Download Expression of gene encoding immunodominant merozoite surface

Indian Journal of Biotechnology
Vol 7, April 2008, pp 200-203
Expression of gene encoding immunodominant merozoite surface protein of
Theileria annulata in Escherichia coli
C Rajendran, D D Ray* and G C Bansal
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243 122, India
Received 8 January 2007; revised 6 July 2007; accepted 10 October 2007
The asexual blood stage “merozoites” of an Indian strain of the tick-borne cattle haemoprotozoa, Theileria annulata,
was generated in in vitro culture and the gene encoding the merozoite surface protein (Tams 1) was amplified from cDNA
by using primers designed from T. annulata (Ankara strain). The amplified gene was cloned into pPROExHT b plasmid
vector and expressed as fusion protein in Escherichia coli, DH5α strain. The recombinant protein was purified using NINTA affinity chromatography and used to detect antibody response in known anti-Tannulata bovine serum samples in a
ELISA format.
Keywords: Theileria annulata, immunodominant merozoites surface protein, pPROEXHTb, E.coli, Ni-NTA affinity
chromatography, western blot analysis
Introduction
The sporozoites of the tick-borne haemoprotozoa,
Theileria annulata, invade the macrophages/B cells in
the lymph gland of cattle through tick-bite and grow
into intracellular macroschizonts. The macroschizonts
transform the host cells and with the progress of
infection, the infected lymphoblasts are found
throughout lymphoid and reticulo-endothelial tissues
of the host. Merozoites are generated within the
infected cells, which upon release, invade the
circulating erythrocytes to form piroplasms. Infection
is then transmitted to a feeding tick via ingestion of
infected erythrocytes.
Merozoites of T. annulata show the presence of a
major immunodominant surface antigen, which
possesses a molecular weight of either 30 or 32 kDa
in various strains of the parasite1 and could represent
the allelic forms of Tams1 (T. annulata merozoite
surface antigen) gene2. Tams1 has been considered for
inclusion in a recombinant subunit vaccine3 and for
the development of a diagnostic ELISA4 for bovine
tropical theileriosis. This laboratory identified only
one genotype of Tams1 having a molecular mass of
32 kDa in T. annulata (Parbhani strain)5 and
expression of this gene in E. coli vector is
communicated in this paper. The recombinant protein
_______________
*Author for correspondence:
Tel: 91-581-2302368
E- mail: dd_rayvet@ yahoo.co.in
was validated to detect antibody response in known
anti-T. annulata bovine sera.
Materials and Methods
Parasite Strain and in vitro Development of Merozoites
The cell line of bovine lymphoblastoid cells
infected with macroschizonts of T. annulata (Parbhani
strain) was grown as suspension culture in RPMI1640 medium supplemented with 10 per cent bovine
serum at 37°C 6. The culture of infected lymphoblasts
was maintained at higher temperature (41°C) for
differentiation of the macroschizonts to extracellular
merozoites7. The culture was centrifuged at 300 g for
15 min for separating the bulk of undifferentiated
infected lymphoblasts. The merozoites were separated
from the supernatant by centrifuging at 2500 g
for 15 min.
Amplification of Tams1
The cDNA was synthesized from ten (10) pg total
RNA of merozoites. Oligonucleotide primers
[Forward primer: 5′-ATG AGG ATG AAA AGA
AAA AGG AGG AAA AAA AAG ATG T 3′
(position 125-161), Reverse primer: 5′-GCG AAG
ACT GCA AGG GGG GAG AAC T -3′ (position
833-857)] were designed from the published sequence
of T. annulata (Ankara strain, Accession No.
U22887). The PCR amplification of Tams1 was
carried out from 1 ng cDNA in a thermal cycler (MJ
Research PTC 200) with the following conditions:
RAJENDRAN et al: EXPRESSION OF RECOMBINANT T. ANNULATA MEROZOITE SURFACE PROTEIN
initial DNA denaturation at 95°C for 5 min, 30 cycles
at 94°C/1 min, annealing at 55°C/1 min, extension at
72°C/1 min, final extension at 72° C/5 min. A 10 µL
of amplified product was visualized by submarine gel
electrophoresis.
Cloning and Characterization of Tams1
The Tams1 gene was purified from gel and cloned
in pDrive (U/A) vector. The competent E. coli strain
DH5α was transformed with cloned Tams1 gene by
standard technique8. The transformed bacteria were
plated on 1.5% LB agar plate containing ampicillin
(100 µg/mL), X-gal (40 µL of 20 mg/mL) and IPTG
(8 µL of 100 mg/mL). The plates were incubated at
37°C for 12-16 h for colony growth. The recombinant
clones were screened for their ampicillin resistance
and production of white colonies on X-gal/IPTG
plate. Twelve randomly picked white colonies were
grown overnight in LB broth with ampicillin in
separate tubes. All the 12 colonies were subjected to
colony PCR in 25 µL reaction buffer containing 10
pmol each of the product specific forward and reverse
primers under conditions specified before and the
amplified product was visualized by electrophoresis.
Three positive plasmids were subjected
individually to RE analysis in 20 µL mixture
containing 2 µg recombinant plasmid, one unit each
of the restriction enzymes Hpa I and APA I, and 1 x
Tango buffer (MBI Fermentas). The contents were
incubated at 37°C for 1 h and analysed in 1% agarose
gel electrophoresis. The colony showing right
orientation of the insert was named as pDTams1.
Subcloning of Tams1 in pPROExHT b Expression Vector
One µg plasmid DNA of pPROExHTb and 2 µg of
pDTams1 plasmid were digested with the restriction
enzymes
BamH
I
and
Hind
III
(MBI Fermentas). Forty ng of digested pPROExHTb
(4.276 kb) and 30 ng of digested Tams1 (770 bp)
were ligated in a 10 µL reaction mixture of 1x ligation
buffer and 1 Weiss U T4 DNA ligase (MBI,
Fermentas). The ligation reaction was performed
overnight at 22°C and then terminated by heating at
70°C for 10 min.
Two µL of reaction mixture was transformed into
competent DH5α cells and plated on LB agar plates
containing ampicillin (100 µg/mL) at 37°C overnight.
The plasmids isolated from six randomly selected
colonies were analyzed by electrophoresis after
double digestion with BamH I/Hind III. One of the
plasmids was randomly selected for expression.
201
Expression, characterization and purification of Tams1
E. coli DH5α cells harbouring the recombinant
pPROExHT b (named as pPROTams1) were grown in
LB medium containing ampicillin (100 µg/mL) at
37°C and induced by 2 mM IPTG. The culture was
incubated further at 37°C with vigorous shaking and
1 mL of it was taken at hourly intervals for 5 h in
separate tubes to monitor the expression of desired
protein in SDS-PAGE9. The blot of protein was
characterized with serum of bovine, which was
recovered from experimental sporozoite induced
infection of T. annulata in standard immunostaining
procedure.
The positive clones of Tams1 were cultured in bulk
and the expressed protein was purified by metal
chelate affinity chromatography using nickelnitrilotriacetic acid (Ni-NTA)-agarose slurry (Qiagen,
Germany).
Validation of Tams1 Protein in ELISA
The duplicate wells of ELISA plate were coated
separately with Tams1 protein at a concentration of
1,2 or 5 µg per mL of carbonate-bicarbonate buffer
(pH 9.6) and titrated with two positive and two
negative bovine sera diluted 1:50, 1:100 or 1:200 with
PBS (pH 7.2) in standard ELISA format10. The
combination of antigen and dilutions of serum, which
yielded maximum differences in OD values of
negative and positive sera were considered as
optimum for the test. Ten sera of bovine calves
immunized with low dose of sporozoites of
T annulata, showing positive antibody titre of 1:160
in indirect fluorescent antibody test, were used to
validate Tams1. The mean of optical densities of two
negative bovine sera was 0.378±0.64. An OD of
0.506 in Tams1 was considered as positive.
Results and Discussion
The primers were designed to amplify an internal
fragment of Tams1 gene to ensure elimination of both
N and C termini. The size of the product was 733 bp
(Fig.1) and the concentration of the purified product
was 12 ng/µL. A product of 876 bp, comprising full
length of the gene (not shown in Fig. 1) could not be
expressed using the prokaryotic expression system
probably due to the toxicity of the hydrophobic signal
sequences and similar views have been expressed by
other workers12. For confirmation of the orientation of
the insert, three positive plasmids were digested
individually with Hpa I and Apa I restriction
enzymes. Since Hpa I cut in the product at 109 bp and
202
INDIAN J BIOTECHNOL, APRIL 2008
Fig. 1—Lanes: M-100 bp DNA marker; 1- amplification of 733
bp Tams1; 2- RE digestion of pDTams1 with Hpa I and Apa I for
the presence of rightly oriented Tams1.
Fig. 3—SDS-PAGE (Lanes: M-Protein molecular weight marker;
1-Uninduced culture; 2-Expression of 32 kDa Tams1 induced
with 2 mM IPTG; 3- Vector control)
Fig. 2—Lanes: M-100 bp DNA marker; 1- a undigested
pPROExHT, b-recombinant plasmid; 2 -770 bp insert release with
BamH I and Hind III.
Apa I in the pDrive vector at 378 bp, release of
product 685 bp indicated that all clones were in right
orientation (Fig.1). The positive clones were termed
as pDTams1.
On double digestion with restriction enzymes
BamH I and Hind III, pDTams1 released an insert of
770 bp because of the addition of 37 nucleotides from
the pDrive vector. The 770 bp was subcloned between
the BamH I and Hind III sites of pPROExHTb
expression vector. The positive clones were termed as
pPROTams1, which yielded 770 bp insert when
double digested with BamH I and Hind III (Fig. 2).
Fig. 4—SDS-PAGE-Time kinetic analysis of expression of Tams1
protein (Lanes: M-Protein molecular weight marker; 1-Uninduced
culture; 2, 3 & 4-Expression in 2, 4 & 5 h).
The expression of 32 kDa Tams1 protein was
detected in 12% SDS-PAGE (Fig. 3). Expression was
detected from 2 h post induction, with maximum at
5 h (Fig. 4). Recombinant Tams1 was purified by a
one step chromatography on Ni-NTA agarose using
nickel as the chelating agent (Fig. 5). The serum of
bovine calf recovered from experimentally induced
RAJENDRAN et al: EXPRESSION OF RECOMBINANT T. ANNULATA MEROZOITE SURFACE PROTEIN
203
T. annulata infection recognized Tams1 in
immunoblot analysis (Fig. 6). The 10 bovine sera
showed positive antibody response in an optimized
ELISA protocol in which Tams1 was used at a
concentration of 1 µg/mL of buffer and sera were
diluted 1:200.
Acknowledgement
The authors are thankful to Director, Indian
Veterinary Research Institute, Izatnagar for providing
facilities to carry out the research work
References
Fig. 5—SDS-PAGE- Purification of 32 kDa Tams1 protein
(Lanes: M-Protein molecular weight marker; 1-Whole cell lysate;
2-cleared lysate; 3 & 4- Wash 1& 2; 5 & 6- Purified Tams1).
Fig. 6—Western blot analysis of Tams1 (Lanes: M-Protein
molecular weight marker; 1-immunoreactivity of Tams1 with
bovine antiserum).
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