chapter 3 results 3.1 validation of the cdna...
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CHAPTER 3
RESULTS
3.1 VALIDATION OF THE cDNA SYNTHESIZED FROM
F.INDICUS AND P.MONODON.
The total RNA extracted from both Fenneropenaeus indicus and
Penaeus monodon shrimps were converted to cDNA. The quality of the
cDNA was checked and used for amplification of the house keeping gene -
actin. The -actin amplification from Fenneropenaeus indicus produced
686bp amplicon that is representative of the -actin (Figure.3.1A). In parallel,
the amplification of -actin from P.monodon generated 500bp amplicons and
confirms the presence of cDNA (Figure.3.1B).
A B
Figure 3.1 A. Amplification of -actin from cDNA of Fenneropenaeus
indicus
PCR products resolved on 1% Agarose gel showing the amplification
of 686bp amplicon of -actin from cDNA of Fenneropenaeus indicus
Lane 1 – 100bp DNA ladder; lane 2 – Negative control; lane 3- 6 - 686bp -
actin product
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Figure 3.1 B. Amplification of -actin from cDNA of Penaeus monodon
PCR products resolved on 1% Agarose gel showing the amplification of
500bp amplicon of -actin from cDNA of Penaeus monodon
Lane 1 – 100bp DNA ladder; lane 2 – Negative control; lane 3- 5- 500bp
TCTP product.
3.2 CLONING AND CONFIRMATION OF TRANSLATIONALLY
CONTROLLED TUMOR PROTEIN (TCTP)
3.2.1 Amplification of Translationally controlled tumor protein (TCTP)
from the cDNA of Fenneropenaeus indicus and Penaeus monodon.
An amplification corresponding to 507 bp TCTP was observed from
the hemocytes of Penaeus indicus. (Figure.3.2 A). Similarly, the cDNA from
Penaeus monodon exhibited an amplification of 507bp product corresponding
to TCTP. (Figure 3.2 B).
A B
Figure 3.2 A. Amplification of TCTP from cDNA of Penaeus indicus.
PCR products resolved on 1% Agarose gel showing the amplification
of 507bp amplicon of TCTP from cDNA of Penaeus indicus. Lane 1 – 100bp
DNA ladder; lane 2 – Negative control ; lane 3- 7 - 507bp TCTP product
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Figure 3.2 B. Amplification of TCTP from cDNA of Penaeus monodon
PCR products resolved on 1% Agarose gel showing the amplification
of 507bp amplicon of TCTP from cDNA of Penaeus monodon. Lane 1 –
100bp DNA ladder; lane 2, 3 - 507bp TCTP product; lane 4 - Negative
control
3.2.2 Cloning and confirmation of recombinant constructs by PCR
The Translationally controlled tumor protein (TCTP) amplicons were
purified and ligated into the TOPO-TA vector and transformed in to the
maintenance host DH5 . The transformants were screened for the presence of
the TCTP insert. The positive transformants were selected and confirmed for
the presence of insert using gene specific and vector specific primers. The
lysate PCR of the selected transformants showed the amplification of 507bp
TCTP product with the gene specific primers and 707bp product with the
vector specific primers respectively (Figure 3.3).
Figure 3.3 Confirmation of the rTranslationally controlled tumor
protein (rTCTP) clone with gene specific and vector specific primers.
PCR products resolved on 1% Agarose gel showing the amplification
of 507bp amplicon with gene specific primers and 707bp amplicons with
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vector specific primers. Lane 1 - 100bp DNA ladder; lane 2 - Negative
control; lane 3 - 507bp amplicons obtained with gene specific primers; lane 4
- 707bp amplicon obtained with vector specific primers.
3.2.3 Confirmation by Restriction digestion
The recombinant plasmid was further confirmed by restriction
digestion with the restriction enzymes BamHI and EcoRI that results in the
release of 507bp TCTP insert (Figure 3.4). The digestion of rTCTP plasmid
with both the restriction enzymes separately, generated a single linear
product.
Figure 3.4. Confirmation of rTranslationally controlled tumor protein
(rTCTP) clone by Restriction digestion
PCR products resolved on 1% Agarose gel showing the presence of
insert that was released on restriction with the restriction enzymes BamHI and
EcoRI. Lane 1 - 100bp DNA ladder; lane 2 - Uncut rTCTP plasmid; lane 3-
rTCTP plasmid restricted with BamHI only ; lane 4 - rTCTP plasmid
restricted with EcoRI only; lane 5- TCTP plasmid digested with BamHI and
EcoRI showing release of 507bp insert.
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3.2.4 Confirmation of clonesby sequencing.
The rTCTP clones were sequenced (Microsynth, Balgagh, Switzerland)
and the sequences were analyzed.
3.3 SEQUENCE ANALYSIS OF TRANSLATIONALLY
CONTROLLED TUMOR PROTEIN
3.3.1 Nucleotide and Amino acid sequence analysis of rTCTP from F.
indicus
The Nucleotide sequence of TCTP from Fenneropenaeus indicus
consist of 507bp ORF coding for 168 amino acids that corresponds to a
Molecular weight of 18.4kDa. The nucleotide sequence consist a start codon
ATG and ending with the stop codon TAA. The BLASTN analysis revealed
that the nucleotide sequence of Penaeus indicus TCTP shared more than 95%
homology with the TCTP nucleotide sequences from other geographical
isolates available in the GENBANK. The nucleotide sequence was deposited
in the GENBANK with the Accession number FJ890311 (Figure 3.5). The
TBLASTN analysis of the rTCTP sequence revealed 96% homology at the
amino acid level with the available sequences in the database.
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Figure 3.5 Nucleotide and amino acid sequence of recombinant
Translationally controlled tumor protein from Fenneropenaeus indicus
The deduced nucleotide (507bp) and amino acid sequence (168) of
Translationally controlled tumor protein (TCTP) from the Fenneropenaeus
indicus. The small letters represent the nucleotide sequences starting with
ATG and ending with the stop codon (TAA) denoted by asterisk. Capital
letters given below represent the amino acid sequences of the TCTP gene.
3.3.2 Multiple sequence alignment of the rTranslationally controlled
tumor protein from Fenneropenaeus indicus
The Multiple sequence analysis of Fenneropenaeus indicus rTCTP
with the sequences from the homologs revealed the presence of few
substitutions in the nucleotide and amino acid sequence (Figure 3.6).
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Figure 3.6 Multiple sequence alignment of recombinant Penaeus indicus
Translationally controlled tumor protein sequence with their homologs.
Multiple sequence alignment of Fenneropenaeus indicus
Translationally controlled tumor protein (FJ890311) with Fenneropenaeus
merguensis (AAV84282), Penaeus monodon (AAO61938), Litopenaeus
vannamei (ABY55541), Fenneropenaeus chinensis (ABB05535) and
Marsupenaeus japonicus (ABZ90154).
A total of five substitutions were observed at the nucleotide level
compared with other isolates. The substitutions at the positions 162 and198
were randomly distributed among the various geographical isolates. The
nucleotide substitutions pertaining to positions 117, 404 and 445 were highly
specific to the Indian isolate. Penaeus indicus has a total of five substitutions
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at the nucleotide level, of which two were consensus and three contributed to
a change in amino acid. A valine to isoleucine substitution at position 37,
aspartic acid to valine substitution at position 135 and aspartic acid to
histidine substitution at position 149 were highly significant in F. indicus. The
N-terminal region of the protein across all geographical isolates was
conserved, except in the Indian isolate, where a substitution was seen at
positions 37, 135 and 149 (Table 3.1).
Table 3.1 Nucleotide and amino acid substitutions in rTCTP from
Fenneropenaeus indicus.
Residue Number Consensus Change
Nucleotides
5 2 3
Aminoacids
37
135
149
Valine
Asparticacid
Asparticacid
Isoleucine
Valine
Histidine
3.3.3 Phylogenetic analysis of the rTCTP sequence
The phylogenetic tree constructed based on TCTP sequences
demonstrates the presence of TCTP from F. indicus and F. chinensis on a
similar node unlike the others that were distantly located. This signifies the
evolutionary relatedness of the Fenneropenaeus indicus TCTP with the TCTP
from Fenneropenaeus chinensis (Figure 3.7).
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Figure 3.7 Phylogenetic tree for Fenneropenaeus indicus recombinant
Translationally controlled tumor protein sequences showing evolutionary
relatedness.
Phylogenetic tree shows a closer evolutionary relatedness of
Fenneropenaeus indicus rTCTP with TCTP from Fenneropenaeus chinensis
and a distant origin with TCTP from Marsupenaeus japonicus.
3.4 PROTEIN SEQUENCE AND STRUCTURE ANALYSIS OF
rTCTP
3.4.1 Primary structure analysis:
The protein had a molecular weight of 18.4kDa. The analysis of the
TCTP protein sequence was performed with ExPASy proteomics server. The
primary structure analysis revealed that the protein was cationic, with an
isoelectric point (pI) of 5.23.
The rTCTP nucleotide sequence was translated using translational tool
in ExPASy proteomics server. The protein sequence analyzed for signal
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sequence prediction using SignalP software revealed the absence of signal
peptidase cleavage site signifying the secretary nature of the protein (Figure
3.8). TCTP is synthesized in the hemocytes of shrimps. The analysis of the
sequence revealed an absence of glycosylation sites in the protein.
Figure 3.8 Signal peptidase cleavage site prediction in rTCTP sequence of
Penaeus indicus.
Signal peptidase cleavage site prediction by SignalP prediction tool
from ExPasy proteomics server. The figure signifies an absence of N-
terminal signal sequence in the protein.
3.4.2 Secondary structure prediction of rTCTP
The secondary structure was assessed by scanning the sequence in
Prosite/Pfam for identification of the protein family and class. This revealed
the presence of TCTP signatures that are very specific to this class of proteins
(Figure 3.9 A). The motif search for pattern of motif sequence present in the
TCTP protein signified the presence of TCTP specific motif that is specific to
the TCTP class of molecules.
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TCTP signatures
S1 S2
EGANPSAEEA FKDLQFFTGESMVPDGMVVLMDY
1 168
Signature 1 Signature 2
Figure 3.9 A. Propred/Pfam analysis of Fenneropenaeus indicus rTCTP
for the identification of motif.
The figure shows the presence of two signature sequences in the
protein that signifies the protein belonging to the TCTP class of molecules.
3.4.3 Predicting the Antigenic Nature of rTCTP
The secondary structure was analyzed using DNA STAR software for
determining the nature of the protein. Protein sequence analysis suggests the
hydrophilic nature of TCTP with hydrophobicity limited to two regions, as
assessed by DNASTAR using Kyte and Doolittle algorithm (Figure 3.9 B).
The surface probability and antigenic index suggest, TCTP to be highly
antigenic in nature. The protein is highly antigenic in nature as determined by
the James-Wolf plot and consists of two - helices as determined by Garnier-
Robson and Chou-Fasman algorithm.
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10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Alpha, Regions - Garnier-RobA
Alpha, Regions - Chou-FasmA
Beta, Regions - Garnier-RobB
Beta, Regions - Chou-FasmB
Turn, Regions - Garnier-RobT
Turn, Regions - Chou-FasmT
Coil, Regions - Garnier-RobC
Hydrophilicity Plot - Kyte-Do0
4.5
-4.5
Alpha, Amphipathic Regions - *
Beta, Amphipathic Regions - *
Flexible Regions - Karplus-SF
Antigenic Index - Jameson-W0
1.7
-1.7
Surface Probability Plot - Em1
6
0
Figure 3.9 B Secondary structure analysis of rTCTP sequence using
DNASTAR software.
The secondary structure analysis of rTCTP protein sequence using
DNA STAR software signifying the protein to be -helical, hydrophilic, and
highly antigenic in nature.
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3.4.4 Tertiary structure of rTranslationally controlled tumor protein
The tertiary structure of TCTP from Penaeus indicus was identified by
modeling of the TCTP structure using geno3D software in Expasy proteomics
server. The structure is predicted by homology modelling using the human
TCTP structure (PDB ACC No: 3EBM) as the reference. This had a similar
pattern of sheet-helix-sheet identified in the calcium binding protein (Figure
3.10).
Figure 3.10 Tertiary structure of rTCTP from Fenneropenaeus indicus
predicted using geno3D software in ExPasy proteomics server.
3.5 EXPRESSION OF TRANSLATIONALLY CONTROLLED
TUMOR PROTEIN (TCTP) TRANSCRIPTS IN HEMOCYTES
AND TISSUES
3.5.1 Confirmation of WSSV infection in Fenneropenaeus indicus from
the wild
The shrimps collected from wild that were positive for the PCR
amplification of the 366bp viral envelope gene Vp18 were considered
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infected and the animals that were PCR negative formed the uninfected group
(Figure.3.11). The animals showed reddish coloration of body, but were
active and did not show any mortality.
Figure 3.11 Confirmation of WSSV infection in Fenneropenaeus indicus
One percent agarose gel showing the polymerase chain reaction (PCR)
amplification of 366 bp region coding for the viral envelope protein vp18 in
infected shrimp. Lane 1-100bp ladder; lane 2 - Negative control; lanes 3-6 -
DNA from uninfected shrimps showing the absence of white spot syndrome
virus (WSSV) infection by PCR lanes 8-12- DNA from WSSV-infected
shrimps showing WSSV infection by PCR.
3.5.2 Hemocyte count
The average hemocyte counts in the WSSV-infected F. indicus
shrimps from wild (40 x 106 cells/mL) were higher than uninfected shrimps
(30 x 106 cells/mL) and the viability was >98% in all the preparations.
Similarly, the average hemocyte counts in the normal and WSSV-infected P.
monodon shrimps were 23 x 106
and 35 x 106 respectively with cells >96%
viable.
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3.5.3 Expression of TCTP Transcripts in Hemocytes of F. INDICUS
Translationally controlled tumor protein message levels were found to
be elevated in the hemocytes of WSSV-infected F. indicus and P. monodon
shrimps respectively compared with the uninfected ones (Figure 3.12 A and
3.12 C). The expression of house keeping gene -actin was similar in all the
samples (Figure 3.12 B). The average IDVs of TCTP from infected P. indicus
and P. monodon were higher than the average values in corresponding
uninfected shrimps, and the -actin values were almost the same (Figure 3.12
D).
Figure 3.12 Analysis of TCTP transcripts in hemocytes of uninfected
and WSSV infected Fenneropenaeus indicus and Penaeus monodon
One percent agarose gel showing the polymerase chain reaction
amplification of (A) 507 bp Translationally controlled tumor protein (TCTP)
and (B) 686 bp -actin from haemocyte cDNA of uninfected and WSSV
infected Penaeus indicus. (C) 507 bp TCTP amplified from uninfected and
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infected Penaeus monodon. (D) Table 3.2 showing the average integrated
density values (IDV) of the amplicons from uninfected and infected shrimps.
Lane 1- 100 bp DNA ladder; lane 2- Negative control; lanes 3-7-
TCTP and -actin amplified from cDNA of uninfected shrimps; lanes 8-12-
TCTP and -actin amplified from cDNA of WSSV-infected shrimps. (C)
Lane 1-100bp DNA ladder; lane 2 - negative control; lanes 3 and 4- TCTP
amplified from cDNA of uninfected shrimps; lanes 5-6 TCTP amplified from
cDNA of WSSV-infected shrimps (D) average IDV values showing higher
expression of transcripts in infected shrimps than uninfected ones.
3.5.4 Expression of TCTP transcripts in Tissues of Fenneropenaeus
indicus
In contrast, the TCTP expression in tissues (gills, hepatopancreas,
pleopods and tail) was absent in uninfected and infected shrimps of both the
species (F. indicus and P. monodon) (Figure 3.13).
Figure 3.13 Expression of TCTP transcripts in tissues of WSSV infected
Penaeus indicus and Penaeus monodon
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One percent agarose gel showing the polymerase chain reaction
amplification of 507bp Translationally controlled tumor protein (TCTP) from
haemocyte cDNA and its absence in uninfected and infected tissues of
Penaeus indicus (A) and P.monodon (B). Lane 1 -100bp DNA ladder, lane 2 -
negative control; lane 3 - haemocytes; lanes 4 – gills ; lane 5- hepatopancreas;
lane 6- Pleopods; lane 7- tails from WSSV infected shrimps.
3.5.5 Expression of TCTP transcripts in different life stages
The TCTP transcripts were identified in the post larvae, and hemocytes
of juvenile, subadult and adults of Fenneropenaeus indicus (Figure 3.14)
Figure 3.14 Expression of TCTP transcripts at various stages of the life
cycle of Fenneropenaeus indicus
One percent agarose gel showing the amplification of (A) TCTP and
(B) -actin at various stages of the life cycle. Lane 1- 100bp DNA ladder;
lane 2 - negative control; lane 3 - Post larvae 30; lane 4 - juvenile shrimp;
lane 5- subadult ; lane 6- adult
A
B
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3.6 EXPRESSION OF RECOMBINANT TRANSLATIONALLY
CONTROLLED TUMOR PROTEIN IN PROKARYOTIC
EXPRESSION SYSTEM
3.6.1 Subcloning and expression of Translationally controlled tumor
protein (TCTP)
The ORF of TCTP gene was reamplified and sub cloned in to
pET100a-D-TOPO vector and the recombinant constructs of pET100a-TCTP
were transformed in to the prokaryotic expression host BL21(DE3) for high
level expression. Transformants were randomly selected to screen for the
recombinant protein expression. Conditions were optimized with various time
points and IPTG concentrations. Thus the expression was performed by
inducing the overnight grown cultures (OD600 = 0.6) with 1mM IPTG at 37oC
for 3 hours. The protein profiles of uninduced and induced cultures along with
the host BL21(DE3) and vector control were analyzed on 12% SDS-PAGE
(Figure 3.15).
Figure 3.15. SDS-PAGE profile of recombinant TCTP expression
12% SDS-PAGE stained with Coomassie brilliant blue showing the
expression of the recombinant TCTP protein after induction with IPTG. Lane
1 – Medium MW Protein marker; lane 2 - host induced ; lane 3 - vector
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induced ; lane 4 - TCTP uninduced ; lane 5 and 6 - TCTP clones from
colonies 1 and 2 induced with 1mM IPTG.
An expression corresponding to 29.5kDa was observed in the
uninduced and induced E.coli cultures, with higher expression levels in latter
compared to former. However there was no corresponding protein expressed
in the host and vector controls. All the transformants with the recombinant
plasmid (pET100a-TOPO-TCTP), yielded similar levels of recombinant
protein expression. More than 90% of the transformants that were screened
were positive for the expression of recombinant TCTP protein on induction
with 1mM IPTG (Figure 3.16).
Figure 3.16 SDS-PAGE profile of BL21(DE3) transformants showing
recombinant TCTP protein expression
12% SDS-PAGE stained with Coomassie brilliant blue showing the
expression of recombinant TCTP protein in all the colonies after induction
with 1mM IPTG. Lane 1 - Medium MW protein marker; lane 2 - colony 2
Uninduced ; lane 3 - colony 2 induced with 1mM IPTG; lane 4 - colony 3
induced ; lane 5 - colony 4 induced; lane 6 - colony 5 induced; lane 7 -
colony 6 induced with1mM IPTG for over expression of the recombinant
protein.
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3.6.2 Confirmation of recombinant TCTP by Western blotting:
The recombinant TCTP protein expression was subsequently
confirmed by Western blot analysis with anti histidine monoclonal antibodies.
A 29.5kDa reactivity was observed in the samples from induced and
uninduced cultures, with recombinant plasmid. (Figure 13.17). The 29.5kDa
reactivity was absent in host and the vector control induced with 1mM IPTG.
Figure 3.17 Confirmation of recombinant TCTP expression by Western
blot using Anti histidine antibody.
Western blot analysis of induced recombinant TCTP resolved on 12%
SDS-PAGE, transferred onto nitrocellulose membrane, probed with mouse
anti histidine monoclonal antibody (1:20,000) followed by incubation with
goat anti mouse ALP (1:20,000), developed with BCIP and NBT. Lane 1 -
Protein molecular weight marker; lane 2 – host BL21(DE3) induced with
1mM IPTG; lane 3 - induced vector control : lane 4,5,6,7 - induced culture
of BL21(DE3)-pET100a-TCTP probed with Anti histidine antibody:
3.6.3 Large scale expression and purification of recombinant TCTP by
Immobilized Metal Affinity Chromatography (IMAC)
Over night culture of BL21(DE3) + pET100a-TOPO-TCTP grown in
15 ml LB was used as a pre-inoculum. 2% of this pre-inoculum was
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transferred in 100ml LB and grown till the OD600 reached 0.6. The cultures
were induced with the optimised concentration of 1mM IPTG for 3 hrs.
Expression of the 29.5kDa protein was analyzed on a 12% SDS-PAGE.
Expression of proteins in T7 expression system facilitates an easy one step
purification on Ni2+
immobilized columns. The proteins were expressed in
BL21(DE3) as soluble fractions in the cell lysates. The proteins were purified
on IMAC columns. An imidazole-based method was used to elute the bound
recombinant TCTP protein from the columns. The purification profile of the
histidine tagged recombinant protein showed a single band at 29.5kDa on
SDS-PAGE. The rTCTP protein was eluted effectively at 25mM imidazole
gradient (Figure 3.18) and the purified protein fractions were subsequently
confirmed by Western blot with anti histidine monoclonal antibody.
Figure 3.18 Purification of recombinant TCTP by Immobilized metal
affinity chromatography (IMAC)
12% SDS PAGE profile showing the recombinant TCTP purified by
IMAC; total and purified recombinant proteins were resolved and stained with
Coomassie brilliant blue.
Lane 1- Protein Molecular Weight marker ; lane 2- Flow through;
lane 3- Wash; lane 4 - 25mM 2nd
fraction ; lane 5- 50mM 2nd
fraction ; lane
6- 75mM 2nd
fraction; lane 7- 100mM 2nd
fraction.
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3.6.4 Endotoxin Contamination: LAL Assay
The recombinant proteins being expressed in E.coli are liable to be
contaminated with endotoxin. Hence recombinant TCTP was assessed for the
presence of contaminating endotoxin by Limulus Amoebocyte Lysate (LAL)
Assay. Results of the assay showed the absence of LPS, in recombinant TCTP
preparations.
3.6.5 Production of TCTP polyclonal antiserum in mice
Figure 3.19 Western blot analysis of recombinant TCTP using
polyclonal antisera developed in mice.
Western blot analysis of induced and purified rTCTP resolved on 12%
SDS PAGE, transferred onto nitrocellulose membrane, probed with antimouse
polyclonal antibody (1:5000) followed by incubation with goat anti mouse
ALP (1:20,000), developed with BCIP and NBT. Lane 1 - Protein Molecular
wt marker ; lane 2 – normal mouse sera; lane 3 - BL21(DE3)-pET100a-
TCTP induced with 1mM IPTG; lane 4 - 25mM 4th IMAC fraction.
33 kDa
20 kDa
10 kDa
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True bred BALB/c mice was immunized with purified recombinant
TCTP and antisera was raised for over a period of 8 weeks. The final bleed
was made and serum was separated and used for the identification of
recombinant TCTP. The anti mouse antisera was used as primary followed by
the addition of goat anti mouse antibody as secondary in Western blotting
thus identifying a 29.5kDa protein. Reactivity was noticed upto 1: 2000
dilution. The normal mouse serum used as a control did not exhibit any
reactivity.
3.7 FUNCTIONAL CHARACTERIZATION OF THE
RECOMBINANT TRANSLATIONALLY CONTROLLED
TUMOR PROTEIN.
3.7.1 Anti-Oxidant Activity assay (Hydrogen Peroxide Assay)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
O.D
60
0n
m
Antioxidant Activity
E.coli E.coli E.coli E.coli E.coli E.coli E.coli
+ H2O2 + H2O2 + H2O2 + H2O2 + H2O2 + H2O2
+5µg +10µg +15µg +20µg +20µg Vit C
TCTP TCTP TCTP TCTP
Figure 3.20 Anti oxidant activity of recombinant Translationally
controlled tumor protein
Antioxidant activity of recombinant Translationally controlled tumor
protein (TCTP) in the presence of 1.2mM hydrogen peroxide solution,
exhibiting growth of E.coli cells in a dose dependent manner.
94
The antioxidant activity of Fenneropenaeus indicus TCTP was tested
invitro by observing the growth of E.coli cells in hydrogen peroxide
containing media in the presence of various concentrations of recombinant
TCTP. The presence of hydrogen peroxide in the media induced oxidative
burden causing a decline in the growth of E.coli cells. The addition of
recombinant TCTP enhanced the growth of the E.coli cells even in the
presence of 1.2mM hydrogen peroxide. A progressive increase in the growth
of E.coli was observed in the tubes with increased TCTP concentrations (5µg,
10µg, 15µg, and 20µg). The E.coli also exhibited elevated growth in the
presence of antioxidant protein Vitamin.C. The presence of hydrogen
peroxide causes oxidative burden that is neutralized by the TCTP in a dose
dependent manner (Figure 3.20). The p value <0.05 between comparative
groups are considered significant.
E.coli Vs E.coli + H2O2 P< 0.05
E.coli + H2O2 Vs E.coli+ H2O2 + rTCTP P< 0.05
E.coli +5µg rTCTP Vs E.coli +15µg rTCTP P<0.05
E.coli + H2O2 Vs E.coli + H2O2 + Vit.C P<0.05
3.7.2 Efficiency of recombinant TCTP in protecting the shrimps
Fenneropenaeus indicus from WSSV infection.
The shrimp Penaeus indicus of 10 grams were divided in to 4 groups
of 20 animals each. The effect of oral and intramuscular administration of
recombinant TCTP in protecting the shrimp against WSSV infection was
evaluated. Experiment 1, 2, and 3 represents three independent experiments
performed to assess the protection delivered by recombinant TCTP against
WSSV infection. (Table 3.3 A, B and C).
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Table 3.3 Survival of shrimps treated with recombinant Translationally controlled tumor protein (TCTP).
EXPERIMENT 1 DAYS
GROUPS 0 1 2 3 4 5 6 8 10 12 14 15
1X PBS 20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
20/20
100)
19/20
(95)
19/20
(95)
18/20
(90)
18/20
(90)
18/20
(90)
18/20
(90)
1XPBS+WSSV 20/20
(100)
18/20
(90)
16/20
(80)
10/20
(50)
6/20
(30)
1/20
(5)
0 0 0 0 0 0
TCTP(ORAL)+WSSV 20/20
(100)
20/20
(100)
19/20
(95)
17/20
(85)
16/20
(80)
13/20
(65)
13/20
(68)
11/20
(58)
8/20
(44)
7/20
(39)
3/20
(16)
2/20
(11)
TCTP(IM)+WSSV 20/20
(100)
20/20
(100)
20/20
(100)
19/20
(95)
17/20
(85)
17/20
(85)
16/20
(84)
13/20
(68)
11/20
(61)
10/20
(55)
9/20
(50)
8/20
(44)
EXPERIMENT 2 DAYS
GROUPS 0 1 2 3 4 5 6 8 10 12 14 15
1X PBS 20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
19/20
(95)
19/20
(95)
19/20
(95)
19/20
(95)
19/20
(95)
19/20
(95)
19/20
(95)
1XPBS+WSSV 20/20
(100)
19/20
(95)
13/20
(65)
8/20
(40)
3/20
(15)
0 0 0 0 0 0 0
TCTP(ORAL)+WSSV 20/20
(100)
20/20
(100)
19/20
(95)
19/20
(95)
18/20
(90)
12/20
(63)
7/20
(36)
6/20
(31)
3/20
(15)
1/20
(5)
0 0
TCTP(IM)+WSSV 20/20
(100)
20/20
(100)
20/20
100)
18/20
(90)
18/20
(90)
18/20
(94)
17/20
(89)
15/20
(78)
15/20
(78)
12/20
(63)
10/20
(52)
10/20
(52)
95
95
EXPERIMENT 3
GROUPS 0 1 2 3 4 5 6 8 10 12 14 15
1X PBS 20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
20/20
(100)
19/20
(95
19/20
(95
1XPBS+WSSV 20/20
(100
18/20
(90)
16/20
(80)
14/20
(70)
10/20
(50)
7/20
(35)
4/20
(20)
0 0 0 0 0
TCTP(ORAL)+WSSV 20/20
(100)
20/20
(100)
18/20
(90)
18/20
(90)
15/20
(75)
11/20
(55)
9/20
(45)
8/20
(40)
5/20
(25)
5/20
(25)
4/20
(21)
4/20
(21)
TCTP(IM)+WSSV 20/20
(100
20/20
(100
19/20
(95)
18/20
(90)
18/20
(90)
18/20
(90)
18/20
(90)
13/20
(65)
13/20
(65)
10/20
(50)
6/20
(31)
6/20
(31)
The table represents number of shrimp survived WSSV infection on treatment with recombinant TCTP. The figure in
each row represents total number of shrimps survived on the day of experiment and figures given below in brackets are the
respective percentage survival during each day till the completion of the experiment at day 15.
96
97
Figure 3.21 Analysis of WSSV infection in surviving shrimps using Vp18
primers.
One percent agarose gel showing amplification of 366bp vp18 gene in
different group of animals from the protection study. Lane 1- 100bp DNA
ladder; lane 2- Negative control; lane 3,4- shrimps injected with 1XPBS;
Lane 5,6 - shrimps infected with WSSV; lane 7,8,9 - survivors of oral TCTP
group challenged with WSSV; Lane 10,11,12 - survivors of intramuscular
injection of TCTP challenged with WSSV.
The experiment was conducted over a period of 15 days. The control
shrimps injected with 1X PBS appeared normal and healthy. They exhibited
more than 80% survival till day 15. The shrimps injected with WSSV showed
100% mortality on day 9 in all the three experiments. All the shrimps in this
group displayed signs of lethargy, reddishness of the body, and reduced food
intake. More than 80% of shrimps that were injected with recombinant TCTP
only, were surviving and did not show adverse symptoms.
Shrimps given oral supplementation of recombinant TCTP exhibited
15% survival till day 15 while shrimps provided with intramuscular injection
of recombinant TCTP exhibited 42% survival till the completion of
experiment. The shrimps injected with WSSV alone displayed symptoms of
white spot syndrome after 5 days and was positive for WSSV by one step
PCR. The shrimps fed with oral TCTP exhibited symptoms of WSSV
infection after 10 days. The shrimps given intramuscular injection of
recombinant TCTP exhibited lethargy after 12 days post infection. Two
98
shrimps from each group and the shrimps that were surviving were tested for
the presence of WSSV. The surviving shrimps did not exhibit PCR
amplification for viral envelope gene Vp18. The oral recombinant TCTP fed
group displayed weak amplification of Vp18 gene and were positive for the
presence of viral infection. The shrimps that were WSSV infected exhibited a
positive amplification for the viral gene (Figure 3.21).
3.8 RECOMBINANT TRANSLATIONALLY CONTROLLED
TUMOR PROTEIN MEDIATED IMMUNE MODULATION IN
SHRIMP PENAEUS INDICUS DURING WSSV INFECTION
Different alphabets between groups (bars) at a time point indicates the
p values between the groups significant (p<0.05). Similar alphabets between
groups (bars) at a time point indicates the p values between the groups not
significant (p>0.05).
3.8.1 The Total hemocyte count
0
10
20
30
40
50
60
70
0hr 12hr 24hr 48hr 60hr 100hr
TH
C (
X 1
06/m
l )
Time
Total Hemocyte Count
1XPBS
WSSV
10ug TCTP+WSSV
TCTP
a a a aa
aac
aba
bc c
a
abbcc
a
b b
c
a
b b
a
Figure 3.22 Effect of recombinant TCTP on the total hemocyte count of
shrimps at various time intervals post challenge.
The total hemocyte count (THC) were similar in the PBS and TCTP
treated group. Upon infection with WSSV the THC decreases significantly
99
compared to the PBS control. On contrary, shrimps receiving TCTP followed
by WSSV infection exhibited higher levels of total hemocyte count similar to
the TCTP treated group. The infected shrimps treated with TCTP exhibited
similar levels of total hemocyte count to the shrimps injected with TCTP after
60hrs (Figure 3.22).
3.8.2 Total protein concentration
0
20
40
60
80
100
120
140
160
180
0hr 12hr 24hr 48hr 60hr 100hr
con
c.o
f pro
tein
(m
g/m
l)
Time
Total Protein
1XPBS
WSSV
10µg TCTP+WSSV
TCTP
abc c
ab a b
c
aba
b
c
ab ab
c
a a
bc
a a
bc
a
Figure 3.23 Effect of recombinant TCTP on the total protein
concentration of shrimps at different time points post challenge.
The total protein concentration were similar in the PBS and TCTP
treated group. Upon infection with WSSV the total protein increases
significantly compared to the PBS control. Shrimps receiving TCTP followed
by WSSV infection exhibited higher levels of total protein than all other
groups (Figure 3.23).
100
3.9.3 Phenol oxidase activity
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0hr 12hr 24hr 48hr 60hr 100hr
O.D
at
49
0 n
m
Time
PO
1XPBS
WSSV
10µg TCTP+WSSV
TCTP
aa
e
b
a
a
a
a
a
a
c
a
a
c
e
a a
b
c
e
b
c
eb
Figure 3.24 Effect of recombinant TCTP on the total phenol oxidase
activity of shrimps at different time points post challenge.
The total phenol oxidase activity in WSSV infected group were higher
than the PBS injected group. A significant reduction in the phenol oxidase
activity was observed in TCTP treated group compared to the WSSV infected
group after 48hrs post infection was observed in shrimps treated with
recombinant TCTP (Figure 3.24).
101
3.9.4 Respiratory burst
0
0.2
0.4
0.6
0.8
1
1.2
0hr 12hr 24hr 48hr 60hr 100hr
O.D
at
65
5 n
m
Time
Respiratory burst
1XPBS
WSSV
10µg TCTP+WSSV
TCTP
b
ce
aa
d
c b a
cb b
a
db
cd
a
b
a
d
b
d
ac
Figure 3.25 Effect of recombinant TCTP on the respiratory burst of
shrimps at different time points post challenge.
The respiratory burst was higher in shrimps infected with WSSV
compared to the control uninfected group. The injection of recombinant
TCTP followed with WSSV infection displayed a significant decrease in the
respiratory burst and the activity was similar to the levels seen in the control
at 60hrs (Figure 3.25).
102
3.9.5 Clotting time
0
20
40
60
80
100
120
140
160
180
0hr 12hr 24hr 48hr 60hr 100hr
Clot
ting
tim
e (s
ec)
Time
Clotting Time
1XPBS
WSSV
10µg TCTP+WSSV
TCTP
aab b b
a
d
b
a
d
bc
a
d d
b
a
b
c
a a
d
b
a
c
Figure 3.26 Effect of recombinant TCTP on the clotting time of shrimps
hemolymph at different time points post challenge.
The clotting time were similar in the PBS and TCTP treated group at
60hrs. Upon infection with WSSV the clotting time increases significantly
compared to the PBS control. On contrary, shrimps receiving TCTP followed
by WSSV infection exhibited reduced clotting time (Figure 3.26).