71

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

72

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

73

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

74

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.

75

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.

76

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).

77

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

78

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).

79

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

80

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.

81

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.

82

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.

83

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

84

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.

85

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

86

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

87

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

88

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

89

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.

90

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

91

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.

92

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

93

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).

94

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).