Wilson, 1987).
1.
Single colony of P. multocida P52 was inoculated in 2ml of broth
and grown overnight at 37 oC.
2.
From the overnight culture 25 μl was inoculated in 25 ml of broth
and grown overnight at 37 oC.
3.
Cells were harvested at 10,000rpm for 10min. at 4 oC.
4.
Pellet were resuspended in lysis buffer containing 2ml TE (10mm
Tris, 1mM EDTA), 400μl of sodium dodecyl sulphate (10%w/v) and
10μl of proteinase K (20mg/ml) and mixed properly by rapid
pipetting.
5.
Mixture was incubated at 370C for 3 hours.
6.
400μl of 5M NaCl was added in cell lysate and after adding 300μl of CTAB (7.5%) the tubes were kept at 600C in a water bath for 10 min.
7.
Same volume of chloroform was added in tubes, mixed well and the
tubes were centrifuged at 12,000 rpm for 15 minutes.
8.
Twenty μg/ml of RNase was added in supernatant and incubated
at 370C for 30 minutes.
9.
Equal volume of phenol: chloroform to the above reaction mixture
was added, mixed by vortexing and centrifuged at 12,000 rpm for
15 minutes.
10. Aqueous layer was separated and equal volume of Chloroform:
Isoamylalcohol (24: 1) was added. It was mixed thoroughly and
centrifuged at 12,000 rpm for 10 min.
11. In the aqueous layer, double volume of chilled ethanol and 1/10
volume of 3M sodium acetate were added and was kept overnight
at -200C.
12. Mixture was centrifuged at 12,000 rpm for 15 minutes at 4 oC.
13. Supernatant was discarded and the pellet was dissolved in 100μl
TE (10mM Tris and 1mM EDTA, pH 8.0).
3.15.1.1 Quantification of DNA
The absorbance of DNA samples were measured on DU-640
spectrophotometer at 260 and 280 nm. The concentration of DNA was
estimated by using the formula:
Concentration of DNA (μg/ml) = OD260 x 50 x dilution factor
3.15.2 PCR Based Amplification of outer membrane protein genes
For amplification of Omp87 gene:
Oligonucleotide primers were designed from published sequence
of P. multocida serotype A:1 (Ruffolo and Adler, 1996) with linkers at
5’ end having BamHI and HindIII restriction endonucleases sites. The
sequences of primers were as follows:
Primer 1. 5’ CCGGATCCATGAAAAAACTTTTAATTGC 3’
Primer 2. 5’ CAAGCTTTTAGAACGTCCCACCAATGCTG 3’
For amplification of Omp34 gene
Primers were designed from OmpH gene of Pasteurella multocida
serotype D. The sequences of primers were as follows:
Primer 1. 5’ TTAGAAGTGTACGCGTAAACCAA 3’
Primer 2. 5’ GCAACAGTTTACAATCAAGACG 3’
Both the primers were synthesized by Sigma trends Bio-products
Pvt. Ltd.
Reaction mixture for PCR reaction
Total volume
25μl
Assay buffer (10X) with
1.5mM MgCl2
2.5 μl
dNTPs(each)
2μl
Template DNA
5μl (40 ng)
Primers
2 μl + 2 μl (20pmole each)
Taq polymerase
1.0U
Total volume was maintained with sterilized ultra pure water.
Amplification was done using the following programme
Temperature
Time
Initial denaturation
94 oC
5 min
Denaturation
94 oC
1 min
Anealing
51 oC
1 min
Extension
72 oC
1 min
72 oC
10 min
30 cycles from step 2
Final extension
3.15.2.1 Electrophoresis and determination of PCR product
Five μl of amplified product were mixed with 1 μl of 6X loading
dye and loaded on to 1.5% agarose gel in 1X TAE buffer and sample was
run at 5volts/cm for one hour. Gel was observed under ultraviolet light
to visualize the bands. The size of amplified DNA was determined by
comparing
with
photographed
by
standard
gel
molecular
weight
marker
and
were
documentation
system
(AlphaImager
2200
Documentation and Analysis System, Alpha Innotech Corporation,
USA).
3.15.3 Purification of amplified products
Amplified products were analyzed on 1.5% agarose gel and eluted
from gel using QIA quick gel extraction kit (Qiagen) as per the
manufacturer’s recommendations.
1.
Band of interest from gel was excised with a clean, sharp scalpel.
2.
Three volumes of buffer QG was added and mixture was incubated
at 50oC for 10 minutes untill the gel slice has been completely
dissolved.
3.
DNA/agarose solution was applied to a minelute column assembled
in 2 ml collection tube and centrifuged at 10,000 rpm for 10
minutes.
4.
Flow through was discarded and 750 μl of PE buffer was added to
the column and centrifuged at 10,000 rpm for 10 minutes.
5.
Column was centrifuged for an additional 1 minute at 13,000 rpm.
6.
Column was placed in 1.5ml microcentrifuge tube and 30 μl of EB
(10mM Tris-Cl, pH 8.5) was used for elution of DNA.
3.15.4 Molecular Cloning
Cloning of the purified PCR products were carried out using
pGEM-T Easy vector (Promega, USA) as per the manufacturer’s
recommendations (Fig 3.1). Various steps and procedures used for
cloning are as follows:
Fig 3.1: Map of pGEM-T Easy vector
3.15.4.1 Ligation
Concentration of amplified gene eluted from agarose gel was
measured by spectrophotometer at 260 nm and 280 nm. Amount of
interest was calculated using the following formula and mixed with vector.
Vector to insert ratio 
Size of PCR product (bp)
 50 ng
3000 bp (size of vector)
Reaction mixture
Test
Control(+)
2X rapid ligation buffer
5 μl
5 μl
pGEM-T Easy Vector (50ng)
1 μl
1 μl
PCR product
3 μl
_
_
2 μl
T4 DNA ligase(3 Weiss units/ μl)
1 μl
1 μl
Deionized water (to make up 10 μl)
10μl
10 μl
Control insert DNA
Ligation product was incubated at 40C for overnight and transformed in
competent cells
3.15.4.2 Preparation of Competent cells
Competent cells were prepared by the method described by Sambrook
et al., (1989). The method involved following steps:
1.
Single colony of E.coli DH5 α was inoculated in 5ml of L.B. broth
and incubated at 370C overnight in an orbital shaker.
2.
From the overnight grown culture, 50 μl cells were inoculated into
5 ml fresh LB broth and allowed to grow with gentle shaking in the
orbital shaker at 200 rpm. After 2.5 to 3 hours of the incubation,
when the culture was in the log phase of growth cycle and the cell
density was enough to give an OD of 0.6 at A600 (absorbance at 600
nm),the culture was cooled on ice.
3.
The cooled cells were centrifuged at 8,500 rpm for 10 minutes at
40C for pelleting.
4.
Supernatant was decanted and pellet was resuspended in 750 μl
ice-cold 0.1M CaCl2.
5.
Cells were again centrifuged at 8,500 rpm for 10 minutes at 40C.
6.
Supernatant was decanted and cell pellet was again resuspended
in 200 μl of ice cold 0.1M CaCl2 and incubated in ice for 1 hour.
Prepared competent cells were stored at -200C till further use or
used for fresh transformation.
3.15.4.3 Preparation of LB agar plates with Amp/IPTG/X-Gal
Before transformation, LB agar plates were prepared containing
ampicillin
(Promega,
(HiMedia,
USA)
India),
and
IPTG
X-Gal
(Isopropyl-β-D
thiogalactoside)
(5-bromo-4-chloro-3-indolyl-
β-D-
galactopyranoside) (Promega, USA). LB agar medium was prepared,
autoclaved and allowed to cool to 50 0C. The media was poured into
petri plates and allowed to solidify. Then 100 μl of 100mM IPTG and 20
μl of 50 mg/ml X-gal were spread over the surface of a plate and
allowed to absorb for 30 minutes at 37 0C prior to use.
3.10.8.4 Transformation
1.
Total 10 μl of ligated mixture was added in 200 μl of competent
cells.
2.
The reaction mixture was incubated in ice (40C) for 30 minutes.
3.
Heat shock was given to the mixture at 420C for 90 seconds in a
swirling water bath and immediately returned to 40C for 5 minutes.
4.
One ml of LB for the survival of competent cells was then added,
mixed and incubated at 370C for 2 hours.
5.
The mixture was then centrifuged at 7,500 rpm for 8 minutes and
supernatant was decanted leaving about 100 μl in the tubes.
6.
The pellet was then resuspended in 100μl and total contents were
spreaded on LB plates containing 100μg/ml ampicillin with X-Gal
and IPTG.
The plates were incubated at 370C for 24 hrs and colonies were
observed.
Control was also processed in the same way but it was without
any DNA and plated on both Amp+ and Amp- LB plates.
3.15.4.5 Screening of Recombinant Clones
The recombinant clones were screened by PCR and confirmed by
restriction enzyme analysis of the desired insert.
3.15.4.6 Colony PCR
The recombinant colonies were screened by PCR using the same
set of primers to detect the presence of desired insert as described in
section of PCR by cell-lysis method. Some recombinant colonies were
selected from the LB agar plate containing ampicillin, IPTG and X-gal.
1.
Single isolated white colonies were picked up from the plates and
inoculated in 5 ml LB broth with 100 μg/ml ampicillin and
incubated overnight at 370C.
2.
Pellet was obtained by centrifugation of culture at 8000 rpm for 10
minutes.
3.
Pellet was resuspended in 100μl of sterilized triple distilled water.
4.
Then the tubes were boiled at 1000C for 10 minutes and chilled
immediately in ice.
5.
The tubes were centrifuged at 5000 rpm for 10 minutes to remove
cell debris.
6.
From the supernatant 5μl was used as template for PCR reaction.
3.15.4.7 Isolation of Plasmid DNA
The plasmid DNA were isolated from PCR positive clones and also
from non recombinant clone i.e. from blue colony ( acted as negative
control). An improved alkali lysis method for miniprep plasmid isolation
was used to isolate the plasmid DNA (Sambrook et al., 1989).
1.
A single colony was inoculated in 5 ml of LB broth containing
100μg/ml of ampicillin and cells were grown overnight at 370C.
2.
1.5 ml culture was pelleted in eppendorf at 10,000 rpm for 10
minutes at 4 oC.
3.
The pellet was suspended in 100μl of ice cold solution I, vortexed
well and kept at room temperature for 5 minutes.
4.
Freshly prepared lysis solution I (200μl) was added, mixed by
vortexing and kept at room temperature for 5 minutes.
5.
Solution III (150μl) was added and mixed by inverting the tube.
6.
The tubes were incubated on ice and then centrifuged at 10,000
rpm for 10 min.
7.
Supernatant was transferred in fresh eppendorf and equal volume
of Phenol: Chloroform: isoamyl alcohol (25:24:1) was added and
mixed well. The tubes then centrifuged at 12,000 rpm for 10 min.
8.
The upper aqueous phase was transferred to a fresh tube.
9.
Double volume of ice cold ethanol was added, supernatant and
contents were mixed by inversion and incubated at -200C for 2 hrs.
10. The contents were centrifuged at 14,000 rpm to pellet the
plasmid(s) 40C for 10 min. and supernatant is poured off.
11. The pellet was washed with 70% ethanol.
12. Pellet was dried and suspended in 50μl TE containing RNase A (20
μg/ml) and tubes were kept at 37 oC for RNase treatment and then
stored at -20 oC.
3.15.4.7.1 Agarose gel electrophoresis of plasmid DNA
Five μl plasmid DNA was mixed with 1 μl of 6X loading dye and
subjected to electrophoresis for 2.5 hrs in 1% agarose gel at 5volt/cm.
Gels were stained in ethidium bromide(0.5 μg/ml) and photographed by
gel documentation system.
3.15.4.8 Restriction Enzyme Digestion
To confirm the insert in clones, plasmid were digested with Not I
enzyme. The digestion was carried out in 10 μl of reaction mixture
containing the following:
Plasmid
7.5 μl
Assay buffer
1.5 μl
Not I
1 μl
The eppendorf was given a brief spin and incubated at 37 0C for 3
hrs. The digested mixture was loaded on 0.8% gel along with molecular
weight marker and visualized on transilluminator.
3.16 SEQUENCING AND HOMOLOGY SEARCH
The cloned PCR products in pGEMT-Easy vector were subjected
to sequencing using Universal T7 and SP6 primers at the DBT
Sponsored
National
DNA
sequencing
facility
of
Department
of
Biochemistry, University of Delhi, South Campus, New Delhi.
The observed data for nucleotide sequence of omp34 gene and
omp87 gene were compared with the reported sequence of ompH gene of
P. multocida CU vaccine strain serotype A: 3,4 and omp87 gene of P.
multocida serotype A:1 respectively using Laser gene DNASTAR
software. Analysis of both the sequences at amino acid level were also
analyzed by same software.
Homologies of the sequences obtained after sequencing were
searched on the website www.ncbi.nlm.nih.gov by using BLAST (Basic
Local Alignment Search Tool) and FASTA programme.
3.16.1 Sequence Alignment
Nucleotides sequences of outer membrane protein genes of
P. multocida P52 studied were analyzed and amino acid sequences were
deduced by using ‘EditSeq’ Programme of Lasergene (DNASTAR Inc,
USA) software. Both nucleotide and amino acid sequences were aligned
separately by using ClustalW method of ‘MegAlign’ programme. For
comparison, nucleotide sequences of known serotypes were included in
the study. Sequences for omp34 were retrieved from EMBL databank.
Serotype B:2 accession number- EU162755, serotype
3,4-
U52213.1, serotype 10- U52207, serotype 12- U52209, serotype 11U52208, serotype D- AY864815.1, serotype 9- U52206, serotype D:4AY603962, serotype 15- U52212, serotype 14- U52211, serotype
1- AF416986, serotype A1- U50907, serotype 7- U52204, serotype
6- U52203, serotype 13-U52210 were used for sequence alignment and
further in silico analysis study.
3.16.2 Phylogenetic Analysis
Aligned nucleotide sequences were subjected to phylogenetic
analysis using ‘MegAlign’ programme of Lasergene software to derive the
ancestral relationship among the sequences of different serotypes.
3.17 STRUCTURAL ANALYSIS OF OUTER MEMBRANE PROTEINS
Amino acid sequences deduced from the nucleotide sequence of
outer membrane proteins were further used for its structural analysis.
Primary and secondary structure analysis of the proteins were done by
using different online server,viz., ProtParam (ExPASy Server: ProtParam
(Gasteiger et al., 2005) Pfam (http://www.pfam.janelia.org/cgi.bin/)
and PDBsum (http://www.ebi.ac.uk/pubsum), respectively.
Results
Chapter 4
Results
4.1
REVIVAL AND CHARACTERIZATION P. multocida P52 STRAIN
Freeze dried culture of P. multocida P52 was revived on Brain
Heart Infusion (BHI) broth and blood agar. Characteristic nonhaemolytic colonies were obtained which were gram-negative cocobacilli
that showed oxidase positive and indole negative reactions.
4.2
PATHOGENICITY TEST OF THE ORGANISM
Intraperitoneally inoculated mice with 0.5ml of 10-5 dilution of
18h old broth culture of P52 died within 24-48 hours. The re-isolated
colonies showed characteristic of P. multocida.
4.3
P. multocida (B:2) SPECIFIC PCR
Under the standard PCR conditions, as described in Materials
and Methods B: 2 specific PCR was done using specific primers and P.
multocida P52 DNA as template. Five μl of amplified product was
analyzed by electrophoresis in 1% agarose gel stained with ethidium
bromide. The size of amplicon was compared with DNA ladder and
found to be of approximately 620 bp (Fig 1).
4.4 PURIFICATION OF OUTER MEMBRANE PROTEINS
The P. multocida P52 outer membrane proteins were obtained by
the method as described by Choi-kim et al., (1991). Fig (2)
demonstrated the presence of eight Omps bands in P. multocida P52.
The molecular weights of the major polypeptide bands were in the range
of 16 to 87 kDa. The major bands were of 16, 27, 30, 34, 37, 44, 68 and
87 kDa.
4.5
AGAR GEL IMMUNODIFFUSION TEST
Immunization of rabbits with whole cell antigen of P. multocida
P52 strain apparently stimulated the production of antiserum. A
positive immunodiffusion test was observed when this hyperimmune
sera was tested with whole cell antigen (Fig 3). It produced three
precipitin lines in all three sets indicating the presence of at least three
major immunogens in sonicated extract of P. multocida.
4.6
ANALYSIS OF IMMUNOGENIC POLYPEPTIDES BY WESTERN
BLOT
Potential immunogens of P. multocida P52 were identified by
electroblot immunoassay. Immunoblotting was performed using the
hyperimmune serum raised against the whole cell antigen in order to
detect immunogenic proteins in outer membrane proteins. Polypeptides
separated on 12% SDS-PAGE were transferred to nitrocellulose
membrane by semi-dry system. Presence of seven polypeptides of 16,
30, 34, 37, 44, 68 and 87 kDa size was reported showing reaction with
antiserum (Fig 4).
4.7
CLONING OF OUTER MEMBRANE PROTEIN GENES
In this study we approached PCR cloning method. On the basis of
available database of Omp genes in other serotypes of P. multocida,
primers were designed by using DNASTAR software.
4.7.1 Genomic DNA extraction
Genomic DNA was extracted from overnight grown culture of P.
multocida P52 and visualized on 0.8% agarose gel (Fig5). For
quantification of DNA, absorbance was taken at 260 and 280nm. The
yield of genomic DNA was 1.8 which confirms the purity of the isolated
DNA sample. Agarose gel electrophoresis of the isolated DNA revealed
that DNA was relatively intact and without RNA.
4.7.2 PCR Amplification of Outer Membrane Genes:
Under the standard PCR conditions, as described in Materials
and Methods Omp gene(s) were amplified using specific primers and P.
multocida P52 DNA as template. Five μl of amplified product was
analyzed by electrophoresis on 1% agarose gel stained with ethidium
bromide. The size of amplicon was compared with DNA ladder.
Amplified products of Omp87 and Omp34 were 2373bp and 942bp
respectively (Fig 6 and 7).
4.7.3 Elution, Ligation and Transformation
Both the amplicons (amplified products of 2372 and 942) were
eluted from agarose gel and ligated individually in pGEM-T vector by
keeping for overnight at 40 and subsequently transformed in E.coli DH5
α cells. Transformants were plated on LB media containing 100 μg/ml
ampicillin and IPTG/X-Gal and incubated at 37
0C
for 24 hrs.
Competent cells were also plated on Amp+ and Amp- plates, which
served as positive and negative control respectively. Competent cells did
not appear on Amp+ plates but grew on Amp- plates when incubated for
24 hrs.
4.7.4 Screening of recombinants
After incubation, recombinant and non-recombinant colonies
appeared as white and blue respectively (Fig 8 and 9). A total of 20
white colonies were picked up from plates and inoculated in LB medium
with 100 μg/ml ampicillin and were grown overnight
4.7.5 PCR based screening of recombinant clones
Recombinant colonies were screened for the amplification of the
desired inserts by PCR using the same primer pair that was used to
amplify the gene. Plasmid DNA(s) were isolated from recombinant clones
(white colonies) and subjected to polymerase chain reaction. A negative
control (plasmid from blue colony) was also used to check any non
specific amplification. Single amplified product of the expected size were
found only in positive samples (Fig 10 and 11). Plasmid DNA was
isolated from each of the PCR positive colonies grown in LB ampicillin
broth for 12-16 hrs by alkaline lysis method (Fig 12).
4.7.6 Insert release of both the genes
PCR positive clones were further subjected to RE digestion for
the confirmation of the correct size of the inserts. The recombinant
plasmids were subjected to single digestion with NotI enzyme at 37 0C
for overnight and visualized on 1.5% agarose along with DNA ladder.
Insert of 2373 bp was released on digestion of recombinant plasmid of
omp87 with NotI (Fig 13). Restriction analysis showed a fragment of
approximate 1 kb and vector size of 3 kb. Undigested recombinant
plasmid showed a band of 4 kb, while non recombinant (control)
plasmid was showing a single band of 3 kb (Fig 14). These results
confirmed the insertion of the desired omp genes into the vector.
4.8
SEQUENCING OF OMP GENES
The cloned omp genes were sequenced at the DNA sequencing
facility of University of Delhi, South campus, New Delhi. Sequencing
results revealed complete open reading frame (ORF) of 2121bp and
942bp for omp87 and 34 kDa respectively. Sequences were analysed in
‘Editseq’ programme (DNASTAR) for further analysis. The nucleotide
and deduced amino acid sequences of both the genes are shown in
appendix (Annexure I).
4.8.1 Sequence analysis of omp87 gene
Cloning and sequencing of omp87 gene revealed the length of
sequence to be 2121 bp. The sequence analysis of coding region of
omp87 gene showed GC content of 40-46%. Nucleotide sequence of P.
multocida P52 showed 94.8% similarity with that of P. multocida
serotype A:1 strain while the homology at amino acid level was 95.2%.
The results of data alignment are presented as residue substitutions of
ClustalW in Table 4.1.
Fig 16 : Alignment of nucleotide sequence of omp87
10
1
1
20
30
40
A T G A A A A A A C T T T T A A T T G C G A G C T T A T T A T T T G G G T C A A omp87FCA1
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - omp87HSB2
50
60
70
80
41
1
C C A C T G C A T T T G C T G C G C C G T T T G T A G T G A A A G A C A T T C G omp87FCA1
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - omp87HSB2
81
1
T G T T G A C G G T G T T C A A G C A G G T A C A G A A G G A A G T G T A T T A omp87FCA1
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - omp87HSB2
121
1
G C T A C G C T T C C T G T T C G T G T T G G G C A G C G A G C A A C A G A T A omp87FCA1
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - omp87HSB2
161
1
A C G A T A T T G C T A A T G T G G T A C G A A A A T T A T T C C T G A G T G G omp87FCA1
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - omp87HSB2
90
130
170
210
100
140
180
220
110
150
190
230
120
160
200
240
201
1
G C A A T A T G A T G A T G T G A A A G C A A G T C G C G A A G G G A A T A C T omp87FCA1
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - omp87HSB2
241
1
T T A G T T G T G A C A G T C A T G C C T A A A C C T G T T A T T T C A A A C G omp87FCA1
- - - - - - - - - - - - - - - A T G C C T A A A C C T G T T A T T T C A A A C G omp87HSB2
281
26
T C G T G A T T G T C G G T A A T A A A T C G A T T C C T G A T G A A G C A A T omp87FCA1
T C G T G A T T G A C G G T A A T A A A T C G A T T C C T G A T G A A G C A A T omp87HSB2
321
66
T A A A C A A A A C T T A G A T G C G A A T G G C T T T A A A G T T G G T G A T omp87FCA1
T A A A C A A A A C T T A G A T G C G A A T G G C T T T A A A G T C G G T G A T omp87HSB2
250
290
330
370
260
300
340
380
270
310
350
390
280
320
360
400
361
106
G T A T T A A A C C G T G C T A A A T T A G A A G A A T T C C G T A A A G G G A omp87FCA1
G T A T T A A A C C G T G C T A A A T T A G A A G A A T T T C G G A A A G G G A omp87HSB2
401
146
T T G T T G A A C A C T A C A A C A G T G T C G G T C G C T A T A A T G C G A A omp87FCA1
T T A T C G A A C A C T A C A A T A G T G T C G G T C G C T A T A A T G C G A A omp87HSB2
410
420
430
440
450
441
186
460
470
480
A G T T G A T G C T A T C G T G A A T A C A T T A C C A A A T A A T A G T G C A omp87FCA1
G G T A G A G G C T A T C G T G A A T A C A C T A C C A A A T A A T A G C G C G omp87HSB2
490
500
510
520
481
226
G A A A T T A A A A T T C A A A T T A A T G A A G A T G A T G T G G C A C T C T omp87FCA1
G A A A T T A A A A T T C A A A T T A A T G A A G A T G A T G T T G C A C T A T omp87HSB2
521
266
T T A A A G A A A T T A C G T T T G A A G G T A A C G A A G C G T T T A G T A G omp87FCA1
T T A A A G A A A T T A T T T T T G A A G G T A A T C A A G C A T T T A G C A G omp87HSB2
561
306
C G G A A A A T T A G C C G A T C A G A T G G A G T T A C A A A C C G A T T C G omp87FCA1
C A G T A A A T T A G A A G A T C A A A T G G A G C T T C A A A C A G A T G C A omp87HSB2
601
346
T G G T G G A A A C T G T T T G G C A A T A A A T T T G A T C A A A C C C A A T omp87FCA1
T G G T G G A A A T T G T T T G G T A A C A A A T T T G A T C A A A C C C A A T omp87HSB2
530
570
610
650
540
580
620
660
550
590
630
670
560
600
640
680
641
386
T C A A T A A A G A T T T A G A A A C C T T A C G T A G C T A T T A T T T A G A omp87FCA1
T C A A T A A A G A T T T A G A G A C C T T A C G T A G C T A T T A T T T A G A omp87HSB2
681
426
T C G T G G T T A C G C G C A A T T T C A G A T T C T T G A T A C G G A T G T C omp87FCA1
T C G T G G T T A C G C G C A A T T C C A A A T T T T A G A T A C T G A T A T C omp87HSB2
721
466
A A A T T A A G T G A T G A T A A A A A A G A A C C G T G T C T T A T - - - - - omp87FCA1
A A A T T A A G T G A T G A T A A A A A A G A A G C G C G T G T C A T T A T T A omp87HSB2
756
506
A A G T G A A - G A A G G T G A C T T A T A T A C G G T G A A A - A C G C G C G omp87FCA1
A A G T G A A A G A A G G T G A C T T A T A T A C A G T G A A A T G C G C G C G omp87HSB2
690
730
770
810
700
740
780
820
710
750
790
830
720
760
800
840
794
546
T A T C T G G G G G G A T G T G G G G T G G C A T G T C A G C A G A A C T T G C omp87FCA1
T A T T C T G G G G G A T G T G G G - T G G C A T G T C A G C A G A A C T T G C omp87HSB2
834
585
C C C G A T T T T A G A G A C G A T T C A A T T G A A T G G T C T T T T C C G T omp87FCA1
T C C G A T T T T A G A T A C G A T T C A A C T A A A T G G T C T T T T C C G T omp87HSB2
850
860
870
880
850
834
585
860
870
880
C C C G A T T T T A G A G A C G A T T C A A T T G A A T G G T C T T T T C C G T omp87FCA1
T C C G A T T T T A G A T A C G A T T C A A C T A A A T G G T C T T T T C C G T omp87HSB2
890
900
910
920
874
625
C G C A C A A G T G T A T T G G A A G T A G A A C A A C G C A A T A A A T C G A omp87FCA1
C G C G C A A A C G T A T T G G A A G T T G A A C A A C G C A T T A A A T C G A omp87HSB2
914
665
A G T T A G G T G A A A G A G G T T A T G C A A C T G C G C A A G T C A A T G T omp87FCA1
A G T T A G G T G A A A G A G G T T A T G C G A C T G C G C A A G T C A A T G T omp87HSB2
954
705
T C A C C C G A C A T T T G A C G A A C A A G A T A A A A C G A T T T C G T T A omp87FCA1
T C A C C C G A C A T T T G A C G A A C A A G A T A A A A C G A T T T C G T T A omp87HSB2
994
745
G A T T T T A T T G T T G A A G C A G G C A A A A G T T A T A C G G T T C G C C omp87FCA1
G A T T T T A T T G T T G A A G C A G G C A A A A G T T A T A C G G T T C G C C omp87HSB2
930
970
1010
1050
940
980
1020
1060
950
990
1030
1070
960
1000
1040
1080
1034 A A A T T C G T T T T G A A G G C A A T A C A A G T A G T G C A G A T A G C A C omp87FCA1
785
A A A T T C G T T T T G A A G G C A A T A C A A G T A G T G C A G A T A G C A C omp87HSB2
1090
1100
1110
1120
1074 C T T G C G T C A G G A A A T G C G T C A A C A A G A A G G C G C T T G G T T A omp87FCA1
825
C T T A C G T C A G G A A A T G C G T C A A C A A G A A G G C G C T T G G C T A omp87HSB2
1130
1140
1150
1160
1114 T C C T C G G A G T T G G T T G A G T T A G G T A A A T T A C G T T T A G A T C omp87FCA1
865
T C C T C G G A G T T G G T T G A G T T A G G T A A A T T A C G T T T A G A T C omp87HSB2
1170
1180
1190
1200
1154 G T A C G G G T T A C T T T G A A A G C G T A G A A A C C A A A A C A G A A G C omp87FCA1
905
G T A C G G G G T T C T T T G A G A G T G T A G A A A C C A A A A C A G A A G C omp87HSB2
1210
1220
1230
1240
1194 T A T C C C G G G T T C T G A T C A A G T C G A T G T G A T T T A T A A G G T C omp87FCA1
945
T A T C C C G G G T T C T G A T C A A G T C G A T G T G A T T T A T A A A G T C omp87HSB2
1250
1260
1270
1280
1234 A A A G A G C G T A A T A C G G G T A G C A T T A A C T T T G G T A T T G G T T omp87FCA1
985
A A A G A G C G T A A T A C G G G T A G C A T T A A C T T T G G T A T T G G T T omp87HSB2
1290
1300
1310
1320
1274 A T G G T A C A G A A A G T G G G T T G A G T T A C C A A G C C A G T A T T A A omp87FCA1
1025 A T G G T A C A G A A A G T G G G T T G A G C T A C C A A G C C A G T A T T A A omp87HSB2
1290
1300
1310
1320
1274 A T G G T A C A G A A A G T G G G T T G A G T T A C C A A G C C A G T A T T A A omp87FCA1
1025 A T G G T A C A G A A A G T G G G T T G A G C T A C C A A G C C A G T A T T A A omp87HSB2
1330
1340
1350
1360
1314 A C A G G A T A A C T T C T T A G G A A T G G G A T C T T C C A T T A G T T T A omp87FCA1
1065 A C A G G A T A A C T T C T T A G G A A T G G G A T C T T C T A T T A G T T T A omp87HSB2
1370
1380
1390
1400
1354 G G T G G G A C G C G T A A T G A T T A C G G T A C T A C G G T G A A T C T C G omp87FCA1
1105 G G T G G G A C G C G T A A T G A C T A C G G T A C T A C A A T C A A T C T T G omp87HSB2
1410
1420
1430
1440
1394 G T T A T A A T G A G C C G T A C T T T A C G A A A G A T G G T G T G A G C C T omp87FCA1
1145 G T T A T A A T G A G C C G T A C T T T A C C A A A G A T G G T G T G A G C C T omp87HSB2
1450
1460
1470
1480
1434 C G G T G G T A A T G T T T C C T T T G A A G A A T A T G A T A G T T C A A A A omp87FCA1
1185 C G G T G G C A A T G T T T T C T T T G A A G A A T A T G A T A G T T C C A A A omp87HSB2
1490
1500
1510
1520
1474 A G T A A T A C C T C T G C G G G C T A T G G A C G G A C T A G C T A T G G T G omp87FCA1
1225 A G T A A T A C C T C T G C G G C C T A T G G A C G G A C T A G C T A T G G T G omp87HSB2
1530
1540
1550
1560
1514 G T A A T T T A A C A C T A G G C T T C C C A G T G A A T G A G A A T A A C T C omp87FCA1
1265 G T A A T T T G A C A C T A G G C T T T C C G G T G A A T G A G A A T A A C T C omp87HSB2
1570
1580
1590
1600
1554 A T A T T A T C T T G G T G T A G G C T A T A C G T A T A A T A A A T T G A A G omp87FCA1
1305 A T A T T A T C T T G G T G T G G G C T A T A C G T A T A A T A A A T T G A A G omp87HSB2
1610
1620
1630
1640
1594 A A T A T C G C G C C G G A A T A T A A T C G T G A T T T A T A T C G C C A A T omp87FCA1
1345 A A T A T C G C G C C G G A A T A T A A T C G T G A T T T A T A T C G C C A A T omp87HSB2
1650
1660
1670
1680
1634 C G A T G A A A T A T A A T G A T T C T T G G A C C T T T A A A T C G C A C G A omp87FCA1
1385 C A A T G A A A T A T A A T G A T T C T T G G A C C T T T A A A T C G C A C G A omp87HSB2
1690
1700
1710
1720
1674 T T T T G A T T T G T C T T T T G G T T G G A A T T A T A A C A G T C T T A A C omp87FCA1
1425 T T T T G A T T T G T C T T T T G G T T G G A A T T A T A A C A G C C T T A A C omp87HSB2
1730
1740
1750
1760
1714 C G C G G C T A T T T C C C A A C C A A A G G G G T A C G T G C C A A T A T T G omp87FCA1
1465 C G T G G C T A T T T C C C A A C T A A A G G G G T A C G T G C C A A T A T T G omp87HSB2
1770
1780
1790
1800
1754 G A G G A C G A G T G A C C A T T C C G G G C T C A G A T A A T A A A T A T T A omp87FCA1
1505 G T G G A C G A G T G A C C A T T C C G G G C T C A G A C A A T A A A T A T T A omp87HSB2
1810
1820
1830
1840
1794 T A A A C T C A A T G C A G A A G C A C A A G G G T T C T A T C C G T T A G A T omp87FCA1
1545 T A A A C T C A A T G C A G A A G C A C A A G G G T T C T A T C C G T T A G A T omp87HSB2
1850
1860
1870
1880
1834 C G T G A A C A T G G T T G G G T A C T T T C A A G C C G T A T T A G T G C C T omp87FCA1
1585 C G T G A A C A T G G T T G G G T A C T T T C A A G C C G T A T T A G T G C C T omp87HSB2
1890
1900
1910
1920
1874 C T T T T G C G G A T G G A T T T A G C G G T A A G C G T T T G C C G T T C T A omp87FCA1
1625 C T T T T G C T G A T G G A T T T G G T G G T A A G C G T T T G C C G T T C T A omp87HSB2
1930
1940
1950
1960
1914 T C A A T A T T A T A G C G C A G G C G G T A T C G G G A G T T T A C G T G G C omp87FCA1
1665 T C A A T A T T A T A G C G C A G G C G G T A T C G G G A G T T T A C G T G G C omp87HSB2
1970
1980
1990
2000
1954 T T T G C C T A T G G T G C G A T T G G A C C A A A T G C A A T T T A T C G C A omp87FCA1
1705 T T T G C C T A T G G T G C G A T T G G A C C A A A T G C A A T T T A T C G T A omp87HSB2
2010
2020
2030
2040
1994 C A C G T C A A T G T C C T G A C A G C T A T T G T T T A G T C A G T A G T G A omp87FCA1
1745 C A C G T C A A T G T C C T G A C A G C T A T T G T T T A G T C A G T A G C G A omp87HSB2
2050
2060
2070
2080
2034 T G T G A T T G G G G G G A A T G C A A T G G T C A C C G C C A G T A C C G A A omp87FCA1
1785 T G T G A T T G G G G G G A A T G C A A T G G T C A C C G C C A G T A C C G A A omp87HSB2
2090
2100
2110
2120
2074 C T C A T T G T T C C A A C A C C A T T T G T C G C A G A T A A A A A T C A A A omp87FCA1
1825 C T C A T T G T C C C A A C A C C A T T T G T C G C A G A T A A A A A T C A A A omp87HSB2
2130
2140
2150
2160
2114 A C T C A G T G A G A A C T T C T C T G T T T G T G G A T G C G G C T A G T G T omp87FCA1
1865 A C T C A G T A A G A A C T T C T T T G T T T G T G G A T G C C G C A A G T G T omp87HSB2
2170
2180
2190
2200
2154 G T G G A A T A C G C G T T G G A A A G C A G A G G A T A A A G C A A A A T T T omp87FCA1
1905 G T G G A A T A C G C G T T G G A A A G C A G A G G A T A A A G C A A A A T T T omp87HSB2
2210
2220
2230
2240
2194 G C A A A A T T A A A T G T G C C C G A T T A C A G T G A T C C A A G T C G C G omp87FCA1
1945 G C A A A A T T G A A T G T G C C A G A T T A C A G T G A C C C A A G T C G C G omp87HSB2
2250
2260
2270
2280
2234 T T C G T G C T T C A G C T G G G G T A G C G C T T C A A T G G C A A T C G C C omp87FCA1
1985 T T C G T G C T T C A G C T G G G G T G G C G C T T C A A T G G C A A T C G C C omp87HSB2
2290
2300
2310
2320
2274 A A T T G G A C C G T T A G T G T T C T C T T A T G C G A A A C C C C T T A A G omp87FCA1
2025 A A T T G G A C C G T T G G T G T T C T C T T A T G C G A A A C C T C T T A A G omp87HSB2
2330
2340
2350
2360
2314 A A A T A C C A A G G C G A T G A A A T T G A G C A G T T C C A A T T C A G C A omp87FCA1
2065 A A A T A C C A A G G C G A T G A A A T T G A G C A G T T C C A A T T C A G C A omp87HSB2
2370
2354 T T G G T G G G A C G T T C T A A
2105 T T G G T G G G A C G T T C T A A
omp87FCA1
omp87HSB2
Fig 17 : Alignment of amino acid sequence of omp87
10
1
1
20
30
40
M K K L L I A S L L F G S T T A F A A P F V V K D I R V D G V Q A G T E G S V L omp87FCA1
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - omp87HSB2
50
60
70
80
41
1
A T L P V R V G Q R A T D N D I A N V V R K L F L S G Q Y D D V K A S R E G N T omp87FCA1
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - omp87HSB2
81
1
L V V T V M P K P V I S N V V I V G N K S I P D E A I K Q N L D A N G F K V G D omp87FCA1
- - - - - M P K P V I S N V V I D G N K S I P D E A I K Q N L D A N G F K V G D omp87HSB2
90
130
100
140
110
150
120
160
121 V L N R A K L E E F R K G I V E H Y N S V G R Y N A K V D A I V N T L P N N S A omp87FCA1
36
V L N R A K L E E F R K G I I E H Y N S V G R Y N A K V E A I V N T L P N N S A omp87HSB2
170
180
190
200
161 E I K I Q I N E D D V A L F K E I T F E G N E A F S S G K L A D Q M E L Q T D S omp87FCA1
76
E I K I Q I N E D D V A L F K E I I F E G N Q A F S S S K L E D Q M E L Q T D A omp87HSB2
210
220
230
240
201 W W K L F G N K F D Q T Q F N K D L E T L R S Y Y L D R G Y A Q F Q I L D T D V omp87FCA1
116 W W K L F G N K F D Q T Q F N K D L E T L R S Y Y L D R G Y A Q F Q I L D T D I omp87HSB2
250
260
270
280
241 K L S D D K K E P C L I S - - E E G D L Y T V K T R V S G G M W G G M S A E L A omp87FCA1
156 K L S D D K K E A R V I I K V K E G D L Y T V K C A R I L G D V G G M S A E L A omp87HSB2
290
300
310
320
279 P I L E T I Q L N G L F R R T S V L E V E Q R N K S K L G E R G Y A T A Q V N V omp87FCA1
196 P I L D T I Q L N G L F R R A N V L E V E Q R I K S K L G E R G Y A T A Q V N V omp87HSB2
330
340
350
360
319 H P T F D E Q D K T I S L D F I V E A G K S Y T V R Q I R F E G N T S S A D S T omp87FCA1
236 H P T F D E Q D K T I S L D F I V E A G K S Y T V R Q I R F E G N T S S A D S T omp87HSB2
370
380
390
400
359 L R Q E M R Q Q E G A W L S S E L V E L G K L R L D R T G Y F E S V E T K T E A omp87FCA1
276 L R Q E M R Q Q E G A W L S S E L V E L G K L R L D R T G F F E S V E T K T E A omp87HSB2
410
420
430
440
399 I P G S D Q V D V I Y K V K E R N T G S I N F G I G Y G T E S G L S Y Q A S I K omp87FCA1
316 I P G S D Q V D V I Y K V K E R N T G S I N F G I G Y G T E S G L S Y Q A S I K omp87HSB2
450
460
470
480
439 Q D N F L G M G S S I S L G G T R N D Y G T T V N L G Y N E P Y F T K D G V S L omp87FCA1
356 Q D N F L G M G S S I S L G G T R N D Y G T T I N L G Y N E P Y F T K D G V S L omp87HSB2
490
500
510
520
479 G G N V S F E E Y D S S K S N T S A G Y G R T S Y G G N L T L G F P V N E N N S omp87FCA1
396 G G N V F F E E Y D S S K S N T S A A Y G R T S Y G G N L T L G F P V N E N N S omp87HSB2
530
540
550
560
519 Y Y L G V G Y T Y N K L K N I A P E Y N R D L Y R Q S M K Y N D S W T F K S H D omp87FCA1
436 Y Y L G V G Y T Y N K L K N I A P E Y N R D L Y R Q S M K Y N D S W T F K S H D omp87HSB2
570
580
590
600
559 F D L S F G W N Y N S L N R G Y F P T K G V R A N I G G R V T I P G S D N K Y Y omp87FCA1
476 F D L S F G W N Y N S L N R G Y F P T K G V R A N I G G R V T I P G S D N K Y Y omp87HSB2
610
620
630
640
599 K L N A E A Q G F Y P L D R E H G W V L S S R I S A S F A D G F S G K R L P F Y omp87FCA1
516 K L N A E A Q G F Y P L D R E H G W V L S S R I S A S F A D G F G G K R L P F Y omp87HSB2
650
660
670
680
639 Q Y Y S A G G I G S L R G F A Y G A I G P N A I Y R T R Q C P D S Y C L V S S D omp87FCA1
556 Q Y Y S A G G I G S L R G F A Y G A I G P N A I Y R T R Q C P D S Y C L V S S D omp87HSB2
690
700
710
720
679 V I G G N A M V T A S T E L I V P T P F V A D K N Q N S V R T S L F V D A A S V omp87FCA1
596 V I G G N A M V T A S T E L I V P T P F V A D K N Q N S V R T S L F V D A A S V omp87HSB2
730
740
750
760
719 W N T R W K A E D K A K F A K L N V P D Y S D P S R V R A S A G V A L Q W Q S P omp87FCA1
636 W N T R W K A E D K A K F A K L N V P D Y S D P S R V R A S A G V A L Q W Q S P omp87HSB2
770
780
790
759 I G P L V F S Y A K P L K K Y Q G D E I E Q F Q F S I G G T F .
676 I G P L V F S Y A K P L K K Y Q G D E I E Q F Q F S I G G T F .
omp87FCA1
omp87HSB2
Table 4.1: Residue substitutions
Accurate, IUB)
A
A
of
Untitled
C
G
T
6
30
10
7
42
C
6
G
30
7
T
10
42
ClustalW
(Slow/
10
10
4.8.2 Sequence analysis of omp 34 gene
Clones showing positive results after plasmid profiling and
restriction digestion were sequenced. Sequencing data revealed the length
of this gene to be 942 nucleotides with termination at stop codon
940TAA942.
The sequence analysis of coding region of omp gene revealed GC
content of 40-45%. The predicted primary protein is composed of 313
amino acids without signal sequence. The mature protein had molecular
mass of 33.7 kDa. Further sequence comparison of omp nucleotide
sequence of P. multocida P52 showed 98.3% similarity while the homology
at amino acid level was 97.5%. The results of this data alignment are
presented as residue substitutions of ClustalW in Table 4.2.
Table 4.2: Residue substitutions
Accurate, IUB)
A
A
of
Untitled
C
G
T
2
8
1
0
6
C
2
G
8
0
T
1
6
0
0
ClustalW
(Slow/
Fig 18 : Alignment of nucleotide sequence of omp34
10
20
30
40
1
1
G C A A C A G T T T A C A A T C A A G A C G G T A C A A A A G T T G A T G T A A ompHCUA3
G C A A C A G T T T A C A A T C A A G A C G G T A C A A A A G T T G A T G T A A omp34HSB2
41
41
A T G G T T C T G T A C G T T T A A T C C T T A A A A A A G A A A A A A A T G A ompHCUA3
A T G G T T C T G T A C G T T T A A T C C T T A A A A A A G A A A A A A A T G A omp34HSB2
81
81
G C G C G G T G A T T T A G T G G A T A A C G G T T C A C G C G T T T C T T T C ompHCUA3
G C G C G G T G A T T T A G T G G A T A A C G G T T C A C G C G T T T C T T T C omp34HSB2
50
90
130
60
100
140
70
110
150
80
120
160
121 A A A G C A T C T C A T G A C T T A G G C G A A G G T T T A A G C G C A T T A G ompHCUA3
121 A A A G C A T C T C A T G A C T T A G G C G A A G G T T T A A G C G C A T T A G omp34HSB2
170
180
190
200
161 C T T A C G C A G A A C T T C G T T T C A G C A C A A A A G T T A A A A A A A C ompHCUA3
161 C T T A C G C A G A A C T T C G T T T C A G C A C A A A A G T T A A A A A A A C omp34HSB2
210
220
230
240
201 A G T T A A A G A A G G T C C T A A C C A A G T A G A A C G C A C A T A T G A A ompHCUA3
201 A G T T A A A G A A G G T C C T A G C C A A G T - - - - - - - - - - - - - - - - omp34HSB2
250
260
270
280
241 G T T G A G C G T A T C G G T A A T G A T G T T C A C G T A A A A C G T C T T T ompHCUA3
225 - - T G A G C G T A T C G G T A A T G A T G T T C A C G T A A A A C G T C T T T omp34HSB2
290
300
310
320
281 A T G C G G G T T T C G C G T A T G A A G G T T T A G G A A C A T T A A C T T T ompHCUA3
263 A T G C G G G T T T C G C G T A T G A A G G T T T A G G A A C A T T A A C T T T omp34HSB2
330
340
350
360
321 C G G T A A C C A A T T A A C T A T C G G T G A T G A T G T T G G T G T G T C T ompHCUA3
303 C G G T A A C C A A T T A A C T A T C G G T G A T G A T G T T G G T G T G T C T omp34HSB2
370
380
390
400
361 G A C T A C A C T T A C T T C T T A G G T G G T A T C A A C A A T C T T C T T T ompHCUA3
343 G A C T A C A C T T A C T T C T T A G G T G G T A T C A A C A A T C T T C T T T omp34HSB2
410
420
430
440
401 C T A G C G G T G A A A A A G C A A T T A A C T T T A A A T C T G C A G A A T T ompHCUA3
383 C T A G C G G T G A A A A A G C A A T T A A C T T T A A A T C T G C A G A A T T omp34HSB2
450
460
470
480
441 C A A C G G T T T C A C A T T T G G T G G T G C G T A T G T G T T C T C T G C G ompHCUA3
423 C A A C G G T T T C A C A T T T G G T G G T G C G T A T G T G T T C T C T G C G omp34HSB2
490
500
510
520
481 G A T G C A G A C A A A C A A G C A C C A C G T G A T G G T C G C G G T T T C G ompHCUA3
463 G A T G C A G A C A A A C A A G C A C C A C G T G A T G G T C G C G G T T T C G omp34HSB2
530
540
550
560
521 T T G T A G C A G G T T T A T A T A A C A G A A A A A T G G G C G A T G T T G G ompHCUA3
503 T T G T A G C A G G T T T A T A T A A C A G A A A A A T G G G C G A T G T T G G omp34HSB2
570
580
590
600
561 T T T C G C A C T T G A A G C A G G T T A T A G C C A A A A A T A T G T A A C A ompHCUA3
543 T T T C G C A C T T G A A G C G G G T T A T A G C C A A A A A T A T G T A A C A omp34HSB2
610
620
630
640
601 G C A G C A G C T A A A C A A G A A A A A G A A A A A G C C T T T A T G G T T G ompHCUA3
583 G T A G C G - - - - A - - - - - A A C A A G A A A A A G C C T T T A T G G T T G omp34HSB2
650
660
670
680
641 G T A C T G A A T T A T C A T A T G C T G G T T T A G C A C T T G G T G T T G A ompHCUA3
614 G T A C T G A A T T A T C A T A C G C T G G T T T A G C A C T T G G T G T T G A omp34HSB2
690
700
710
720
681 C T A C G C A C A A T C T A A A G T G A C T A A C G T A G A A G G T A A A A A A ompHCUA3
654 C T A T G C A C A A T C T A A A G T G A C T A A C G T A G A A G G T A A A A A A omp34HSB2
730
740
750
760
721 C G C G C A C T T G A A G T A G G T T T A A A C T A T G A C A T T A A T G A C A ompHCUA3
694 C G C G C A C T T G A A G T G G G T T T A A A C T A T G A T A T T A A T G A C A omp34HSB2
770
780
790
800
761 A A G C A A A A G T T T A C A C T G A C T T G A T T T G G G C A A A A G A A G G ompHCUA3
734 A A G C G A A A G T T T A C A C T G A C T T G A T T T G G G C A A A A G G A G G omp34HSB2
810
820
830
840
801 T C C A A A A G G T G C G A C T A C A A G A G A T C G T T C T A T C A T C T T A ompHCUA3
774 T C C A A A A G G T G C G A C T A C A A G A G A T C G T T C T A T C A T C T T A omp34HSB2
850
860
870
880
841 G G T G C G G G C T A C A A G C T T C A C A A A C A A G T T G A A A C C T T T G ompHCUA3
814 G G T G C G G G C T A C A A G C T T C A C A A A C A A G T T G A A A C C T T T G omp34HSB2
890
900
910
920
881 T T G A A G G T G G C T G G G G C A G A G A G A A A G A T G C T A A T G G C G T ompHCUA3
854 T T G A A G G T G G C T G G G G C A G A G A G A A A G A T G C T A A T G G C G T omp34HSB2
930
940
950
960
921 A A C A A C A A A A G A T A A C A A A G T T G G T G T T G G T T T A C G C G T A ompHCUA3
894 A A C A A C A A A A G G C A A T G T C G T T G G T G T T G G T T T A C G C G T A omp34HSB2
961 C A C T T C T A A
934 C A C T T C T A A
ompHCUA3
omp34HSB2
Fig 19 : Alignment of amino acid sequence of omp34
10
20
30
40
1
1
A T V Y N Q D G T K V D V N G S V R L I L K K E K N E R G D L V D N G S R V S F ompHCUA3
A T V Y N Q D G T K V D V N G S V R L I L K K E K N E R G D L V D N G S R V S F omp34HSB2
41
41
K A S H D L G E G L S A L A Y A E L R F S T K V K K T V K E G P N Q V E R T Y E ompHCUA3
K A S H D L G E G L S A L A Y A E L R F S T K V K K T V K E G P S Q V E R - - - omp34HSB2
81
78
V E R I G N D V H V K R L Y A G F A Y E G L G T L T F G N Q L T I G D D V G V S ompHCUA3
- - - I G N D V H V K R L Y A G F A Y E G L G T L T F G N Q L T I G D D V G V S omp34HSB2
50
90
130
60
100
140
70
110
150
80
120
160
121 D Y T Y F L G G I N N L L S S G E K A I N F K S A E F N G F T F G G A Y V F S A ompHCUA3
115 D Y T Y F L G G I N N L L S S G E K A I N F K S A E F N G F T F G G A Y V F S A omp34HSB2
170
180
190
200
161 D A D K Q A P R D G R G F V V A G L Y N R K M G D V G F A L E A G Y S Q K Y V T ompHCUA3
155 D A D K Q A P R D G R G F V V A G L Y N R K M G D V G F A L E A G Y S Q K Y V T omp34HSB2
210
220
230
240
201 A A A K Q E K E K A F M V G T E L S Y A G L A L G V D Y A Q S K V T N V E G K K ompHCUA3
195 V A K - - - Q E K A F M V G T E L S Y A G L A L G V D Y A Q S K V T N V E G K K omp34HSB2
250
260
270
280
241 R A L E V G L N Y D I N D K A K V Y T D L I W A K E G P K G A T T R D R S I I L ompHCUA3
232 R A L E V G L N Y D I N D K A K V Y T D L I W A K G G P K G A T T R D R S I I L omp34HSB2
290
300
310
320
281 G A G Y K L H K Q V E T F V E G G W G R E K D A N G V T T K D N K V G V G L R V ompHCUA3
272 G A G Y K L H K Q V E T F V E G G W G R E K D A N G V T T K G N V V G V G L R V omp34HSB2
321 H F .
312 H F .
ompHCUA3
omp34HSB2
4.9 SEQUENCE ALIGNMENT
4.9.1 Homology search
The Omp34 gene sequences of B:2 serotype obtained in this study
along with the published sequences of P.multocida serotypes were
aligned by ClustalW method. Sequences of all serotypes were retrieved
from EMBL sequence database. The nucleotide and amino acid
alignments of different isolates/strains are shown in fig 16 and 17
respectively. The pairwise nucleotide sequence identity/divergence was
also analyzed using laser gene software (DNASTAR).
4.9.2 Phylogenetic analysis
The phylogenetic relationship among all the isolates was studied
on the basis of their nucleotide as well as amino acid sequences which
revealed slightly different results.
Phylogenetic tree was constructed using omp gene sequences
downloaded from the NCBI database in order to compare the
relationship of P52 with other serotypes. The phylogenetic analysis
based on nucleotide sequences revealed 5 major clusters. Serotype 1
showed an entirely different lineage from the starting (Fig 18). Serotype
B2 found in major cluster had more number of the isolates and was
close to serotype 3, 4. Serotype 6, 7 and 13 formed a different cluster.
Serotype 10 and 12 were found to be 100% similar.
The pairwise identity/divergence (Table 4.3) was also done using
DNASTAR. P. multocida serotype B:2 revealed 98.2% homology with
Serotype11
SerotypeD
SerotypeB2
SerotypeA34
SerotypeD4
Serotype15
Serotype9
serotype10
Serotype12
Serotype7
Serotype6
Serotype13
Serotype14
SerotypeA1
Serotype1
18.8
18
16
14
12
10
8
6
Nucleotide Substitutions (x100)
4
Fig. 20 : Nucleotide sequences based phylogenetic tree
2
0
Table 4.3 : Pairwise nucleotide sequernce identity/ divergences
serotype 3,4and only 72.2% with serotype 1. P52 showed 88.3% similarity
with serotype 10 and 11 and 83% with serotypes 13, 14 and 15.
4.10 OUTER MEMBRANE PROTEIN ANALYSIS
Analysis of outer membrane proteins were done by using
computational biology. Amino acid sequences were deduced from the
nucleotide
sequences
and
imported
to
the
ExPAsy
server
(http://www.expasy.org/tool/) for primary structure analysis, Pfam
(http://www.pfam.janelia.org/cgi.bin/) for functional domain and
protein
family
study
and
PDBsum
server
(http://www.
ebi.ac.uk/pubsum) for secondary structure prediction and analysis.
For the prediction of transmembrane helix TMpred server was used.
In silico translation and primary structure of the outer membrane
protein were done using on line bioinformatics tools (ExPASy Server:
ProtParam, Gasteiger et al., (2005). Theoretical pI, Aliphatic index and
Grand average of hydropathicity (GRAVY) of the omp proteins of P.
multocida P52 are given in table 4.4.
Table 4.4: Summary of primary structure of omp proteins
S.No.
Amino Mol. weight Theoritical Aliphatic
Charge GRAVY
acid
(kDa)
pI
Index
Omp34
313
33.760
9.16
80.64
+6.59
.0.318
Omp87
790
87.57
5.94
79.97
-3.46
-0.387
The instability index of omp34 and 87 were computed to be
13.93 and 30.11 respectively which classified both the proteins as
stable. The half-life of omp34 was estimated to be 4.4 hours
(mammalian reticulocytes, in vitro), >20 hours (yeast, in vivo), >10
hours (in Escherichia coli, in vivo). Information on primary structure
supported the possibility of expression of omp gene in E. coli. The
details of the results are given in Annexure II.
Analysis of Pfam results showed that the protein belong to Pfam
A family. One major functional domain i.e. porin-1 was found in the
omp34 sequence and six domains i.e. five surface antigen variable
number repeats and one bacterial surface antigen were found in omp87
sequence by trusted matches. The detailed results of the study are
given in the table 4.5.
Table 4.5: Details of conserved domains of omp proteins
Omp 34
Omp 87
Domain
Porin_1
Start
6
End
313
Surf Ag VNR
21
88
Surf Ag VNR
89
169
Surf Ag VNR
172
258
Surf Ag VNR
261
340
Surf Ag VNR
343
416
Bac surface Ag
442
790
The Pfam study also showed more domains other than these
domains based on potential matches of the sequences. Omp34 showed
three domains i.e. Opacity, Transposase 11 and porin_1 and omp87
showed
four
domains
i.e.
Bacillus
PapR,
two
POTRA2
and
Autotransporter. The details of the results are given in table 4.6 and in
annexure
Table 4.6: Showing the domains based in potential matches.
Domain
Start
End
Opacity
211
313
Porin_1
7
52
Tranposase 11
218
284
Porin_1
269
313
Bacillus PapR
1
10
POTRA 2
21
43
POTRA 2
343
370
Autotransporter
495
532
Omp 34
Omp87
A total of five and ten transmembrane helices were found in
omp34 and omp87 respectively. Only scores above 500 are considered
significant. There is no helix formed in omp34 having score above 500
so the details of the results of omp 87 are given in table 4.7.
Table 4.7: Prediction of transmembrane helix
Omp87
From
To
Inside to outside
4
23
644
664
4
23
640
656
675
695
Outside to inside
Discussion
Chapter 5
Discussion
Haemorrhagic septicaemia accounts for the largest proportion of
mortality in cattle and buffaloes in India. Although several vaccines are
used to control the disease, yet occurrence of the disease in many
regions of the country has been reported. It has always been a challenge
for the researcher to improve the existing vaccines, while overcoming
the drawbacks of existing vaccines.
At present, two types of vaccines against HS in use. The first type
are killed whole cells (bacterins) and the second type are live attenuated
vaccines. Killed whole cells stimulate protection only against P.
multocida strains of the same serotype and only for short periods. The
most effective bacterin is the oil adjuvant- one dose provides protection
for 9-12 months; so it should be administered annually. The oil
adjuvant vaccine has not been popular because of difficulty in syringing
and occasional adverse local tissue reactions. To overcome from these
problems there is an urgent need to search a conserved immunogenic
protein as vaccine candidate.
Outer membrane proteins of P. multocida are reported to be
immunogenic and protective (Srivastava, 1998, Basagoudanavar et
al., 2006). Some of the major outer membrane proteins like porins are
reported to be highly immunogenic. These are pore forming outer
membrane
proteins
and
possess
beta
barrel
structure
having
extracellular, transmembrane and intracellular domain. They are
conserved in gram-negative bacteria showing high homology in primary
amino acid sequence and secondary structure (Jeanteur et al., 1991).
They form large channels allowing the diffusion of hydrophilic molecules
into periplasmic space. They are strong immunogen and have been
demonstrated to induce protective immunity in animal models against
gram-negative bacterial infections (Luo et al., 1999).
The outer membrane proteins of P. multocida P52 were isolated
by the method described by Choi-Kim et al. (1991). The organism was
disrupted
by
sonication
and
large
particles
were
removed
by
centrifugation at low speed. The OMPs were separated into detergent
insoluble and detergent soluble fractions. Sonicated antigen was used
to induce antibody production. Antiserum raised against whole cell
protein was used for agar gel precipitation test and in western blot
analysis. It was possible to identify immunodominant outer membrane
protein(s) and recombinant clones containing specific omp gene.
According to Confer et al. (1996) the detergent (sodium lauryl
sacosinate)
insoluble
fraction
was
almost
identical
with
other
membrane fraction purified by sucrose gradient. Vasfi Marandi and
Mittal (1997) also reported the presence of outer membrane proteins in
detergent insoluble fraction.
SDS-PAGE analysis of OMP preparation of P. multocida P52
strain revealed the presence of eight polypeptide bands. The molecular
weight of these polypeptides varied between 16 kDa to 97 kDa. With
little difference, Pati et al. (1996) reported ten polypeptides bands of
molecular weight 25 to 88 kDa of the same preparation. Kedrak and
Opacka (2002) reported protein bands of 22 to 86 kDa in the OMP
profiles of bovine strains (serotype B:2). The polypeptides of 31, 34 and
37 kDa molecular weights gave prominent bands on SDS-PAGE
indicating that the same were synthesized in abundance corroborating
the observations made by Tomer et al. (2002).
Present results are in close agreement with the results of other
workers with minor differences. Wasnik (1998) and Tomer et al.
(2002) detected 13 and 20 polypeptidess bands respectively in profiles
of outer membrane proteins. Anshu et al. (2005) revealed the presence
of 11 protein fractions with two major OMPs of 32 and 35 kDa in
capsular type B isolates. Arora et al. (2007) have reported a
homogenous outer membrane profile of 17 different P. multocida isolates
of bovine origin comprising 23 polypeptides ranging in MW from 13 to
94 kDa. On the basis of band thickness and stain intensity 32kDa
protein appeared to be the major protein followed by 25 and 28 kDa.
Apart from this, other significant protein bands observed were 13, 34,
44.5, 46, 80 and 84 kDa. They also considered that 32kDa protein band
represented a type specific marker for the Asian HS isolates so it might
be act as a candidate antigen for a subunit HS vaccine and can be
exploited in immunodiagnosis of HS.
Immunization of rabbits with whole cell antigen of P. multocida
P52 strain apparently stimulated the production of antiserum. When
this hyperimmune sera was tested with whole cell antigen using
immunodiffusion test, it showed positive results. It produced three
precipitin lines in all three sets indicating the presence of at least three
major immunogens in sonicated extract of P. multocida.
The results of western blot profiles of major immunogens
indicated that all the major protein bands appeared immunogenic;
however 34 kDa protein was found to be most immunodominant among
them. In the present study, seven immunodominant outer membrane
proteins of 87, 68, 44, 37, 34, 31 and 16 kDa molecular weights were
identified. The results differed little from those reported by Pati et al.
(1996) who encountered faint signals of only three outer membrane
proteins of 44, 37, and 33 kDa reacting to hyperimmune sera.
According to Arora et al. (2007), 32 kDa protein band was found to be
immunodominant along with 25 kDa in all the P. multocida isolates of
bovine origin.
The original objective of this study was to isolate and characterize
the genes encoding immunopotent outer membrane proteins of P.
multocida B:2 P52 strain. Although, about eight (polypeptide) bands of
outer membrane proteins were resolved on SDS-PAGE, all these
peptides were not equally immunopotent and reacted differently on
immunoblotting. With previous observations, the large size 87 kDa
outer membrane protein was imagined as potential immunogen. It was
found to react strongly on western blot with antisera raised against
sonicated antigen. The results strengthened the hypothesis that 87 kDa
OMP was an immunodominant antigen and might serve as potent
immunogen in vaccine preparation. Proteins of 87 and 34 kDa were
found to be immunodominant antigens.
For further study we have cloned genes encoding outer membrane
proteins of 87 and 34 kDa using PCR based techniques. PCR
techniques have already been employed for cloning of ompH gene of P.
multocida A:1 by Luo et al., (1997). The distribution of Omp genes
among different serotypes of P. multocida has been reported. A 16 kDa
Omp gene was found to be present in all the serotypes of P. multocida
(Goswami et al., 2004).
For the amplification of omp87 gene primers were designed from
the available sequence of P. multocida serotype A:1 (Ruffolo and Adler,
1996). Restriction endonucleases sites for BamHI and HindIII were
incorporated into the primer specific 5’ and 3’ end of coding strand.
These two enzymes were selected because of lack of restriction sites for
these two enzymes inside the omp87 gene. Incorporation of restriction
sites facilitated directional cloning of the gene which avoided orientation
problem.
The major immunodominant protein in several strains of P.
multocida has been reported to show general properties of other
bacterial porins. We assumed that P.
multocida P52 major outer
membrane protein with a molecular mass of 34kDa was a porin. This
protein also showed to have inherent properties to be expressed in vivo
at a very high level because of its promoter.
Bacterial porins genes are sometimes difficult to clone in E. coli,
because of their lethal effect on host cells (Luo et al., 1997). This is
due to the fact that whole omp34 gene posses signal peptide at its Nterminal and after expression in E. coli it translocates the protein to E.
coli membrane that may causing osmotic imbalance and ultimately cell
lysis. Keeping the above view in our mind, we have designed primer
excluding the signal sequence. For the amplification of omp34 gene
primers were designed from the available sequence of ompH gene of P.
multocida CU vaccine strain of serotype A:3,4 causing fowl cholera.
Genomic DNA was isolated from P. multocida P52 and quantified
by taking reading at 260 and 280nm. Ratio between the O.D. at two
wavelengths, provide an estimate of purity of the nucleic acid. Pure
preparation of DNA and RNA has O.D. of 1.8 and 2.0 at 260/280
respectively. If there is protein or phenol contamination, O.D. 260/280
will be significantly less than 1.0 (Sambrook et al., 1989). The yield of
genomic DNA was 1.8 confirms the purity of isolated DNA sample.
Agarose gel electrophoresis of the isolated DNA revealed that DNA was
relatively intact and without RNA. Genomic DNA from P. multocida P52
was used as template at a concentration of 40ng per reaction. Negative
control was always kept to check any non specific amplification. Both
the genes were successfully amplified through polymerase chain
reaction.
Both the genes of P. multocida serotype B:2 were cloned in
pGEMT-Easy
vector
and
recombinant
clones
were
screened
by
blue/white screening. The pGEM-Z vectors containing a sequence
coding for the lac α peptide were interrupted by a multiple cloning sites.
Non recombinants plasmids produce a functional α-peptide, which by
complementing the product of host cells lacZ-M15 gene, leads to the
production of functional β-galactosidase. Bacterial colonies harbouring
lacZ-M15 gene and pGEM-Z vectors were blue in colour when plated on
indicator plates containing IPTG and X-Gal. However, when the lac αpeptide was disrupted by cloning in pGEM-Z multiple cloning region
complementation did not occur and no β-galactosidase activity was
observed. Therefore, bacterial colonies harbouring recombinant pGEMZ vectors constructs were white (Sambrook et al., 1989).
Clones showing positive results after plasmid profiling and
restriction digestion, were sequenced by the DNA sequencing facility at
UDSC, Department of Biochemistry, University of Delhi, South Campus,
New Delhi.
Sequence analysis of omp 87 gene
Sequencing of omp87 gene revealed the length of sequence was
2121bp. The C-terminus of Oma87 reveals typical characteristic of
OMPs. The last residue at the C-terminus, phenylalanine, was highly
conserved among outer membrane proteins and is essential for stability
and correct assembly of protein into the outer membrane. According to
Ruffolo and Adler (1996) the last 10 amino acid residues of membrane
proteins,
including
the
terminal
phenylalanine,
have
conserved
hydrophobic residues which are important for incorporation of the
protein in the membrane. The last 10 residues Q-F-Q-F-S-I-G-G-T-F, of
Oma87 contain the conserved hydrophobic residues at positions 2, 4, 6
and 10.
Although the glycine at position 8 is not hydrophobic, given the
overall conservation of the other residues, particularly the terminal
phenylalanine it is most probable that these residues form a membrane
spanning region, with the C-terminus of Oma87 possibly directed
towards the periplasm. The conserved C-terminal amino acids are also
present in the H. influenzae D15 surface antigen.
At nucleotide level, a total of 105 substitutions could be observed
in the omp87 sequence. Out of these 105 substitutions, Adenine into
Cytosine, Adenine into Guanine, Adenine into Thymine, Cytosine into
Guanine, Cytosine into thymine were present at 6, 30, 10, 7, 42 and
10th
positions
respectively.
These
substitutions
might
play
an
important role in proper packaging of the gene with in the genome of P.
multocida. These substitutions might be providing functional stability to
the organism in terms of protein structure.
At amino acid level a total of 37 substitutions were found. Out of
these, 3 substitutions were from aromatic to aliphatic amino acid and
rest were within aliphatic amino acids. At position 149 and 284 aspartic
acid is substituted into glutamic acid and glutamic acid is converted in
aspartic acid so both substitutions nullify each other.
The residue substitution of amino acid from aliphatic to aliphatic
or aromatic to aromatic or even change of amino acid with similar
structures and functions into one another makes no difference in the
overall biophysical property of a protein like isoelectric pH, conductivity
and charge interactions. However, substitutions at some critical
positions may alter the structure of the molecular scaffold of proteins
resulting into slightly altered properties.
Sequence analysis of omp 34 gene
The sequence analysis of coding region of omp34 gene revealed
the presence of 942bp. This stretch of 942 nucleotides was devoid of the
signal sequence, which is reported to comprise of 60 nucleotides (Luo
et al., 1997).
On comparison, it is clearly evident that at nucleotide level, a
total of 17 substitutions could be recorded. Out of these 17
substitutions, adenine was ubstituted by cytosine at two positions (619
and 939). Adenine was substituted by guanine at eight positions 218,
576, 606, 735, 765, 797, 932 and 937. Besides this, there was only one
substitution of adenine to thymine at 938 position. Out of the rest six
substitutions, cytosine was substituted by thymine at four places and
thymine by cytosine at two places. Thymine and cytosine being
pyrimidines had smaller structure than purines (adenine and guanine).
On alignment of the protein sequences, we have observed no
significant difference at amino acid level because all substitutions were
within aliphatic amino acids. At amino acid position 203 alanine is
substituted by lysine whereas, it is interesting to note that at amino
acid position 313 lysine was substituted to valine. Here it can be
emphasized that valine and alanine both are aliphatic amino acid with
similar properties. So it can be claimed that these two substitutions at
positions 203 and 313 may not result in significant changes in the
protein.
At positions 266 and 311, glutamic and aspartic acids were
substituted by glycine. Isoelectric point of acids was low i.e. around one
or two in comparison to glycine i.e. to 6. So we concluded that
isoelectric point of the protein formed by gene ompH of B:2 becomes
slightly high in comparison to ompH of A:1 due to loss of two acids in
the protein sequence. Three other substitutions also occurred i.e.
asparagine with serine, alanine with valine and lysine with glutamine at
positions 73, 201, and 207 respectively.
The Laser gene software (DNA SATR) analysis of omp34 gene
predicted a protein having the characteristics of typical gram-negative
bacterial porins. The amino acid sequence showed similarities to outer
membrane proteins of other bacterial species indicating the conserved
nature of the omp gene in gram-negative bacteria. Amino acid
composition also indicated that it was non specific bacterial porin in its
highly negative hydropathy index with high glycine, low proline and
lacking in cysteine content (Luo et al., 1999).
Based on amino acid composition and hydropathy profile we
proposed that omp34 was membrane associated protein. Hydrophilic
domain corresponding to cell surface-exposed domain were highly
variable in their amino acid composition. A number of hydrophilic
peaks of omp34 corresponded the predicted surface exposed domain.
Primary
and
secondary
structure
of
the
deduced
protein
sequences were also studied with the help of PROTPARAM, Pfam and
PDBsum tools of ExPAsy server. Pfam analysis showed the presence of
one conserved domain in omp 34 i.e. Porin_1 and six domains in omp
87 i.e. five Surface Antigen Variable number repeats and one Bacterial
surface Antigen. Surface Ag VNR is found primarily in bacterial surface
antigen normally as variable number repeats at the N-terminus. The Cterminus of these proteins is normally represented by Bacterial Surface
Antigen.
Omp34 belongs to beta-barrel protein superfamily. The outer
membrane of Gram-negative bacteria acts as a molecular filter for
hydrophilic compounds. Proteins known as porins are responsible for
the molecular sieve properties of the outer membrane. Porin is a major
antigenic outer membrane protein of P. multocida and has high
immunogenicity in antibody production (Lee et al., 2007).
Transmembrane helices of both the genes were also predicted by
Tmpred server. Five helices were found in omp34 and best path of
transmembrane helices is from 89-108 residues at amino acid level. In
omp87, 10 helices were found from inside to outside and outside to
inside.
Summary
Chapter 6
Summary
Pasteurella multocida serotype B:2 is the causative agent of
Haemorrhagic septicaemia (HS), a fatal disease of cattle and buffaloes.
It is one of most fatal disease in India due to the high mortality of
susceptible populations. Control of HS has always remained a problem
because current vaccines are not sufficiently effective and require
repeated administration. At the present, formalin inactivated whole cell
bacterin is used to prepare vaccines in India. This vaccine has several
limitations such as short term immunity, poor syringibility and
production problem. To overcome from these problems there is an
urgent need of search of a conserved immunogenic protein as a vaccine
candidate. To achieve the objective, gene encoding omp87 and 34 are
targeted for production r-DNA vaccine of HS in the present study. The
recombinant subunit vaccine should produce longer lasting protection
against multiple P. multocida serotypes with no possibility of reverting
to virulence. This represents a new vaccine, which has potentially
broader specificity against the infectious agent, resulting in a superior
safety and efficacy profile when compared to other vaccine or antibiotic
approaches providing a competitive alternative for animal health
industry.
In r-DNA vaccine preparation, the most important criteria is to
target an immunopotent gene. Outer membrane proteins of P. multocida
are reported to be immunogenic and protective. So the present study
was undertaken to identify and clone the P. multocida P52 (serotype
B:2) gene(s) encoding 87 and 34 kDa outer membrane proteins in E.
coli and to evaluate their immunogenicity. Nucleotide sequence analysis
with phylogenetic analysis of both the proteins was performed to derive
ancestral relationship between different serotypes. Further amino acid
sequences were deduced from the nucleotide sequences and primary
and secondary structures were also studied. The results of the present
study are summarized below:

P. multocida B:2 was revived and found to be pathogenic to mice,
causing 100% mortality with in 24 hrs .

P. multocida (B:2) specific PCR was done to check the purity of
P52 strain using KT-61 and KT-72 oligoes and positive result with
a band size of 620bp was found.

Results of SDS-PAGE analysis of P. multocida B:2 outer
membrane proteins revealed the presence of eight bands. The
molecular weight of the major polypeptide bands ranged from 16
to 87 kDa. The major bands were of 16, 27, 30, 34, 37, 44, 68
and 87 kDa.

Polyclonal antibodies were raised in rabbit against sonicated
antigen. Three precipitin lines were produced between whole cell
antigen
and
hyperimmnune
immunodiffusion test.
sera
when
checked
by

To identify the immunogenic polypeptides, western blotting was
done with hyperimmune serum. Polypeptides of 16, 30, 34, 37,
44, 68 and 87 kDa showed positive reaction with antiserum and
were found to be immunogenic.

DNA from P.multocida B:2 was isolated by using C-TAB method
which was used as a template for amplification of genes encoding
both the outer membrane proteins. Genes were amplified by PCR
using different sets of primers. The amplification product of
omp87 and omp34 were found to be 2373 bp and 942bp
respectively.

The purified PCR products were cloned into pGEM-T Easy vector.
The transformed colonies were screened by blue-white screening
on LB agar plates with ampicillin, IPTG and X-gal. Few colonies
were selected and screened by PCR using omp specific primers.
Plasmid DNA was isolated from the PCR positive colonies and
recombinant plasmids were screened for the presence of desired
inserts by restriction enzyme analysis with NotI enzyme to verify
the size. The results confirmed a product of 2373 bp from clone of
omp87 and a product of 942 bp from the clone of omp34.

Both the cloned omp genes were sequenced and were submitted
to NCBI gene database and provided the accession numbers
EU570212 and EU162755 for the omp87 and omp34 respectively.

The sequencing data revealed the length of omp34 was 942
nucleotides with termination at stop codon
940TAA942.
The
predicted primary protein is composed of 313 amino acid without
a signal sequence. The mature protein had molecular mass of
33.7 kDa.

Further comparison of nucleotide sequence of omp34 of P.
multocida B:2 showed 98.3% similarity with serotype B 3,4 while
homology at amino acid level was 97.1%. Phylogenetic analysis
confirmed that serotype B:2 could be grouped with serotype B
3,4.

Sequencing of omp87 gene revealed the presence of 2121 bp. The
predicted primary protein is composed of 707 amino acid without
a signal sequence.

Nucleotide sequence of omp87 of P. multocida B:2 showed 94.8%
similarity with that of P. multocida serotype A:1 strain while the
homology at amino acid level was 95.2%.

Primary and secondary structures of both the outer membrane
proteins were also studied by using different servers. Functional
domain and protein family were studied through Pfam server,
which revealed the presence of one conserved domain in omp 34
i.e. porin_1 and six domains in omp 87 i.e. five surface Antigen
Variable number repeats and one bacterial surface antigen.

Transmembrane helices of both the genes were also predicted by
Tmpred server. Ten and five transmembrane helices were found
in omp87 and omp34 respectively. In omp34 the best path of
transmembrane helices is from residues 89-108.
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Appendix
APPENDIX-I
(A) MEDIA USED
Luria-Bertani (LB) medium
Bacto tryptone
10.0
Yeast extract
5.0
NaCl
5.0
Agar
2%
pH
7.0
(B) GENERAL BUFFERS AND REAGENTS
1. Stock solutions
a. Phosphate buffered saline (PBS) (pH-7.2)
Sodium chloride (NaCl)
8.0 gm
Disodium hydrogen phosphate (Na2HPO4)
1.16 gm
Potassium chloride (KCl)
0.2 gm
Potassium dihydrogen phosphate (KH2PO4)
0.2 gm
Add distilled water to make 1000 ml. Sterilized by autoclaving for
20 min.
b. Hepes buffer 10mM (pH 7.4)
Hepes buffer 0.476 gm was dissolved in 200ml distilled water and
filtered it through .2μm filter paper.
c. 2 % (w/v) sodium lauryl sarcosinate
2 gm of sodium lauryl sarcosinate was dissolved in 100ml
distilled water. Freshly prepared.
d. Ampicillin stock solution (50mg/ml)
Ampicillin (sodium salt)
500mg
Distilled water
10ml
Sterilize by filtration.
e. Kanamycin (15mg/ml)
Kanamycin
200mg
Distilled water
10ml
Sterilize by filtration.
f. Isopropyl β-D-thiogalactoside (IPTG) (1M)
IPTG
238 mg
Distilled water
1 ml
Sterilized by filtration, store at -200C.
(C) REAGENTS FOR SDS-PAGE
1. Stock solutions
a. 30% Acrylamide
Acrylamide
29.2g
N-N methylene Bis-acrylamide
0.8 g
Triple distilled water (TDW)
100 ml
Fiters through whatmann No.1 and stored at 4 0C in dark bottles.
b. Separating gel buffer 2M TRIS (pH 8.8)
Tris buffer 24.2g was dissolved in 75ml of TDW. Adjust pH with
conc. HCl and volume was made 100ml.
c. Stacking gel buffer 0.5 M TRIS (pH 6.8)
Tris buffer 6.057 was dissolved in 75 ml of TDW. Adjust pH with
conc. HCl and volume was made 100ml.
d. 10% Ammonium per sulphate
In 1 ml Triple Distilled Water 0.1 gm APS was dissolved.
e. 10% SDS
1.0gm of SDS was dissolved in 10 ml TDW.
f. Sample buffer
Component
ml
0.5 M Tris (pH 6.8)
1.2
10% SDS
2.0
Glycerol
1.0
TDW
4.8
0.5% Bromophenol blue (W/V)
0.5
Β- mercaptoethanol
0.5
g. Electrode buffer (pH8.2)
Component
g/l
Tris buffer
3.025
Glycine
14.509
SDS
1.0
h. Staining Solution
In 25 ml TDW 0.5g Coomassie blue R-250 was dissolved. This
solution was mixed with 720 ml TDW containing 180ml methanol
and 60 ml glacial acetic acid, filtered through Whatman No. 1
filter paper and stored at room temperature.
i. Destaining Solution
10% Glacial aceteic acid and 35% methanol was used as
destaining solution.
2. Working solution
a. Separating gel
Distilled water
2 ml
Resolving buffer (pH 8.8)
1.4 ml
Acrylamide (30%)
2.5 ml
10% SDS
50 μl
10% APS
50 μl
TEMED
4 μl
b. Stacking gel
Distilled water
Stacking buffer
2 ml
(pH 6.8)
622μl
Acrylamide (30%)
310 μl
10% SDS
25 μl
10% APS
25μl
TEMED
4 μl
(D) SOLUTIONS FOR WESTERN BLOTTING
a. Blocking buffer
Wash buffer
100ml
BSA
1gm
b. Substrate buffer
50mM Tris-Cl (pH 7.6)
10ml
Diamino benzidine
6mg
Hydrogen peroxide
6μl
Freshly prepared just before use and Diamino benzidine store at 20 0C.
c. Wash buffer
PBS (7.4)
1000ml
Tween-20
500μl
d. Transfer buffer
Tris
12.1g
Glycine
56.64g
Methanol
800ml
Final volume was made 4 liter of with TDW and stored at 40C
after autoclaving.
(E) REAGENTS FOR GENOMIC DNA ISOLATION
NaCl
5M
Phenol: Chloroform
1:1
Chloroform: isoamyl alcohol
24 : 1
a. RNase (10 mg/ml)
RNase
10 mg
Distilled water
1 ml
Heated in boiling water bath for 15 minutes. Stored at -200C.
b. Proteinase K (20 mg/ml)
Proteinase K
20 mg
Distilled water
1 ml
Stored at -200C.
c. Sodium acetate (3M)
Sodium acetate
24.6 gm
Distilled water
100 ml
d. Tris EDTA (pH 8.0)
Tris Cl
10mM
EDTA
1mM
(F) REAGENTS FOR AGAROSE GEL ELECTROPHORESIS
a. TAE (50X) (pH 7.8)
Tris
24.29 g
0.5M EDTA
10mM
Glacial acetic acid
5.7 ml
Final volume
100 ml
Working solution is 1X
b. Gel loading dye
Bromophenol blue
0.25%
Sucrose in water
40% (W/V)
c. Intercalating Dye
Ethidium bromide
10 mg
Distilled water
1 ml
(G) REAGENTS FOR PLASMID ISOLATION
a. Resuspension buffer
Glucose
50mM
Tris-Cl(pH 8.0)
25mM
EDTA (pH 8.0)
10mM
Autoclaved for15 min at 15-lb/sq. Stored at 40C.
b. Lysis buffer I
NaOH
0.2N
SDS
1.0%
c. Lysis buffer II
5M Potassium acetate
60ml
Glacial acetic acid
11.5ml
Distilled water
28.5ml
The resulting solution will be 3M with respect to potassium and
5M with respect to acetate.
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Reverse complemented strand Features:
1: EU570212. Reports Pasteurella multo...[gi:171740840]


sequence
Refresh
Links
Features
Sequence
LOCUS
EU570212
2396 bp
DNA
linear
BCT
06-APR-2008
DEFINITION Pasteurella multocida outer membrane protein 87 gene,
partial cds.
ACCESSION
EU570212
VERSION
EU570212.1 GI:171740840
KEYWORDS
.
SOURCE
Pasteurella multocida
ORGANISM Pasteurella multocida
Bacteria; Proteobacteria; Gammaproteobacteria;
Pasteurellales;
Pasteurellaceae; Pasteurella.
REFERENCE
1 (bases 1 to 2396)
AUTHORS
Saxena,A., Yadav,A., Saxena,M.K., Rao,V.D.P. and Sharma,B.
TITLE
Pateurella multocida OMP87 immunogenicity
JOURNAL
Unpublished
REFERENCE
2 (bases 1 to 2396)
AUTHORS
Saxena,A., Yadav,A., Saxena,M.K., Rao,V.D.P. and Sharma,B.
TITLE
Direct Submission
JOURNAL
Submitted (15-MAR-2008) Biochemistry, Indian Veterinary
Research
Institute, Bareilly, U.P. 243122, India
FEATURES
Location/Qualifiers
source
1..2396
/organism="Pasteurella multocida"
/mol_type="genomic DNA"
/db_xref="taxon:747"
CDS
<1..2364
/note="OMP87"
/codon_start=1
/transl_table=11
/product="outer membrane protein 87"
/protein_id="ACB54932.1"
/db_xref="GI:171740841"
/translation="LIASLLFGSTTAFAAPFVVKDIRVDGVQAGTEGSVLATLPVRVG
QRATDNDIANVVRKLFLSGQYDDVKASREGNTLVVTVMPKPVISNVVIDGNKSIPDEA
IKQNLDANGFKVGDVLNRAKLEEFRKGIIEHYNSVGRYNAKVEAIVNTLPNNSAEIKI
QINEDDVALFKEIIFEGNQAFSSSKLEDQMELQTDAWWKLFGNKFDQTQFNKDLETLR
SYYLDRGYAQFQILDTDIKLSDDKKEARVIIKVKEGDLYTVKCARILGDVGGMSAELA
PILDTIQLNGLFRRANVLEVEQRIKSKLGERGYATAQVNVHPTFDEQDKTISLDFIVE
AGKSYTVRQIRFEGNTSSADSTLRQEMRQQEGAWLSSELVELGKLRLDRTGFFESVET
KTEAIPGSDQVDVIYKVKERNTGSINFGIGYGTESGLSYQASIKQDNFLGMGSSISLG
GTRNDYGTTINLGYNEPYFTKDGVSLGGNVFFEEYDSSKSNTSAAYGRTSYGGNLTLG
FPVNENNSYYLGVGYTYNKLKNIAPEYNRDLYRQSMKYNDSWTFKSHDFDLSFGWNYN
SLNRGYFPTKGVRANIGGRVTIPGSDNKYYKLNAEAQGFYPLDREHGWVLSSRISASF
ADGFGGKRLPFYQYYSAGGIGSLRGFAYGAIGPNAIYRTRQCPDSYCLVSSDVIGGNA
MVTASTELIVPTPFVADKNQNSVRTSLFVDAASVWNTRWKAEDKAKFAKLNVPDYSDP
SRVRASAGVALQWQSPIGPLVFSYAKPLKKYQGDEIEQFQFSIGGTF"
ORIGIN
1 ttaattgcga gcttattatt tggttcaacc actgcatttg ctgcgccgtt
tgtagtgaaa
61 gacattcgtg ttgacggtgt tcaagcaggt acagaaggaa gtgtattagc
tacacttcct
121 gttcgtgttg ggcagcgagc aacagataac gatattgcta atgtggtacg
aaaattattc
181 ctgagtgggc aatatgatga tgtgaaagca agtcgcgaag ggaatacttt
agttgtgaca
241 gtcatgccta aacctgttat ttcaaacgtc gtgattgacg gtaataaatc
gattcctgat
301 gaagcaatta aacaaaactt agatgcgaat ggctttaaag tcggtgatgt
attaaaccgt
361 gctaaattag aagaatttcg gaaagggatt atcgaacact acaatagtgt
cggtcgctat
421 aatgcgaagg tagaggctat cgtgaataca ctaccaaata atagcgcgga
aattaaaatt
481 caaattaatg aagatgatgt tgcactattt aaagaaatta tttttgaagg
taatcaagca
541 tttagcagca gtaaattaga agatcaaatg gagcttcaaa cagatgcatg
gtggaaattg
601 tttggtaaca aatttgatca aacccaattc aataaagatt tagagacctt
acgtagctat
661 tatttagatc gtggttacgc gcaattccaa attttagata ctgatatcaa
attaagtgat
721 gataaaaaag aagcgcgtgt cattattaaa gtgaaagaag gtgacttata
tacagtgaaa
781 tgcgcgcgta ttctggggga tgtgggtggc atgtcagcag aacttgctcc
gattttagat
841 acgattcaac taaatggtct tttccgtcgc gcaaacgtat tggaagttga
acaacgcatt
901 aaatcgaagt taggtgaaag aggttatgcg actgcgcaag tcaatgttca
cccgacattt
961 gacgaacaag ataaaacgat ttcgttagat tttattgttg aagcaggcaa
aagttatacg
1021 gttcgccaaa ttcgttttga aggcaataca agtagtgcag atagcacctt
acgtcaggaa
1081 atgcgtcaac
taaattacgt
1141 ttagatcgta
cccgggttct
1201 gatcaagtcg
taactttggt
1261 attggttatg
ggataacttc
1321 ttaggaatgg
tactacaatc
1381 aatcttggtt
tggcaatgtt
1441 ttctttgaag
acggactagc
1501 tatggtggta
ttatcttggt
1561 gtgggctata
tgatttatat
1621 cgccaatcaa
tgatttgtct
1681 tttggttgga
ggtacgtgcc
1741 aatattggtg
actcaatgca
1801 gaagcacaag
aagccgtatt
1861 agtgcctctt
atattatagc
1921 gcaggcggta
aaatgcaatt
1981 tatcgtacac
gattgggggg
2041 aatgcaatgg
cgcagataaa
2101 aatcaaaact
gaatacgcgt
2161 tggaaagcag
cagtgaccca
2221 agtcgcgttc
tggaccgttg
2281 gtgttctctt
gcagttccaa
2341 ttcagcattg
agccat
//
aagaaggcgc ttggctatcc tcggagttgg ttgagttagg
cggggttctt tgagagtgta gaaaccaaaa cagaagctat
atgtgattta taaagtcaaa gagcgtaata cgggtagcat
gtacagaaag tgggttgagc taccaagcca gtattaaaca
gatcttctat tagtttaggt gggacgcgta atgactacgg
ataatgagcc gtactttacc aaagatggtg tgagcctcgg
aatatgatag ttccaaaagt aatacctctg cggcctatgg
atttgacact aggctttccg gtgaatgaga ataactcata
cgtataataa attgaagaat atcgcgccgg aatataatcg
tgaaatataa tgattcttgg acctttaaat cgcacgattt
attataacag ccttaaccgt ggctatttcc caactaaagg
gacgagtgac cattccgggc tcagacaata aatattataa
ggttctatcc gttagatcgt gaacatggtt gggtactttc
ttgctgatgg atttggtggt aagcgtttgc cgttctatca
tcgggagttt acgtggcttt gcctatggtg cgattggacc
gtcaatgtcc tgacagctat tgtttagtca gtagcgatgt
tcaccgccag taccgaactc attgtcccaa caccatttgt
cagtaagaac ttctttgttt gtggatgccg caagtgtgtg
aggataaagc aaaatttgca aaattgaatg tgccagatta
gtgcttcagc tggggtggcg cttcaatggc aatcgccaat
atgcgaaacc tcttaagaaa taccaaggcg atgaaattga
gtgggacgtt ctaaaagctt ggatcttgct gaaaaactcg
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Hide:
sequence
Reverse complemented strand Features:
1: EU162755. Reports Pasteurella multo...[gi:161138207]


Refresh
Links
Features
Sequence
LOCUS
EU162755
942 bp
DNA
linear
BCT
03-DEC-2007
DEFINITION Pasteurella multocida strain P:52 adhesive protein (ompH)
gene,
partial cds.
ACCESSION
EU162755
VERSION
EU162755.1 GI:161138207
KEYWORDS
.
SOURCE
Pasteurella multocida
ORGANISM Pasteurella multocida
Bacteria; Proteobacteria; Gammaproteobacteria;
Pasteurellales;
Pasteurellaceae; Pasteurella.
REFERENCE
1 (bases 1 to 942)
AUTHORS
Yadav,A., Saxena,M.K., Rao,V.D.P. and Sharma,B.
TITLE
OMPH of Pasterella multocida vaccine strain: in vitro
expressed
protein immunogenicity
JOURNAL
Unpublished
REFERENCE
2 (bases 1 to 942)
AUTHORS
Yadav,A., Saxena,M.K., Rao,V.D.P. and Sharma,B.
TITLE
Direct Submission
JOURNAL
Submitted (19-SEP-2007) Biochemistry, Indian Veterinary
Research
Institute, Bareilly, U.P. 243122, India
FEATURES
Location/Qualifiers
source
1..942
/organism="Pasteurella multocida"
/mol_type="genomic DNA"
/strain="P:52"
/db_xref="taxon:747"
gene
<1..942
/gene="ompH"
CDS
<1..942
/gene="ompH"
/note="OmpH"
/codon_start=1
/transl_table=11
/product="adhesive protein"
/protein_id="ABX58059.1"
/db_xref="GI:161138208"
/translation="ATVYNQDGTKVDVNGSVRLILKKEKNERGDLVDNGSRVSFKASH
DLGEGLSALAYAELRFSTKVKKTVKEGPSQVERIGNDVHVKRLYAGFAYEGLGTLTFG
NQLTIGDDVGVSDYTYFLGGINNLLSSGEKAINFKSAEFNGFTFGGAYVFSADADKQA
PRDGRGFVVAGLYNRKMGDVGFALEAGYSQKYVTVAKQEKAFMVGTELSYAGLALGVD
YAQSKVTNVEGKKRALEVGLNYDINDKAKVYTDLIWAKGGPKGATTRDRSIILGAGYK
LHKQVETFVEGGWGREKDANGVTTKGNVVGVGLRVHF"
ORIGIN
1 gcaacagttt acaatcaaga cggtacaaaa gttgatgtaa atggttctgt
acgtttaatc
61 cttaaaaaag aaaaaaatga gcgcggtgat ttagtggata acggttcacg
cgtttctttc
121 aaagcatctc atgacttagg cgaaggttta agcgcattag cttacgcaga
acttcgtttc
181 agcacaaaag ttaaaaaaac agttaaagaa ggtcctagcc aagttgagcg
tatcggtaat
241 gatgttcacg taaaacgtct ttatgcgggt ttcgcgtatg aaggtttagg
aacattaact
301 ttcggtaacc aattaactat cggtgatgat gttggtgtgt ctgactacac
ttacttctta
361 ggtggtatca acaatcttct ttctagcggt gaaaaagcaa ttaactttaa
atctgcagaa
421 ttcaacggtt tcacatttgg tggtgcgtat gtgttctctg cggatgcaga
caaacaagca
481 ccacgtgatg gtcgcggttt cgttgtagca ggtttatata acagaaaaat
gggcgatgtt
541 ggtttcgcac ttgaagcggg ttatagccaa aaatatgtaa cagtagcgaa
acaagaaaaa
601 gcctttatgg ttggtactga attatcatac gctggtttag cacttggtgt
tgactatgca
661 caatctaaag tgactaacgt agaaggtaaa aaacgcgcac ttgaagtggg
tttaaactat
721 gatattaatg acaaagcgaa agtttacact gacttgattt gggcaaaagg
aggtccaaaa
781 ggtgcgacta caagagatcg ttctatcatc ttaggtgcgg gctacaagct
tcacaaacaa
841 gttgaaacct ttgttgaagg tggctggggc agagagaaag atgctaatgg
cgtaacaaca
901 aaaggcaatg tcgttggtgt tggtttacgc gtacacttct aa
//
Disclaimer | Write to the Help Desk
NCBI | NLM | NIH
ExPASy ProtParam tool
ExPASy Home page Site Map Search ExPASy Contact us Proteomics tools
Swiss-Prot
Search for
ProtParam
User-provided sequence:
10 20 30 40 50 60
M KKLLIASLL FGSTTAFAAP FVVKDIRVDG VQAGTEGSVL
ATDNDIANVV
70 80 90 100 110 120
R KLFLSGQYD DVKASREGNT LVVTVMPKPV ISNVVIDGNK
LDANGFKVGD
130 140 150 160 170 180
V LNRAKLEEF RKGIIEHYNS VGRYNAKVEA IVNTLPNNSA
VALFKEIIFE
190 200 210 220 230 240
G NQAFSSSKL EDQMELQTDA WWKLFGNKFD QTQFNKDLET
AQFQILDTDI
250 260 270 280 290 300
K LSDDKKEAR VIIKVKEGDL YTVKCARILG DVGGMSAELA
LFRRANVLEV
310 320 330 340 350 360
E QRIKSKLGE RGYATAQVNV HPTFDEQDKT ISLDFIVEAG
EGNTSSADST
370 380 390 400 410 420
L RQEMRQQEG AWLSSELVEL GKLRLDRTGF FESVETKTEA
ISQERNTGSI
430 440 450 460 470 480
N FGIGYGTES GLSYQASIKQ DNFLGMGSSI SLGGTRNDYG
YFTKDGVSLG
490 500 510 520 530 540
G NVFFEEYDS SKSNTSAAYG RTSYGGNLTL GFPVNENNSY
LKNIAPEYNR
550 560 570 580 590 600
D LYRQSMKYN DSWTFKSHDF DLSFGWNYNS LNRGYFPTKG
IPGSDNKYYK
610 620 630 640 650 660
L NAEAQGFYP LDREHGWVLS SRISASFADG FGGKRLPFYQ
RGFAYGAIGP
670 680 690 700 710 720
N AIYRTRQCP DSYCLVSSDV IGGNAMVTAS TELIVPTPFV
SLFVDAASVW
730 740 750 760 770 780
N TRWKAEDKA KFAKLNVPDY SDPSRVRASA GVALQWQSPI
LKKYQGDEIE
790
Q FQFSIGGTF
References and documentation are available.
Please note the modified algorithm for extinction coefficient.
Number of amino acids: 790
Molecular weight: 87571.1
Theoretical pI: 5.94
http://www.expasy.ch/cgi-bin/protparam (1 of 3)1/11/2008 12:31:22 PM
ATLPVRVGQR
SIPDEAIKQN
EIKIQINEDD
LRSYYLDRGY
PILDTIQLNG
KSYTVRQIRF
IPGSDQVDVI
TTINLGYNEP
YLGVGYTYNK
VRANIGGRVT
YYSAGGIGSL
ADKNQNSVRT
GPLVSSYAKP
Swiss-Prot/TrEMBL Go Clear
ExPASy ProtParam tool
Amino acid composition:
Ala (A) 55 7.0%
Arg (R) 40 5.1%
Asn (N) 49 6.2%
Asp (D) 50 6.3%
Cys (C) 3 0.4%
Gln (Q) 34 4.3%
Glu (E) 43 5.4%
Gly (G) 72 9.1%
His (H) 4 0.5%
Ile (I) 46 5.8%
Leu (L) 61 7.7%
Lys (K) 49 6.2%
Met (M) 8 1.0%
Phe (F) 40 5.1%
Pro (P) 25 3.2%
Ser (S) 66 8.4%
Thr (T) 43 5.4%
Trp (W) 9 1.1%
Tyr (Y) 38 4.8%
Val (V) 55 7.0%
Asx (B) 0 0.0%
Glx (Z) 0 0.0%
Xaa (X) 0 0.0%
Total number of negatively charged residues (Asp + Glu): 93
Total number of positively charged residues (Arg + Lys): 89
Atomic composition:
Carbon C 3910
Hydrogen H 6063
Nitrogen N 1059
Oxygen O 1207
Sulfur S 11
Formula: C3910H6063N1059O1207S11
Total number of atoms: 12250
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm
measured in water.
Ext. coefficient 106245
Abs 0.1% (=1 g/l) 1.213, assuming ALL Cys residues appear
as half cystines
http://www.expasy.ch/cgi-bin/protparam (2 of 3)1/11/2008 12:31:22 PM
CSV format
ExPASy ProtParam tool
Ext. coefficient 106120
Abs 0.1% (=1 g/l) 1.212, assuming NO Cys residues appear as
half cystines
Estimated half-life:
The N-terminal of the sequence considered is M (Met).
The estimated half-life is: 30 hours (mammalian
reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).
Instability index:
The instability index (II) is computed to be 30.11
This classifies the protein as stable.
Aliphatic index: 79.97
Grand average of hydropathicity (GRAVY): -0.387
ExPASy Home page Site Map Search ExPASy Contact us Proteomics tools
Swiss-Prot
Search for
http://www.expasy.ch/cgi-bin/protparam (3 of 3)1/11/2008 12:31:22 PM
ProtParam
User-provided sequence:
10
20
30
40
50
60
ATVYNQDGTK VDVNGSVRLI LKKEKNERGD LVDNGSRVSF KASHDLGEGL SALAYAELRF
70
80
90
100
110
120
STKVKKTVKE GPSQVERIGN DVHVKRLYAG FAYEGLGTLT FGNQLTIGDD VGVSDYTYFL
130
140
150
160
170
180
GGINNLLSSG EKAINFKSAE FNGFTFGGAY VFSADADKQA PRDGRGFVVA GLYNRKMGDV
190
200
210
220
230
240
GFALEAGYSQ KYVTVAKQEK AFMVGTELSY AGLALGVDYA QSKVTNVEGK KRALEVGLNY
250
260
270
280
290
300
DINDKAKVYT DLIWAKGGPK GATTRDRSII LGAGYKLHKQ VETFVEGGWG REKDANGVTT
310
KGNVVGVGLR VHF
References and documentation are available.
Please note the modified algorithm for extinction coefficient.
Number of amino acids: 313
Molecular weight: 33761.0
Theoretical pI: 9.12
Amino acid composition:
Ala (A) 27
8.6%
Arg (R) 14
4.5%
Asn (N) 16
5.1%
Asp (D) 19
6.1%
Cys (C)
0
0.0%
Gln (Q)
8
2.6%
Glu (E) 17
5.4%
Gly (G) 43
13.7%
His (H)
4
1.3%
Ile (I)
9
2.9%
Leu (L) 25
8.0%
Lys (K) 28
8.9%
Met (M)
2
0.6%
Phe (F) 15
4.8%
Pro (P)
3
1.0%
Ser (S) 16
5.1%
Thr (T) 18
5.8%
Trp (W)
2
0.6%
Tyr (Y) 15
4.8%
Val (V) 32
10.2%
Asx (B)
Glx (Z)
0
0
0.0%
0.0%
CSV format
Xaa (X)
0
0.0%
Total number of negatively charged residues (Asp + Glu): 36
Total number of positively charged residues (Arg + Lys): 42
Atomic composition:
Carbon
Hydrogen
Nitrogen
Oxygen
Sulfur
C
H
N
O
S
1509
2369
417
459
2
Formula: C1509H2369N417O459S2
Total number of atoms: 4756
Extinction coefficients:
Extinction coefficients are in units of
water.
Ext. coefficient
Abs 0.1% (=1 g/l)
cystines
M-1 cm-1, at 280 nm measured in
33350
0.988, assuming ALL Cys residues appear as half
Estimated half-life:
The N-terminal of the sequence considered is A (Ala).
The estimated half-life is: 4.4 hours (mammalian reticulocytes, in
vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).
Instability index:
The instability index (II) is computed to be 13.93
This classifies the protein as stable.
Aliphatic index: 80.64
Grand average of hydropathicity (GRAVY): -0.318
[313 residues]
Trusted matches - domains scoring higher than the gathering threshold (A)
Domain Start End
Bits
Evalue
Alignment Mode
Porin_1
-90.90
0.00072
Align
6
313
ls
Matches to Pfam-B
Domain
Start End
Pfam-B_121714 260
Alignment
313 Align
Potential matches - Domains with Evalues above the cutoff
Domain
Start End
Bits
Evalue
Alignment Mode
Opacity
211
313
-70.40 0.89
Align
ls
Porin_1
7
52
0.90
3.2
Align
fs
Transposase_11
218
284
3.50
0.45
Align
fs
Porin_1
269
313
7.50
0.048
Align
fs
Alignments of Pfam-A domains to HMMs
Format for fetching alignments to seed
Jalview Java alignment view er
Alignment of Porin_1 vs UNKNOWN-QUERY/6-313
UNKNOWN-QU
*->KdGNklDlygkvvglhyfsddtgtdgddtYaRiGFKGeTqindqLtG
dG k+D g v
+ ++++ d d ++R+ FK + +++++L +
6
QDGTKVDVNGSVRLILKKEKNERGDLVDNGSRVSFKASHDLGEGLSA
52
UNKNOWN-QU
101
yGQwEynvs......vngtEgeqsnqwGs..gTRlaFaGLKfGdyGsfDy
E ++s++ +++v+ + ++ + G++ ++
aG ++ + G++
53 LAYAELRFStkvkktVKEGPSQ-VERIGNdvHVKRLYAGFAYEGLGTLTF
UNKNOWN-QU
140
UNKNOWN-QU
180
UNKNOWN-QU
217
UNKNOWN-QU
257
UNKNOWN-QU
296
UNKNOWN-QU
GRnygvlyDveawtDmlPefgGdtyasvaqtDnfmtgrangvaTRYRNpd
G
++ +Dv+
D
+f
+++n+ + ++ +
+ +
102 GNQLTIGDDVG-VSD-YTYFL-------GGINNLLSSGEKAINF--KSAE
FFGLVdGLnFalqyqGkNesrtrnngrdvrkqNGDGfgasltY..dngGf
F G+ G +
+ +
+G Gf ++ Y++++g
141 FNGFTFGGAYVFSADADKQA----------PRDGRGFVVAGLYnrKMGDV
gfsyggaYansdrtddQklelkqtllgngdkaeawrlgaKYDaNnvYlAv
gf++ + Y++
t
++ a+ +g
++ l v
181 GFALEAGYSQKYVTVA-------------KQEKAFMVGTELSYAGLALGV
aYaqtrnmtpygggnadntvesdsgfanKtqnfEvvAqyqFDFGNLrPsv
Yaq +
+ g++
+ + + + + v+
G
218 DYAQSKVTNVEGKKR----ALEVGLNYDINDKAKVYTDLIWAKG-----sYlqsKgkdlngkkgdnDlvkYVdVGatYYFNKNmStyVdYkiNlldknd
+ ++ ++
d
++ Ga Y
K
t+V +++ ++++ +
258 ----GPKGATTR---DR----SIILGAGYKLHKQVETFVEGGWGREKDAN
dftkaaGiatd.divaVGLvYqF<-*
G++t +++v+VGL+ F
297 ------GVTTKgNVVGVGLRVHF
313
Align to seed
Alignment of Transposase_11 vs UNKNOWN-QUERY/218-284
UNKNOWN-QU
264
UNKNOWN-QU
284
*->eyv.v.d.q.g.k.r.r.vyrkv.r.lke.py.k.kw.ilrrvvvvk
+y+++++++ ++k+r +v
+++++k ++y+ +w ++
++
218
DYAqSkVtNvEgKkRaLeVGLNYdInDKAkVYtDlIWaKGGPKGATT
errkiklvaqkskkgketplyvtnlltelsaeeiaelyrlRwqvErv<-*
++r+i
++ + y+l++qvE++
265 RDRSI---------------------------ILGAGYKLHKQVETF
Align to seed
Alignments of Pfam-B domains to best-matching to Pfam-B sequence
Format for fetching alignments to Pfam-B families:
Hypertext linked to sw isspfam
Query Query/260-313 matching Pfam-B_121714
temp 260 KGATTRDRSIILGAGYKLHKQVETFVEGGWGREKDANGVTTKGNVVGVGL 309
KGATTRDRSIILGAGYKLHKQVETFVEGGWGREKDANGVTTKGNVVGVGL
Query 260 KGATTRDRSIILGAGYKLHKQVETFVEGGWGREKDANGVTTKGNVVGVGL 309
temp 310 RVHF 313
RVHF
Query 310 RVHF 313
Align to family
TMpred output for unknown
min=17 | max=33 | html | | plain_text |
MKKLLIASLLFGSTTAFAAPFVVKDIRVDGVQAGTEGSVLATLPVRVGQRATDNDIAN
VVRKLFLSGQYDDVKASREGNT%
0D%
0ALVVTVMPKPVISNVVIDGNKSIPDEAIKQNLDANGFKVGDVLNRAKLEEFRKGIIEH
YNSVGRYNAKVEAIVNTLPNNSA%
0D%
0AEIKIQINEDDVALFKEIIFEGNQAFSSSKLEDQMELQTDAWWKLFGNKFDQTQFNKD
LETLRSYYLDRGYAQFQILDTDI%
0D%
0AKLSDDKKEARVIIKVKEGDLYTVKCARILGDVGGMSAELAPILDTIQLNGLFRRANV
LEVEQRIKSKLGERGYATAQVNV%
0D%
0AHPTFDEQDKTISLDFIVEAGKSYTVRQIRFEGNTSSADSTLRQEMRQQEGAWLSSELV
ELGKLRLDRTGFFESVETKTEA%
0D%0AIPGSDQVDVIISQERNTGSINFGIGYGTESGLSYQASIKQDNFLGMGSSISLGGTR
NDYGTTINLGYNEPYFTKDGVSLG
%0D%
0AGNVFFEEYDSSKSNTSAAYGRTSYGGNLTLGFPVNENNSYYLGVGYTYNKLKNIAP
EYNRDLYRQSMKYNDSWTFKSHDF
%0D%
0ADLSFGWNYNSLNRGYFPTKGVRANIGGRVTIPGSDNKYYKLNAEAQGFYPLDREHG
WVLSSRISASFADGFGGKRLPFYQ
%0D%
0AYYSAGGIGSLRGFAYGAIGPNAIYRTRQCPDSYCLVSSDVIGGNAMVTASTELIVPTP
FVADKNQNSVRTSLFVDAASVW%
0D%0ANTRWKAEDKAKFAKLNVPDYSDPSRVRASAGVALQWQSPIGPLVSSYAKPLK
KYQGDEIEQFQFSIGGTF.
TMpred output for unknown
[ISREC-Server] Date: Fri Jan 11 8:04:57 Europe/Zurich 2008
Sequence: MKK...GTF, length: 790
Prediction parameters: TM-helix length between 17 and 33
1.) Possible transmembrane helices
The sequence positions in brackets denominate the core region.
Only scores above 500 are considered significant.
Inside to outside helices : 5 found
from to score center
4 ( 4) 23 ( 23) 1732 13
265 ( 267) 283 ( 283) 40 275
644 ( 644) 664 ( 664) 552 654
672 ( 675) 695 ( 692) 266 684
748 ( 748) 765 ( 765) 473 756
Outside to inside helices : 5 found
from to score center
4 ( 4) 23 ( 23) 1469 13
497 ( 502) 525 ( 520) 9 512
635 ( 640) 659 ( 656) 537 648
675 ( 675) 701 ( 695) 502 685
748 ( 750) 768 ( 768) 143 758
2.) Table of correspondences
http://www.ch.embnet.org/cgi-bin/TMPRED_form_parser (1 of 3)1/11/2008 12:34:35 PM
TMpred output for unknown
Here is shown, which of the inside->outside helices correspond to which of the outside->inside
helices.
Helices shown in brackets are considered insignificant.
A "+"-symbol indicates a preference of this orientation.
A "++"-symbol indicates a strong preference of this orientation.
inside->outside | outside->inside
4- 23 (20) 1732 ++ | 4- 23 (20) 1469
( 265- 283 (19) 40 ++ ) |
|( 497- 525 (29) 9 ++ )
644- 664 (21) 552 | 635- 659 (25) 537
( 672- 695 (24) 266 ) | 675- 701 (27) 502 ++
( 748- 765 (18) 473 ++ ) |( 748- 768 (21) 143 )
3.) Suggested models for transmembrane topology
These suggestions are purely speculative and should be used with extreme caution since they
are based on the assumption that all
transmembrane helices have been found.
In most cases, the Correspondence Table shown above or the prediction plot that is also created
should be used for the topology
assignment of unknown proteins.
2 possible models considered, only significant TM-segments used
*** the models differ in the number of TM-helices ! ***
-----> STRONGLY prefered model: N-terminus outside
3 strong transmembrane helices, total score : 2523
# from to length score orientation
1 4 23 (20) 1469 o-i
2 644 664 (21) 552 i-o
3 675 701 (27) 502 o-i
------> alternative model
2 strong transmembrane helices, total score : 2269
# from to length score orientation
1 4 23 (20) 1732 i-o
2 635 659 (25) 537 o-i
http://www.ch.embnet.org/cgi-bin/TMPRED_form_parser (2 of 3)1/11/2008 12:34:35 PM
TMpred output for unknown
You can get the prediction graphics shown above in one of the following formats:
l GIF-format
l Postscript-format
l numerical format
Back to ISREC home page
http://www.ch.embnet.org/cgi-bin/TMPRED_form_parser (3 of 3)1/11/2008 12:34:35 PM
min=17 | max=33 | html | OMP34 | plain_text |
ATVYNQDGTKVDVNGSVRLILKKEKNERGDLVDNGSRVSFKASHDLGEGLSA
LAYAELRFSTKVKKTVKEGPSQVERIGN%0D%0ADVHVKRLYAGFAYEGLGTL
TFGNQLTIGDDVGVSDYTYFLGGINNLLSSGEKAINFKSAEFNGFTFGGAYVFS
ADADKQA%0D%0APRDGRGFVVAGLYNRKMGDVGFALEAGYSQKYVTVAK
QEKAFMVGTELSYAGLALGVDYAQSKVTNVEGKKRALEVGLNY%0D%0ADIN
DKAKVYTDLIWAKGGPKGATTRDRSIILGAGYKLHKQVETFVEGGWGREKDA
NGVTTKGNVVGVGLRVHF
TMpred output for OMP34
[ISREC-Server] Date: Mon Jan 7 14:40:28 Europe/Zurich 2008
Sequence: ATV...VHF, length: 313
Prediction parameters: TM-helix length between 17 and 33
1.) Possible transmembrane helices
The sequence positions in brackets denominate the core region.
Only scores above 500 are considered significant.
Inside to outside
from
87 ( 89) 108 (
133 ( 135) 156 (
202 ( 202) 219 (
helices :
3 found
to
score center
108)
201
98
153)
200
143
219)
348
210
Outside to inside
from
111 ( 111) 131 (
202 ( 202) 220 (
helices :
2 found
to
score center
131)
246
120
220)
9
212
2.) Table of correspondences
Here is shown, which of the inside->outside helices correspond to which of the outside>inside helices.
Helices shown in brackets are considered insignificant.
A "+"-symbol indicates a preference of this orientation.
A "++"-symbol indicates a strong preference of this orientation.
(
(
(
inside->outside | outside->inside
87- 108 (22) 201 ++ ) |
|( 111- 131 (21) 246 ++ )
133- 156 (24) 200 ++ ) |
202- 219 (18) 348 ++ ) |( 202- 220 (19)
9
)
3.) Suggested models for transmembrane topology
These suggestions are purely speculative and should be used with extreme caution
since they are based on the assumption that all transmembrane helices have been found.
In most cases, the Correspondence Table shown above or the prediction plot that is also
created should be used for the topology assignment of unknown proteins.
2 possible models considered, only significant TM-segments used
!!! probably no transmembrane protein - no possible model found !!!
You can get the prediction graphics shown above in one of the following formats:



GIF-format
Postscript-format
numerical format
Back to ISREC home page
Protein: noname
Length: 313
N-terminus: IN
Number of transmembrane helices: 1
Transmembrane helices: 89-108
Total entropy of the model: 17.0120
Entropy of the best path: 17.0128
The best path:
seq ATVYNQDGTK VDVNGSVRLI LKKEKNERGD LVDNGSRVSF KASHDLGEGL
pred IIIIIIIIII IIIIIIIIII IIIIIIIIII IIIIIIIIII IIIIIIIIII
50
seq SALAYAELRF STKVKKTVKE GPSQVERIGN DVHVKRLYAG FAYEGLGTLT
pred IIIIIIIIII IIIIIIIIII IIIiiiiiii iiiiiiiiHH HHHHHHHHHH
100
seq FGNQLTIGDD VGVSDYTYFL GGINNLLSSG EKAINFKSAE FNGFTFGGAY
pred HHHHHHHHoo oooooooooo oooOOOOOOO OOOOOOOOOO OOOOOOOOOO
150
seq VFSADADKQA PRDGRGFVVA GLYNRKMGDV GFALEAGYSQ KYVTVAKQEK
pred OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO
200
seq AFMVGTELSY AGLALGVDYA QSKVTNVEGK KRALEVGLNY DINDKAKVYT
pred OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO
250
seq DLIWAKGGPK GATTRDRSII LGAGYKLHKQ VETFVEGGWG REKDANGVTT
pred OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO
300
seq KGNVVGVGLR VHF
pred OOOOOOOOOO OOO
313
Vita
The authoress, Archana Yadav was born on April 25, 1977 in Haridwar,
Uttarakhand. She passed her high school and intermediate examination with first
division in 1993 and 1995, respectively, from U. P. Board. She completed her B.Sc.
degree in Zoology, Botany and Chemistry with first division from C. C. S.
University, Meerut in 1998. She did her M.Sc. in Microbiology with first division
from Gurukul Kangri Vishwavidyalaya, Haridwar in 2000.
She joined College of Basic Sciences and Humanities, G.B.P.U.A & T.,
Pantnagar for her Ph.D. degree in Microbiology in July 2004.
She was qualified National Eligibility Test for lecturership. During her Ph.D.
programme she was joined DBT project as Senior Research Fellow for a period of
two years.
Permanent Address:
Q No. 61, Type II, Sector 4
B.H.E.L. Ranipur Haridwar
Uttarakhand 249403.
Email: [email protected]
ABSTRACT
Name
:
Archana Yadav
Id. No.
:
31741
Sem. & year
of admission
:
1st,
Degree
:
Ph.D.
Major
:
Microbiology
Minor
:
Molecular Biology & Genetic Engineering
Thesis Title
: “Cloning and characterization of outer membrane
protein(s) of Pasteurella multocida serotype B:2
(P52)”
Advisor
:
2004-05
Department :
Microbiology
Dr. Anita Sharma
Pasteurella multocida serotype B:2 is a causative agent of Haemorrhagic
septicaemia (HS), a fatal disease of cattle and buffaloes. Formalin inactivated
whole cell bacterin is frequently used to prepare vaccines in India against HS.
This vaccine has several limitations such as short term immunity, poor
syringibility and production problem. To overcome from these problems there
is an urgent need of search of a conserved immunogenic protein as vaccine
candidate. Outer membrane proteins of P. multocida are reported to be
immunogenic and protective. SDS-PAGE analysis of outer membrane proteins
of vaccine strain P52 revealed the presence of eight polypeptides with
molecular weigh ranging from 16 to 87 kDa.
Antiserum raised against whole cell antigen showed positive result by
agar gel immunodiffusion test. Western blot analysis revealed the presence of
seven immunodominant outer membrane proteins. Out of seven, omp87 and omp34
were selected as potent antigens for further study.
Primers sets were designed from available sequences in NCBI Gene
Bank. Both genes (omp87 and omp34) were amplified using DNA as template
by PCR. The amplified fragments were cloned in pGEMT-Easy vector. After
sequencing, size of the cloned omp87 gene was assessed and found to be 2121
bp accounting for 707 amino acid residues and size of omp34 was 942 bp
accounting for 313 amino acid residues. Both the sequences were analysed
using online bioinformatics tools (ExPASY server). On comparison of
nucleotide sequence, omp87 showed 94.8% similarity with serotype A:1 while
homology at amino acid level was 95.2%. Similarly at nucleotide level 98.3%
and at amino acid level 97.5% similarity has been observed in omp34.
The sequences were submitted to NCBI Gene Bank and accession
number EU570212 and EU162755 were obtained for omp87 and omp34
respectively. On phylogenetic analysis it was found that omp34 of serotype B:2
clustered with serotype 3,4. Probable secondary structures and number of
transmembrane helices were also predicted.
(Anita Sharma)
Advisor
(Archana Yadav)
Authoress