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388
Advances in Environmental Biology, 6(1): 388-396, 2012
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
The Exposure Immunogenic Protein of Viral Nervous Necrotic on Humpback Grouper
That Influences to Proliferation and Expression of Immune Cells (Interferon γ and NFKb Cell)
1
Uun Yanuhar, 2Ery Gusman, 1Diana Arfiati
1
2
Faculty of Fisheries and Marine Science, Brawijaya University, Malang, Indonesia.
Faculty of Fisheries, Borneo University, East Kalimantan, Indonesia.
Uun Yanuhar, Ery Gusman, Diana Arfiati; The Exposure Immunogenic Protein of Viral Nervous
Necrotic on Humpback Grouper That Influences to Proliferation and Expression of Immune Cells
(Interferon γ and NF-Kb Cell)
ABSTRACT
The main topic of this study are to find out of the proliferation and expression of IFN- γ and NF-kB cell to
Humpback grouper that exposured by immunogenic protein of Viral Nervous Necrosis (VNN). The methods
used on this study were exploration by doing isolation to Humpback grouper organs both normal and infected by
immunogenic protein of VNN, coat protein isolation, protein fractionation by SDS-PAGE, separation and
cutting of protein bands from SDS-PAGE result, coat protein measured by Nano drop spectrophotometer,
Hemagglutination (HA) Test of VNN immunogenic protein, and IFN- γ and NF-kB cell proliferation and
expression qualitative detection by Immunohistochemistry (IHC).The results of this research confirm that HA
test was positive. It showed that 45,9 kDa VNN immunogenic protein had been reacted by agglutination
reaction between 45.9 kDa VNN immunogenic protein and erythrocytes of humpback grouper with sensitivity
of 0,015625. Positive concentration result was up to 1/64 or its sensitivity of 0.015625. It means antigen
determinant (epitope) from immunogenic protein have highly sensitivity from range 0.006 to 0.06. IHC test
result show that 45,9 kDa VNN protein immunogenic epitope have ability to induce humpback grouper tissue
receptors by creating immune cell qualitatively, there are IFN γ and NF-kB cell immune to humpback grouper
specific tissue organ, eye, brain and kidney after labeled by secondary antibody anti IFN γ and NF-kB cell anti
mouse . ELISA test using the secondary antibody that same with examination test of IHC were showed that
proliferation and expression of IFN γ and NF-kB cell quantitatively had been confirmed value positive around
0.9 mg/ml concentration. The conclusion of this studies showed that immunogenic protein of VNN with weight
molecule of 45.9 kDa have ability to induce proliferation and expression of immune cells of humback grouper
as like IFN γ and NF-kB cells both qualitative and quantitative on tissue organ especially eye, brain, and
kidney organ.
Key words: Humpback grouper, immunogenic protein, IFNγ, NF-kB, VNN
Introduction
One of the constraints in the cultivation of
Humpback grouper (Cromileptes altivelis) is a
limited supply of seed due to pathogen infection. The
Infectious pathogens in farmed fish can cause the
deaths of more than 80% even up to 100% [4]. Viral
Nervous Necrosis (VNN) is the virus that causes
disease and encephalopathy retinopathy which has a
wide host range.
In Indonesia reported that the VNN has invaded
most of the grouper fish cultivation by 100%
mortality rate. Symptoms are the fish circling or
whirling, there sleeping or dead fish at the bottom
like the dead and the presence of symptoms of fish
behavior that is not fair [16].
A detailed knowledge about the molecules
associated with immune system can also be used for
further and deeper subsequent applications in
aquaculture, among others, 1) for the development of
biomarkers, 2) for the development of vaccines or
adjuvants, and 3) development of transgenic fish.
VNN is an RNA virus which has genetic material in
the form of single-stranded RNA (+). Interferon
(IFN) is protein or glycoprotein having ability to
prevent viral replication. IFN-γ activates the cells to
the immune response including natural killer cells
(NK) and macrophages. Nuclear Factor-kappa lightchain-enhancer of activated B cells (NF-kB) is found
in almost all types of animal cells and is also related
to the cellular response to stimuli such as stress,
cytokines, free radicals, ultraviolet radiation, LDL
Coresponding Author:
Uun Yanuhar, Faculty of Fisheries and Marine Science, Brawijaya University, Malang, Indonesia
E-mail: [email protected],
Mob: 081252495924
389
Adv. Environ. Biol., 6(1): 388-396, 2012
oxidation and bacterial or viral antigens. Tues IFN γ
is expressed on B cells is the result of differentiation
of the B220 isoform population which is a
modulation in the immune response produced by B
cells and T cells.
NF-kB is a protein complex that controls
transcription of the DNA. NF-kB is found in almost
all types of animal cells and is also related to the
cellular response to stimuli such as stress, cytokines,
free radicals, ultraviolet radiation, LDL oxidation
and bacterial or viral antigens. NF-kB plays an
important role in the regulation of immune responses
to infection. Conversely, errors on NF-kB regulation
is closely related to cell proliferation, inflammation
and autoimmune diseases, septic shock, viral
infection, and development of the immune system
that is not appropriate [2].
NF-kappaB is a transcription factor that is
critical for innate and adaptive immunity. Recently, a
noncanonical pathway for NF-kappaB activation has
emerged. NF-KappaB (Nuclear Factor-KappaB) is a
heterodimeric protein composed of different
combinations of members of the Rel family of
transcription factors. The Rel/ NF-KappaB family of
transcription factors are involved mainly in stressinduced, immune, and inflammatory responses. In
addition, these molecules play important roles during
the development of certain hemopoietic cells,
keratinocytes, and lymphoid organ structures. NFKappaB (Nuclear Factor-KappaB) is a heterodimeric
protein composed of different combinations of
members of the Rel family of transcription factors.
The Rel/ NF-KappaB family of transcription factors
are involved mainly in stress-induced, immune, and
inflammatory responses. In addition, these molecules
play important roles during the development of
certain hemopoietic cells, keratinocytes, and
lymphoid organ structures. More recently, NFKappaB family members have been implicated in
neoplastic progression and the formation of neuronal
synapses. NF-KappaB is also an important regulator
in cell fate decisions, such as programmed cell death
and proliferation control, and is critical in
tumorigenesis [3].
NF-KappaB can be activated by exposure of
cells to LPS (Lipopolysaccharides) or inflammatory
cytokines such as TNF (Tumour Necrosis Factor) or
IL-1 (Interleukin-1), growth factors, lymphokines,
oxidant-free radicals, inhaled particles, viral infection
or expression of certain viral or bacterial gene
products, UV irradiation, B or T-Cell activation, and
by other physiological and non physiological stimuli.
The most potent NF-KappaB activators are the
proinflammatory cytokines IL-1 and TNF, which
cause rapid phosphorylation of KappaBs at two sites
within their N-terminal regulatory domain. TNF,
which is the best-studied activator, binds to its
receptor and recruits a protein called TRADD (TNFAssociated Receptor Death Domain). TRADD binds
to the TRAF2 (TNF Receptor-Associated Factor-2)
that recruits NIK (NF-KappaB-Inducible Kinase).
Both IKK1 and IKK2 have canonical sequences that
can be phosphorylated by the MAP (Mitogen
Activated Protein) kinase NIK/MEKK1 and both
kinases can independently phosphorylate I-KappaBalpha or I-KappaB-beta.
The recognition of bacterial and viral products
by Toll-like receptors on cells of the innate immune
system also results in NF-KappaB induction, leading
to the production of proinflammatory cytokines and
the activation of Antigen Presenting Cell for T-Cell
costimulation in the adaptive immune response. Viral
infection leads to the increased expression and
secretion of the cytokine interferon gamma (IFNgamma) from host cells. IFN-gamma activates the
double-stranded RNA (dsRNA)-dependent serinethreonine protein kinase R (PKR). dsRNA produced
during viral replication induces PKR dimerization,
autophosphorylation, and activation of the eIF2alpha kinase activity. When eIF-2alpha is
phosphorylated, cellular and viral protein translation
cannot efficiently occur. Alternatively, bacterial
products or cellular stress can also activate PKR by
an endogenous gene product called PACT. The
binding of PACT to PKR promotes conformational
changes that allow PKR to activate the downstream
signaling pathways leading to the activation of NFKappaB. Several survival and stimulatory factors that
is important in the development and BAFF (B-Cell
activating factor) coordinated response of B and T
lymphocytes also employ NF-KappaB to carry out
their instructive functions. BAFF induces B-Cell
survival and development and requires the specific
BAFF receptor, BAFFR (BR3), as well as NFKappaB2, and the NIK kinase. In both immature and
mature B cells, BAFF induced processing of p100 to
p52, dependent on BAFF-R and NIK, and in addition
this process is independent of the canonical IKK
complex [5].
The interaction of the Antigen Presenting Cell
and T-Cell also causes NF-KappaB activation in both
cell types NF-KappaB activation is triggered in TCells by the engagement of the T cell receptor and
the CD28 receptor with their ligands, MHC class II,
and the costimulatory molecules CD80 and CD86
presented by Antigen Presenting Cells. The T-Cell
receptor and CD28 synergize in induction of the NFKappaB-dependent genes required for T-Cell
activation and proliferation, such as IL-2, IL-2
receptor (IL-2R), and IFN (Interferons). Activated TCells, in turn, elicit NF-KappaB activation in
Antigen Presenting Cells.
The activation of NF-KappaB has been linked to
inflammatory events associated with autoimmune
arthritis, asthma, septic shock, lung fibrosis,
glomerulonephritis, atherosclerosis, and AIDS. In
contrast, complete and persistent inhibition of NFKappaB has been linked directly to apoptosis,
inappropriate immune cell development, and delayed
cell growth.
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Adv. Environ. Biol., 6(1): 388-396, 2012
This study aims to determine the molecular
expression of IFN-γ cells and NF-kB Humpback
grouper (Cromileptes altivelis) as a responsif and
activation of process immunity in the cell on tissue
organ that appears causes inflammatory and
stimulatory reaction in the cell system. The reaction
immunity of the cell on tissue organ of Humback
grouper were including proliferation and expression
of IFN-y and NF-kB after fish induced by VNN
immunogenic proteins of 45,9 kDa qualitatifely.
Material and Methods
Location and Time of Study:
The research was conducted at the Central
Laboratory of Biological Sciences, Central
Laboratory of Biomedical Faculty of Medicine,
Laboratory of Aquatic Sciences and Biotechnology
Faculty of Fisheries and Marine Sciences Brawijaya
University, Malang.
Isolation ofa VNN coat protein:
Coat proteins isolated from Humpback grouper
organs (eyes, brain, and kidney) that were positive
infected by VNN. Brain, eyes, and kidney from
normal Humpback grouper coat protein isolation
were also performed for comparison. Coat protein
isolation method is to perform homogenizing organs
by mortar under laminar flow then added with extract
buffer (1:2w/v). The next homogenates were
centrifuged at 50,000 rpm for 1 hour at 40C and
followed with 150,000 rpm for 3 hours at 40C by
using ultra high centrifuge (Optima TMMAX-XP,
Beckman Coulter, USA). Coat protein VNN and
normal fish stored in deep freezer -800C until for
further test.
Separation of coat protein and cutting the protein
bands:
Coat protein VNN separated using SDS-PAGE
(Biorad) according to the method by Widyarti using
minislab 12.5% and 4% with gel tracking Voltage
90V, 400 mA, for 120 minutes using coomassie
brilliant blue color material and Low molecules
standard by PRO-STAINTM range Marker
(Fermentas). Molecular weight protein bands were
calculated using linear equations from a distance
separating gel. Protein bands from VNN selected for
use as a candidate adhesion proteins on the basis of
the major bands that appear and do not join with
other protein bands (non-dimer).The protein bands
cutting using gel cutter. Gel bands of protein that has
been cut are stored in the running buffer solution for
elution performed.
Electro elution and Dialysis:
The VNN candidate adhesion proteins on the
protein bands were separated by using horizontal
electrophoresis electro elution method (Biorad).
Pieces of gel from each candidate included in the
membrane protein adhesion cellophane then
electrophoresed at 120V, 400 mA for 60 minutes.
Next Proteins dialyzed in PBS solution pH 7.4 at 40C
for 48 hours. The liquid in a cellophane bag then
taken and put into micro tube and precipitated by
incubating the solution of acetone (1:1v/v) overnight
at 40C. Acetone mixture of protein sand then
centrifuged at 10,000 rpm for 20 minutes at 40C.
Proteins found in the form of pellets, dried and
dissolved in 100 mLof 0.5 M tris HCl, pH8.6.
Furthermore, the protein concentration was measured
using a Nanodrop ND1000 spectrophotometry with
an absorbance at 280 nm.
Hemagglutination (HA) Test of VNN Protein
Adhesion:
VNN candidate adhesion proteins performed
hem agglutination (HA) test as directed by Hanne
and Finkelstein. Blood isolated from normal grouper
using 1 ml syringes GX26½" (Therumo) previously
wetted with 10% EDTA solution. Fish blood then
washed twice using PBS, homogenized and then
centrifuged at 3500 rpm for 10 minutes. Erythrocytes
obtained from subsequent centrifugation sediment
was diluted with PBS (1:200) for use in the HA test.
HA test is done by using micro plate V-bottom 96
well. A total of 50m Leach candidate protein VNN
sinks and inserted in a dilution series was made using
PBS solution at a dilution of up to2-10(1 / 1024). For
comparison is used as a negative control wells
containing only PBS. Furthermore, all added
erythrocytes has been diluted with PBS to all wells,
shake it and micro plate hem agglutination reaction
was observed at least after the 20 minute. Positive
reaction was characterized by the absence of the
erythrocyte sedimentation (in the form of dot) in the
bottom of wells.
Clinical test and the isolation of adhesion antiprotein:
The protein shows the highest value of VNN
hemagglutination then performed clinical tests on
grouper for anti-protein antibodies VNN adhesion.
Clinical test conducted in the Central Aquaculture of
Situbondo by using the4 Humpback groupers (size
150-200g) for each VNN protein adhesion and 4
fishes are used for the control (normal fish). Before
the injection treatment, fish acclimatized for 1 week
in advance. Fish maintained in concrete tanks (2x 5x
1.5m3) and each treatment separated by cage net (2x
1x 1.5m3).
Adhesion protein from each sample were
injected intra-peritoneal (ip) with a concentration
of33g/150g of fish with a volume of 0.1 ml/fish.
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Adv. Environ. Biol., 6(1): 388-396, 2012
Inoculation is done by mixing the protein adhesion
with Complete Freund's adjuvant (CFA) (1:1 v/v).
Seven days after first injection carried out the second
injection (booster) with the volume and
concentration of the same protein with the addition
of Incomplete Freund's adjuvant (IFA). Fish
maintained for 14 days with feeding using trash fish
until full (adlibitum) with aeration and water
turnover of about 200% /day. Serum from each
treatment fishes and the control, were obtained by
taking blood at day 14th after the first injection.
Blood were centrifuged at 10,000 rpm for 10 minutes
at 40C to obtain serum. Serum is stored in a-200C to
the next test.
Expression Detection of IFN- and NF-kB cells by
immunohistochemistry technique:
Detection methods of IFN- and NF-kB cell by
immunohistochemistry based on Nanda et al. (2008)
they are brain, eye, and kidney tissue’s, frozen and
placed on a chuck of a previously cooled (-200C) in
a CM3050 cryostat (Leica Corp., Deerfield, IL).
Tissue sections (6 μm) in thaw-mounted on Super
frost Plus glass slides (Fisher Scientific, Itasca, IL).
Preparations eye tissue, brain, and kidneys of
infected humpback grouper by VNN and normal
were fixed in 2% paraformaldehyde (pH 7.3) for 10
minutes, washed, incubated with 2% blocking serum
(serum from the horse or goat), and incubated
overnight at 40C with primary antibody of IFN- and
Marker
NF-kB antibody. Next, countered with secondary
antibodies conjugated with biotin for 30 minutes at
room temperature. Biotin was detected with avidinbiotin
peroxidase
kit
(ABC-Elite,
Vector
Laboratories) using diaminobenzidine tetrachloride
as chromogene. The last stage is a piece of tissue
dehydrated gradually with alcohol, cleaned with
xylene, and coverslip with Permount (Fisher
Scientific). Tissue sections counter stained with
hematoxylin (1:4) as blue counterstain nuclei.
Data Analysis:
Analysis of expression of IFN- and NF-kB
were conducted by using the program of CorelPaint
11 or Corel Draw Graphics Suite X4 to know the size
of the specificity of colored tissue.
Results:
Profile of VNN Immunogenic Protein Bands:
Protein immunogenic derived from infected
Humpback grouper by VNN. Results from VNN
immunogenic protein electrophoresis using Sodium
Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
(SDS-PAGE) at a concentration of 12.5% in profiles
get 15 bands of protein. Results Protein
electrophoresis with SDS-PAGE for Marker and
immunogenic proteins of VNN can be seen in Figure
1.
VNN Immunogenic Protein Gel
Fig. 1: Electrophoresis result of VNN protein bands by SDS-PAGE.
Table 1: Hemagglutination Test Result of VNN Coat Protein.
Sampel
Titer pengenceran
1/2
1/4
1/8
1/16
1/32
1/64
1/128
VNN
+
+
+
+
+
+
_
45.9 kDa
1/256
_
1/512
_
1/1024
_
Kontrol
-
3992
Adv. Enviroon. Biol., 6(1): 3888-396, 2012
The resultts of the calcuulation on thee profile view
w
bands of protein
p
molecu
ular weight marker
m
proteinss
are exposeed to 9 bands with a mollecular weightt
starting froom 9 kDa, 18
8.5 kDa, 26 kD
Da, 37.8 kDa,,
46.2 kDa, 61.5 kDa, 90..5 kDa, 115 kDa, up to 1988
kDa. Proteeins that play a role in virus attachment too
host recepttors is the coaat protein. VNN
N coat proteinn
molecular weight ranges from 37.18 kD
Da and 42 kDaa
[12].
Basedd on previous research that the bands off
proteins that serve as the canndidate VNN
N
i a protein band with a
immunogeenic protein is
molecular weight of 45.99 kDa [15].
Protein dialysis
d
and Electro
E
elutionn SDS-PAGE
E
results
and
Coat
Protein
Concentrationn
Measurement with a Nan
nodrop spectroffotometer:
e
Proteinn
Basedd on the dialysis and Electro elution
SDS-PAGE
E results, it can
c further bee Coat Proteinn
Concentrattion Measurem
ment VNN with
w
Nanodropp
spectrophhotometer. S
Spectrophotom
meter Nanodroop
readings to the concenntrations of sp
pecific adhesioon
45.9 kDa) withh absorbance of
o 0.51 mg/ml.
protein (4
Results of
o Hemagglutinnation (HA) Teest of VNN Cooat
Protein:
Baseed on HA testt results of VN
NN Coat Proteein
to Hum
mpback grouuper (Cromileeptes altiveliis)
erythrocy
ytes obtained thhe highest dilu
ution titer at 1/664
(Table 1), which meanns that the coaat protein is abble
to aggluttinating VNN red blood cellls (erythrocytees)
normal Humpback
H
groouper up to 64
4 times dilutioon.
This is clearly seen in tthe absence off red color on thhe
s
6. Positivve reactions alsso occurred daata
bottom sinks
dilution 1/2, 1/4, 1/8, 11/16 and 1/32, because of Cooat
Protein VNN
V
can aggluutinating erythhrocytes does nnot
occur as indicated by the
t dot at the bottom
b
of wellls.
NN immunogeenic protein 455.9
HA test results that VN
kDa aggainst Humpbback grouperr (Cromilepttes
altivelis)) erythrocytes can
c be seen in Table
T
1.
n Test Result of
o VNN Coat P
Protein against Humpback groouper erythrocy
ytes.
Fig. 2: Hemagglutination
The concentration
c
o HA with a dilution 1/644
of
showed thhat the agglutin
nation reactionn between thee
protein imm
munogenic VN
NN 45 kDa an
nd erythrocytess
of Humpbaack grouper sh
howed levels of sensitivity too
concentratiions are 0,0155625. Goldsby
y [6] said thatt
the agglutiination reactioon which show
wed a positivee
hemagglutinin directly give the value
v
of thee
sensitivity of 0.3ug/m
ml, results inn a positivee
hemagglutination admin
nisteredat a concentration off
0.006 to 0.06. In this researcch producedd
ve(+)on the haemagglutinin
h
n
concentratiions of positiv
test up to
o a concentraation1 / 64or the level off
sensitivity of 0.015625
5 which meeans that thee
determinattion of anntigen (epitoope) of ann
immunogeenic protein haas a high sen
nsitivity in thee
range of 0..006 to 0.06.
Accorrding to figu
ures of haem
maglutinin testt
results shoowed that the concentration of dilution upp
to 1/128 iss formed whichh point indicattes no bindingg
between the immunoggenic proteins VNN andd
erythrocytees
(negative))
grouper
to
thee
dilution1/11024, while in much smaller concentrationss
are not forrmed point, thiss means that thhere is bindingg
between thhe immunogen
nic proteins wiith erythrocytee
cell groupper, as said byy previous ressearchers, thiss
suggests that the protein
p
is haemaglutinin.
h
.
Haemagluttinin protein found on VNN viruss
glycoproteein is a residue that is harmfull to fish [15].
The resuults of immunohistochemistryy test on speciffic
tissue off Humpback grouper
g
on th
he expression of
IFN-:
results
of
immunnohistochemisttry
The
examinattion performed on grouperr that has beeen
clinically
y tested with a VNN immuunogenic proteein
45.9 kDaa and has beenn tested has an
nti-grouper IgM
M
antibody
y response. Im
mmunohistochhemistry test is
performeed on humpbacck grouper tisssue covering thhe
eye, braiin and kidneyss. Results of teesting performeed
to detectt expression off IFN- which is an immunne
cell that is formed as a response to the presence of
feration detectioon
antibodiees anti IFN- . IFN - prolife
can be done
d
by lookiing at the bond between thhe
antigen (45.9 kDa off VNN immunnogenic proteiin)
and IFN
N -
monooclonal antiboody anti-mouuse
secondarry antibody biootin conjugate.
The results indicaate that expresssion of antigeen
NN
determinnants (epitopes) of 45.99 kDa VN
immunog
genic proteinss specificablee to induce an
a
immune cell receptorrs through thhe formation of
FN - cells of
proliferattion and exprression on IF
Humpback grouper. IF
FN - antiboddy used showeed
similaritiies to regionss that are reccognized by thhe
mouse, so
s to see the reaction of antiggen and antiboddy
in induccing an immunne response cellular
c
can be
b
done.
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Adv. Environ. Biol., C(): CC-CC, 2012
Some results of the expression of IFN - cell
positively confirmed by reaction of gold brown
colour on tissue organ of grouper . That was used to
A
seeing the expression of immune cells in tissues that
have been tested grouper with VNN immunogenic
proteins 45.9 kDa, as shown in Figure 3.
B C
D E
F Fig. 3: The results of the molecular expression of IFN - cells in tissues grouper with immunohistochemistry
examination of the organs of the eye, M100x (A), M1000x (B); brain, M100x (C), M1000x (D), eye,
M100x (E), M1000x (F), indicated by brown color.
The results demonstrated the expression of IFN  cells by the presence of browncolor (indicated by
arrows), brownish color because of the peroxidese
enzyme
reaction
with
the
DAB
and
Hematoxilenkromogen eosin. The reaction that forms
a golden brown color, indicating a strong bond
between the epitopes of the VNN immunogenic
protein of 45.9 kDa with anti- IFN - antibody
identify specific parts of the protein immunogenic, if
at grouper tissue did not test the cells expressing the
IFN -
golden brown color as the antigen and
antibody IFN - cells will not beformed.
The results of immunohistochemistry test on specific
tissue of Humpback grouper on the expression of
NFkB molecule:
The
results
of
immunohistochemistry
examination in humpback Grouper that have been
tested clinically with VNN immunogenic proteins
45.9 kDa to see the tissue-specific immune responses
394
Adv. Environ. Biol., C(): CC-CC, 2012
through NF-kB cell molecules. Cell immune
responses through the expression of NF-kBis showed
by the presence of anti-rabbit secondary antibody
binding of NF-kB biotin conjugates with antigen
(VNN immunogenic protein of 45.9 kDa).
The results of this expression shows that the
VNN immunogenic protein epitopes of 45.9 kDa
able to induce cell molecule NF-kB in humpback
grouper, where the antibody NF-kB is also in
common with the area which is also recognized by
anti-grouper antibodies (IgM), this by cross-reaction
based on the specific antibody with antigenic
determinants (epitopes) of specific VNN viral.
The results of the expression of immune cells is
NF-kB cell molecule on the tissue expression of
grouper have been tested with a VNN immunogenic
protein of 45.9 kDa as shown in Figure 4.
Fig. 4: Expression of NF-kB cell by immunohistochemistry with anti-rabbit secondary antibody biotin
conjugate in normal organ (kidney (A)); treatment on the organs of the eye (B), brain (C), and kidney
(D). The results indicated a positive expression by the presence of brown color (arrow), brown color
indicates presence of antigen by antibody reaction with peroxidase enzyme and substrate chromogenic
labeling.
Discussion:
VNN infected in fish with the expression of
resistance is very weak immune cells, because
immune cells are used for system protection against
viruses, as well as the fish are not infected by the
virus gained IFN - cell molecule expression as IFNγ and NF-kB cell molecules. The number of immune
cells induced expression differs depending on the
target cells of each organ. To see the expression of
immune cells that is IFN-γ molecules and NF-kB in
the test grouper fish in this study used a VNN
immunogenic protein coating of 45.9 kDa.
The results of the expression of IFN - cells and
NF-kB study are shown in the highest organ of the
brain to NF-kB and the eye for IFN - cells. In a
previous study [15] has conducted a test crossreaction of anti-adhesin algino, suggesting that CD4
and CD8 can be recognized by both the brain organ
Humpback grouper (Cromileptes altivelis). CD4 cells
are formed by the immune response to VNN viral
proteins after proliferation on Th1 cells capable to
stimulate formation of further proliferation of
immune cells ie cells IL4 and IL5. In macrophage
cells were differentiated cell activity to induce IFNgamma plays an important role in chronic
inflammatory. This is reinforced by Goldsby et al [6]
writes that during chronic inflammationin the host
cell is exposed to the antigen material will produce
cytokines, which activates the cytokine IFN-gamma
formed secreted by Th1 cells, NK cells and Tc cells
that play a role the action of the immune system
defenses.
Furthermore, it is said that Crude Protein
adhesion of VNN 45.9 kDa in cells expressing
grouper proved CD4 cells and CD8+ cells and also
IFN- and NF-kB. IFN - and NF-kB has a higher
expression in some organs of the grouper on the
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Adv. Environ. Biol., C(): CC-CC, 2012
brain and eyes, this is due to the proliferation and
expression of immune cells on humback grouper
against the VNN immunogenic protein virus
response is regulated by nerve cells in the center.
This suggests that the organ systems are more likely
to have antibody mediated immunity (AMI) that play
a role in controlling VNN infection compared to cell
mediated immunity (CMI). The cells in the brain
organ responsible for the immune system, among
others, microglia cells that have a variety of receptors
in the immune system such as toll-like receptor
(TLR), complement receptors, immunoglobulin and
lymphokine receptors.
Immune response in Humpback grouper cell
adhesion was triggered by VNN protein of 45.9 kDa
as a protein that is immunogenic and haemaglutinin.
This is indicated by a high value (up to 1/64 dilution)
on the hemagglutination (HA) test of normal
erythrocytes grouper. The ability of VNN
immunogenic proteins of 45.9 kDa was demonstrated
by the ability to agglutination red blood cells
(erythrocytes) normal humpback grouperat up to 64
times dilutions. Viral infection process always begins
with the attachment between the protein adhesion
(ligand) on pathogens with receptors on host [15].
Therefore, not all organs or tissues in the host is a
good place for a particular pathogen. In this case
known as the portal of entry of the host cell or tissue
which is a special target of the entry of pathogens
into the host's body [10].
Two kinds of immune response that developed
host defenses against viral infection is the body that
mediated antibody (antibody mediated immunity,
AMI) and cell-mediated immune (cellular mediated
immunity, CMI). AMI system involves antibodies
that are synthesized by plasma cells that are derived
from B cells To activate B cells in synthesizing
antibodies, antigens enter the body presented by APC
(antigen presenting cells) via MHC II molecules to
CD4+ T cells (T helper cells). Th cells would then
secrete cytokines that provide signals to B cells to
conduct proliferation and differentiation into plasma
cells and memory B cells while the CMI is intended
to antigens contained in the cell.
Antigens will be broken down by proteases into
molecules smaller then bound by MHC I molecules
for presentation to the cell surface to CD8+ T cells
(cytotoxic T cells). Tc cell that activated will seek a
target cell characterized by the expression of cell
surface MHC I in the present foreign antigens. Tc
cell would bind to target cells through Fas and Fas
ligand and secrete perforin and granzyme that
induces apoptosis in target cells.
Humpback Grouper test with a size of about 150
g in a clinical trial is expected that the fish at that
size has a perfect immune system so that the
production of cells and molecules that play a role in
the immune system of grouper fish is done optimally.
In addition, the use of fish with a large size will
facilitate the retrieval of blood and organ tissues.
IFN - and NF-kB cell molecule expression in
grouper that have been tested clinically with
immunogenic proteins showed that the 45.9 kDa
molecular VNN IFN - cells and NF-kB expressed
in organs of eyes, brain, and kidneys. It is shown
from the results of immunohistochemistry test. IFN  cell molecule expression and NF-kB in the kidney
and the brain is also supported from the results of
molecular photoelectrons which showed IFN -
cells and NF-kB on the surface of the kidney and
brain cells. In the study by Graham and Secombes
(1988, 1990), suggesting that leukocytes from
rainbow trout kidneys secrete IFN-γ molecule in the
presence of anti viral and MAF activity when
leukocytes are stimulated with mitogen. Also
described by Lu et al that the ability of molecules of
IFN - cells to induce T cell differentiation (in
particular on T cells with low proliferative capacity,
and produce IFN-γ, IL-10 and TGF-β) can increase
expression of IFN - cells which play a role in the
maintenance tolerance or equipment also imposes
limits on the reactivity of the immune system
cellular.
These results confirm a research that
immunogenic proteins 45.9 kDa an VNN
immunogenic protein capable of triggering an
immune response of humpback grouper fish and have
demonstrated the molecular expression of IFN γ and
NF-kB cell molecules expressed in the organs of the
brain, eyes, kidneys, with expression levels that vary
depending on the cell target organ of grouper.
The existence of a virus infection or antigen
(immunogenic proteins) of several viruses in host
cells that are nature protection inhibits the function
of immune cells. VNN infection by the virus has
proved capable of inhibiting immune cell expression
of MHC class I [15]. In this study has generated
MHC class I that has been measured can be triggered
by exposure to VNN immunogenic protein with 45.9
kDa.
MHC class I expression in this study turned out
after testing cellular expression of immune cell IFN-γ
and NF-kB showed measurable positive qualities.
Positive reaction indicated by the reaction of antigen
with antibody, either by NF-kB and IFN-γ antibodies
labeled by biotin labeling.
Immune reaction that formed through formation
of MHC which further differentiate and proliferate
into CD4+ cells due to exposure to an VNN
immunogenic protein 45.9 kDa occurs rapidly in
cells with the expression levels very quickly after
exposure to immunogenic proteins. Similarly, when
exposed to the VNN virus, the formation of MHC
class I cells and immune cells of IFN-γ and NF-kB
cell also occurs very rapidly, but the amount
produced is very low, because the immune cells are
used to maintain the life of the cell or cell survival
system [1]. At the molecular mechanisms that occur
indicated further that the virus is able to suppressor
reduce the expression of immune cell MHC class I
396
Adv. Environ. Biol., C(): CC-CC, 2012
on the cell surface, it is intended to counter act the
inhibition of antigen presented by CD4 + or CD8 +
depending on the level of virus infection [6]. The
results using VNN immunogenic protein 45.9 kDa
has been known to express CD4 + and CD8+ cells.
Base on molecular technics has been
demonstrated that expression of CD4+ cells have
been formed because differentiation and proliferation
of MHC class I. Thus in this study has been produced
that the expression of IFN-gamma cells and B220
cells can be triggered by treatment with exposure to a
VNN immunogenic protein 45.9 kDa. Several
viruses have been reported also in addition can
reduce the function of MHC class I cells are also able
to reduce MHC class II cell function at the cell
surface, where the function includes a block function
to antigen-specific T helper cells function as an
antiviral, aproper T helper cells capable of
proliferation induce a formation of MHC class II
because of a virus infection, the function to be
reduced and even disappear, causing fish to die [6].
7.
8.
9.
10.
11.
Conclusions:
From the results of this research can take the
conclusion that: VNN immunogenic proteins with
45.9 kDa molecule weight, capable to induce
proliferation and expressi0n of IFN-γ cells and NFkBcell molecules on humback grouper as like
immune system cellular that confirmed qualitatively
uses
immunohistochemistry
staining.
The
recommended for further research needs to be done
for the measurement of expression and proliferation
of immune cell as IFN-y and Nf-kB with
flowcytometry.
12.
13.
14.
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