國立中山大學學位論文典藏.pdf

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1 3C Cloning and expression of Taura syndrome virus 3C protein

2 I

3 Taura syndrome 60 - >90% Taura syndrome virus TSV TSV Dicistroviridae proteinase RNA-dependent RNA polymerase(rdrp) helicase motifs picornavirus TSV coat-polyprotein (protease) TSV 3C protease polyportein TSV picornavirus TSV VP-polyprotein ptsvvp 3C protease ptsv3c protease RdRp-polyprotein(pET3CD) TSV protease (ptsvvp3c) TSV protease precursor (VP-polyprotein) eletroelution VP-polyprotein Anti-VP-polyprotein polyclonal antibody Anti-VP-polyprotein polyclonal antibody VP2+VP1 VP1+VP3 precursor protein VP-polyprotein I

4 Abstract: Taura syndrome virus (TSV), the epidemic agent of Taura syndrome (TS), was tentatively classified in the family Dicistroviridae, caused disastrous losses with high mortality rates from 60 to >90% of affected pond-cultured Litopenaeus vannamei. The amino acid sequence alignments of ORF1 revealed sequence motifs characteristic of a helicase, a protease and an RNA-dependent RNA polymerase, similar to the non-structural proteins of picornavirus, suggesting perhaps a similar to picornavirus polyprotein processing. Proteolytic processing of the TSV polyprotein and protease is still unclear. In order to further understand the role of protease interaction with polyprotein, several clones were constructed. In this study, we constructed several clones that expressed the TSV virus coat protein(vp-polyprotein) ptsvvp, putative 3C protease ptsv3c, protease RdRp-polyprotein(pET3CD) and both VP+3C proteins(ptsvvp3c) in E. coli. The VP-polyprotein band was excised from SDS-polyacrylamide gels and electroeluted for antibody production in mice. Processing of the VP-polyprotein by 3C protease was carried out in the in vitro co-transcription/translation system and detected by Western blot. The results showed there were protein bands corresponding to the sizes of VP2+VP1 and VP1+VP3 indicating that the processing might have been partial or incomplete. II

5 I Abstract VIII 1 4 Taura syndrome virus DNA 11 A mini-preparation B Competent cell C D Total RNA E - (RT-PCR) F RT G (PCR) A ptsvvp B ptsv3c C ptsvvp3c D phis-vp E pgem-a pgem-b pgem-ab pgem-3cd F pet3cd VP-polyprotein A protease VP-polyprotein RdRp - 18 B VP-polyprotein C in vitro protease VP-polyprotein RdRp D SDS-PAGE E electroelution F (Lowry protein assay) G (Bradford protein assay) H Anti-VP-polyprotein I (Antigen-capture dot blotting) J Western blotting K (Sliver stain) 26 III

6 27 RT-PCR VP-ployprotein VP-polyprotein protease anti-6x-his antibody protease poly-his anti-6x-his antibody 6X-His tag VP-polyprotein VP-ployprotein protease ptsvvp3c electroelution VP-polyprotein Anti-VP-polyprotein ptsvvp3c in vitro anti-vp-polyprotein protease RdRp-polyprotein ptsvvp ptsv3cd in vitro anti-vp-polyprotein RT-PCR VP-ployprotein protease VP-polyprotein Anti-VP-polyprotein in vitro VP-ployprotein protease precursor IV

7 Table 1 Table 2 Table 3 Comparison of the putative cleavage sites of the capsid proteins encoded by ORF2 between TSV and the CrPV-like viruses V

8 Fig. 1 Fig. 2 Schematic diagram of the genome organization of TSV ptsvvp Fig. 3 Restriction enzymes mapping profile of ptsvvp vector Fig. 4 ptsv3c Fig. 5 Restriction enzymes mapping profile of ptsv3c vector Fig. 6 ptsvvp3c Fig. 7 Fig. 8 Restriction enzymes mapping profile of ptsvvp3c vector phis-vp Fig. 9 Restriction enzymes mapping profile of phis-vp vector Fig. 10 pet3cd Fig. 11 Restriction enzymes mapping profile of pet-3cd vector.- 65 Fig. 12 TSVorf1F2 orf-1-2r orf-1-2f TSVorf-BR1 TSVorf1F2 TSVorf-BR1 TSV3CDF TSVorf1R Fig. 13 Detection of virus-infected samples by using RT-PCR Fig. 14 Fig. 15 Fig. 16 The SDS-PAGE analysis of protein from the clone ptsvvp The SDS-PAGE analysis of ptsvvp VP-polyprotein solubility The effects of oxygen concentration to VP-polyprotein expression Fig. 17 Fig. 18 SDS-PAGE analysis the VP-polyprotein expressed at 30 or SDS-PAGE analysis of different harvest fraction from superdex 200 dialysised ptsvvp induced production VI

9 Fig. 19 Fig. 20 Fig. 21 Fig. 22 Fig. 23 Fig. 24 Fig. 25 Fig. 26 Fig. 27 SDS-PAGE analysis of different harvest fraction from superdex 200 dialysised ptsvvp induced production The SDS-PAGE analysis of protein from the clone ptsv3c The SDS-PAGE analysis of protein from the clone pqe30 purified by Ni 2+ spin column Western blotting analysis of TSV3C using anti-6x-his antibody The SDS-PAGE analysis of protein from the clone phis-vp The SDS-PAGE analysis of protein from the clone phis-vp purified by Ni 2+ spin column Western blotting analysis of His tag VP-polyprotein using anti-6x-his antibody The SDS-PAGE analysis of protein from the clone ptsvvp3c The SDS-PAGE analysis of VP-polyprotein from electroelution Fig. 28 Dot Blotting test of immobilized VP-polyprotein in nitrocellulose filter. VP-polyprotein Fig. 29 Western blotting analysis of VP-polyprotein Fig. 30 Fig. 31 Fig. 32 Western blotting analysis of vp-polyprotein and putative protease from in vitro transcription and translation The SDS-PAGE analysis of protein from the clone pet3cd Western blotting analysis of vp-polyprotein and putative protease RdRp-polyprotein from in vitro transcription and translation VII

10 . A pet B pqe C ptsvvp D ptsv3c E phis-vp VIII

11 1987 Litopenaeus vannamei Penaeus vannamei Perez-Farfante Kensley 1997 L. vannamei Perez-Farfante Kensley, ppt 1-2 ppt,

12 / Lightner Redman, 1998 Vibrio parahaemolytivus Lightner, 1988 (Monodon-type baculovirus, MBV) (white spot syndrome virus, WSSV)(Lo Kou, 1998) Taura syndrome virus, TSV (Taura syndrome, TS TSV Tu et al.,

13 protease 3

14 Taura syndrome virus 1992 Taura fungicides Anon Taura syndrome TS 1995 Hasson Taura syndrome virus TSV 80-85% Poulos et al., 1999 TSV 1 Hasson Tu et al, Yu & song,

15 molting Red tail disease Brock et al., 1995 buckshot inclusion body 5

16 1-20 µm (hematoxylin) (eosin) Hasson et al., 1995 Tu et al, (Brock et al., 1995 histopathology bioassay immunoassay in situ hybridization reverse transcription polymerase chain reaction, RT-PCR real-time reverse transcription polymerase chain reaction, RT-PCR, real-time RT-PCR 6

17 DIG digoxigenin cdna Mari et al., 1998 RT-PCR real-time RT-PCR Nunan et al.,

18 20 icosahedral non-envelope nm g/ml RNA 3' polya 10kb bps (non-coding regions) (open reading frames, ORF) Fig 2.1 ORF1 ORF2 nonstructural and structural polyprotein polyprotein 1995 RNA (picornaviridae) Dicistroviridae Culley et al.,2003 Mari 2002 ORF1 RNA (Picornaviridae) Cricket parailysis-like viruses proteinase RNA-dependent RNA polymerase(rdrp) helicase motifs RNA 3C protease RNA insect picorna-like virus protease domain GDCC protease 8

19 motif GxCG protease domain RNA (picorna-like virus superfamily) 1400 (histidine) 1440 (aspartic acid) 1538 (cystine) active residues catalytic triad Ryan Flint, 1997 Mari et al., 2002 RNA 3C protease polyprotein ORF2 polyprotein RNA RNA RNA 3C protease polyprotein capsid Bonami 1997 SDS-PAGE kda VP1 VP2 VP3 58 kda Mari (2002 VP1 VP2 VP3 N SKDRDMTKVNA ANPVEIDNFDTT AGLDYSSSDTST ORF2 ORF2 VP2 VP1 VP kda RNA VP2 VP3 9

20 RNA phenylalanine / (Serine) table. 1 ORF1 ORF2 frame RNA in-frame AUG start codon internal ribosome entry site IRES ORF2 (Sasaki & Nakashima, 1999) ORF1 ORF2 intergenic region RNA Sasaki Nakashima Mari (2002 Mfold version 2.3 intergenic region RNA Sasaki Nakashima PSIV stem-loop homology ORF2 IRES in-frame AUG RNA intergenic region internal ribosome entry site element IGR-IRES Shibuya et al., 2004 IRES IRES RNA 10

21 DNA A mini-preparation 1.5 ml Eppendorf centrifuge MiniSpin plus rpm 15 QIAprep Spin Miniprep Kit QIAGEN DNA 20-50µl TE [10 mm Tris-base (ph 8.0) 1 mm EDTA (ph 8.0)] B Competent cell 1 ml SOB 1000 ml Bacto tryptone 2% yeast extract 0.5% NaCl 10 mm KCl 2.5 mm MgCl 2 10 mm E. coli 37 1 ml 100 ml SOB 18 OD ,000 rpm (Eppendorf centrifuge 5415R) ml TB HEPES 10 mm CaCl 2 11

22 15 mm KCl 250 mm ( ph 6.7) MnCl 2 55 mm ml 50% DMSO (dimethyl sulfoxide) polypropylene tubes C competent cell plasmid DNA ml SOC 100 ml SOB 1.8 ml 20% glucose rpm µl ampicillin 100 µg /ml kanamycin 100 µg /ml LB Difco Sambrook et al., 1989 D Total RNA 100 mg 1 ml TRIZOL Reagent invitrogen,

23 ml chloroform 14,000 rpm (Eppendorf centrifuge MiniSpin plus) ml isopropyl alcohol 14,000 rpm (Eppendorf centrifuge MiniSpin plus) 10 1 ml 75% ethanol 14,000 rpm (Eppendorf centrifuge MiniSpin plus) RNA -20 E - (RT-PCR) Ready-To-Go TM RT-PCR (Pharmacia) 46µl DEPC-treated water Ready-To-Go TM RT-PCR beads 2.0 units of Taq DNA polymerase, 10 mm Tris-HCl, 60 mm KCl, 1.5 mm MgCl 2, 200µM of each dntp, M-MuL V reverse transcriptase, RNAguard TM ribonuclease inhibitor RNase/DNase-Free BSA foward primer 1µl 20 µm reverse primer 1µl 20 µm RNA sample 1µl 50 µl PCR Eppendorf Mastercycler personal A (Table 2)

24 F RT RNA SuperScript III reverse transcriptase Invitrogen primer 1µl 20 µm 1µl RNA 1µl dntp 10 mm each datp, dgtp, dctp, dttp 14 µl 65 4µl 5X first-strand Buffer, 1µl 0.1M DTT, 1µl SuperScript III reverse transcriptase PCR Eppendorf Mastercycler personal C (Table 2) -20 G (PCR) TaKaRa Taq DNA polymerase TaKaRa (PCR) Eppendorf Mastercycler personal D (Table 2) -20 Mari 2002 NCBI National Center for Biotechnology Information NCBI, NC_

25 protease Mari 2002 clustal w Robles-Sikisaka 2001 NetPicoRNAV1.0 ( Blom et al., 1996 Altschul et al., 1997 HAV(Hepatitis A virus; NCBI, NC_ Bergmann et al., 1997 Cohen et al., 1987) Poliovirus NCBI, NC_ picornavirus Drosophila C virus (DCV NCBI, AF Johnson, et al., 1998) Cricket paralysis virus (CrPV NCBI, AF Wilson et al., 2000) dicistrovirus A ptsvvp RNA TSVcoatF1 TSVcoatR1 A (Table 2) RT-PCR 3 kb VP-polyprotein BamH I pet28a Nco I Klenow (TaKaRa) BamH I pet28a A PCR T4 ligase (TaKaRa) VP-polyprotein 15

26 ptsvvp Fig.2. B ptsv3c RNA TSV3CF1 TSV3CR1 B (Table 2) RT-PCR 1 kb TSV protease Kpn I Sal I pqe30 ( B PCR T4 ligase (TaKaRa) poly His protease ptsv3c Fig. 4. C ptsvvp3c ptsv3c Xho I Nhe I 1.2 kb protease ptsvvp BamH I protease Klenow (TaKaRa) ptsvvp Alkaline phosphatase (TaKaRa) protease T4 ligase (TaKaRa) protease ptsvvp3c Fig

27 D phis-vp RNA TSVHisVPF1 TSVcoatR1 A (Table 2) RT-PCR 3 kb VP-polyprotein BamH I BamH I pqe30 T4 ligase (TaKaRa) 6X-His tag VP-polyprotein phis-vp Fig. 8. E pgem-a pgem-b pgem-ab pgem-3cd RNA TSVorf1F2 orf-1-2r orf-1-2f TSVorf-BR1 TSVorf1F2 TSVorf-BR1 TSV3CDF TSVorf1R2 A (Table 2) RT-PCR DNA T4 ligase (TaKaRa) pgem-t Easy (Promega) ORF pgem-a pgem-b pgem-ab pgem-3cd 12 Fig. 12. F pet3cd RNA 17

28 TSV3CDF TSVorf1R2 A (Table 2) RT-PCR DNA Nhe I Sal I pet28a PCR T4 ligase (TaKaRa) 6X-His tag protease RdRp pet3cd Fig. 10. VP-polyprotein A protease VP-polyprotein protease+rdrp ptsvvp pet3cd BL21 DE kanamycin 100 µg /ml LB Difco 37 OD mm IPTG 4 ptsv3c phis-vp JM ampicillin 100 µg /ml LB Difco 37 OD mm IPTG 4 ptsvvp3c BL21 DE3 JM109 JM109(DE3) 1 30 kanamycin 100 µg /ml LB 18

29 37 OD mm IPTG 4 B VP-polyprotein ptsvvp VP-polyprotein French press ( psi) rpm (Hitachi, RPR ) mm Tris-HCl (ph 8) 8 M Urea superdex 200 prep grade Pharmacia Biotech 50 cm 1.5 cm 1.2 cm 40 superdex 200 prep grade gel buffer 0.75 ml/min bufferr 5 cm buffer buffer 1 ml buffer 0.75 ml/min bufferr µl Hitachi, U2001 SDS-PAGE C in vitro protease VP-polyprotein RdRp 19

30 ptsvvp pet3cd ptsvvp3c E.coli T7 S30 Extract system (Promega) SDS-PAGE Western blotting D SDS-PAGE 10 µl 2X SDS-PAGE loading buffer (100 mm Tris-HCl (ph 6.8), 200 mm dithiotheitol (DDT), 4% SDS, 0.2% bromophenol blue, 20% glycerol) (12%) ml H 2 O ml 4X 1.5 M Tris-HCl, ph 8.8 (Tris-base g 10% SDS 3.95 ml 80 ml ph ml) 3 ml 30% acrylamide mix (29% acrylamide 1% bis-acrylamide acrylamide 29 g biacrylamide 1 g) 100 ml 5 50 µl 10% APS 5 µl TEMED 2.1 ml H 2 O 0.75 ml 4X 0.5 M Tris-HCl, ph 6.8 (Tris-base g 10% SDS 7.4 ml 80 ml ph ml) 0.5 ml 30% acrylamide mix 5 20

31 30 µl 10% APS 3 µl TEMED well 120 V commassie blue 30 marker band E electroelution Lutsyk(1990) SDS-PAGE ISCO electroeluter sample tank eletroelution buffer 20 mm Tris base, 150 mm glycine, 0.01% SDS Buffer tank electroeluter sample tank buffer tank buffer 120 V commassie blue Centricon YM-30 Amicon, Millipore, 5000Xg 10 mm Tris-HCl buffer F (Lowry protein assay) bovine serum albumin BSA 21

32 0.8 ml digestion reagent A 0.53 ml H 2 O 0.1 ml 10 SDS 0.1 ml 0.8N NaOH 0.05 ml 20 Na 2 CO ml 2 Na, K-Tartrate 4H 2 O, 0.01 ml 1 CuSO4 5H 2 O 0.2 ml phenol reagent 0.16 ml H 2 O 0.04 ml Folin-Ciocalteace reagent OD 700 BSA OD 700 OD 700 G (Bradford protein assay) Bradford Bradford, 1976 Amersco BSA( 0.5 mg/ml) µl 0.15 N NaCl 100 µl 1 ml Bradford Hitachi, U nm 0.15 N NaCl 100 µl 1 ml Bradford G Anti-VP-polyprotein ptsvvp electroelution 100 µl 100 µg/ml 100 µl PBS 200 µl 22

33 Freund s complete adjuvant Sigma ml BALB/cByJ ptsvvp electroelution 100 µl 100 µg/ml 100 µl PBS Freund s incomplete adjuvant Sigma rpm (Eppendorf centrifuge MiniSpin plus) 10 anti-vp-polyprotein H (Antigen-capture dot blotting) anti-vp-polyprotein 1 10 D-PBS (Gibco) 1 cm 1 cm (OSMONICS) (35 mm 35 mm) transfer buffer (Tris 3.0 g glycine 14.4 g ddh 2 O 1 L ) 10 transfer buffer 23

34 20 µl electroelution VP-polyprotein 10 µl BSA VP-polyprotein 3 ml PBS 5 ( PBS ) 4 PBS-Tween 20 5 PBS 5 anti-vp-polyprotein (10 PBS buffer ) shaker 2 anti-vp-polyprotein PBS-Tween 20 5 IgG [Goat anti-mouse IgG (H&L) HRPO Leinco Technologies, PBS] 2 PBS-Tween 20 ddh 2 O 5 5 mg DAB (3,3 -Diaminobenzidine Tetrahydrochloride) (PIERCE) 9.72 ml ddh 2 O 280 µl 35% H 2 O 2 I Western blotting 24

35 SDS-PAGE SDS-PAGE transfer buffer (Tris 3.0 g glycine 14.4 g ddh 2 O 1 L ) 10 SDS-PAGE (BIO-RAD, Model No. Trans-Blot SD cell) SDS-PAGE transfer buffer 25V 380 ma 45 PBS PBS 5% 1 PBS-tween 5 PBS PBS 2 PBS-tween PBS ( IgG [Goat anti-mouse IgG (H&L) HRPO Leinco Technologies, PBS]) 2 PBS-tween PBS 2 PBS-tween 5 5 mg DAB (3,3 -Diaminobenzidine Tetrahydrochloride) (PIERCE) 9.72 ml ddh 2 O 280 µl 35% H 2 O 2 25

36 DAB solution J (Sliver stain) Pharmacia (Pharmacia) Fixation 50 ml 12.5 ml 125 ml Sensitizing 37.5 ml 25 % w/v glutardialdehyde ml 5 % w/v 5 ml 8.5 g 125 ml Silver reaction 2.5% w/v 12.5 ml 37% w/v formaldehyde 0.05 ml 125 ml g 37 % w/v formaldehyde ml 125 ml EDTA-Na 2 2H 2 O g 125 ml 37.5 ml 87% w/w glycerol 5.75 ml 125 ml 26

37 RT-PCR TRIZOL Reagent RNA RNA (Total RNA) TSVF1 TSVR1 Nunan et al., 1998 B RT-PCR 220 bps 220 bps ( Fig. 14) pet28a ptsvvp ptsvvp pet28a T7 promoter IPTG 110 kda ptsvvp (mapping) EcoR I bp Sac I Xho I bp Fig. 3 ptsvvp. C 27

38 ptsvvp BL21 DE3 (Fig. 15) VP-polyprotein (Fig. 16) 50 ml 75 ml 100 ml 150 ml 250 ml 170 (Fig.17) VP-ployprotein VP-ployprotein SSN-1 (Bragard et al., 2000; Wizemann and Brunn, 1999) (Fig. 17) VP-ployprotein 28

39 VP-polyprotein ptsvvp VP-polyprotein French press rpm 15 superdex 200 VP-polyprotein SDS-PAGE Fig. 18, Fig. 19 VP-polyprotein protease protease pqe30 ptsv3c pqe30 T5 promoter IPTG 40 kda ptsv3c Kpn I Sal I bp BamH I bp Fig. 5 (Fig. 20) pqe30 6X-His tag (fusion protein) 29

40 Ni (Fig. 21) anti-6x-his antibody protease ptsv3c 6X-His tag protease SDS-PAGE anti-6x-his antibody protease 40 kda anti-6x-his antibody protease 40 kda smear ptsv3c 6X-His tag protease protease (Fig. 22) poly-his ptsvvp polyprotein (pqe30) polyprotein phis-vp 6X-His tag polyprotein pqe30 T5 promoter phis-vp EcoR I 30

41 bp Sac I Xho I bp Hind III Fig. 9 phis-vp. D phis-vp 4 IPTG 110 kda (Fig. 23) 6X-His tag polyprotein VP-polyprotein (Fig. 24) anti-6x-his antibody 6X-His tag VP-polyprotein phis-vp 6X-His tag polyprotein SDS-PAGE anti-6x-his antibody 6X-His tag VP-polyprotein 110 kda anti-6x-his antibody 6X-His tag VP-polyprotein 110 kda smear phis-vp 6X-His tag VP-polyprotein 6X-His tag VP-polyprotein 31

42 protease Fig. 25 VP-ployprotein protease ptsvvp3c ORF2 ptsvvp ptsv3c Xho I Nhe I 1.2 kb protease ptsvvp BamH I Klenow protease ptsvvp3c ptsvvp3c Xho I bp EcoR I Fig. 7 BL21 DE3 JM109 JM109(DE3) (Fig. 26) protease T5 promoter ORF2 T7 promoter VP-ployprotein protease JM109(DE3) VP-ployprotein protease VP-polyprotein VP-polyprotein 110 kda kda VP1 VP2 VP3 32

43 electroelution VP-polyprotein Ni 2+ VP-polyprotein VP-polyprotein VP-polyprotein SDS-PAGE ISCO electroeluter 120 V 2.5 Fig. 27 SDS-PAGE VP-polyprotein Anti-VP-polyprotein dot blotting Fig. 28 Anti-VP-polyprotein ptsvvp ptsvvp3c electroelution VP-polyprotein SDS-PAGE Fig. 29 ptsvvp ptsvvp3c electroelution VP-polyprotein 110 kda ptsvvp ptsvvp3c 33

44 VP-polyprotein Anti-VP-polyprotein ptsvvp3c in vitro anti-vp-polyprotein VP-ployprotein protease VP-ployprotein protease protease VP-ployprotein ptsvvp3c VP-ployprotein protease anti-vp-polyprotein protease VP-ployprotein E.coli T7 S30 Extract system T7 RNA polymrease ptsvvp3c T7 promoter ptsvvp3c T7 promoter VP-polyprotein E.coli RNA polymerase protease ptsvvp3c T5 promoter protease VP-ployprotein protease SDS-PAGE anti-vp-polyprotein 34

45 Fig 30 VP-ployprotein protease VP-ployprotein VP-ployprotein kda VP1 VP2 VP3 94 kda 78 kda VP2+VP1 VP1+VP3 ptsv3c ptsvvp VP-ployprotein protease RdRp-polyprotein protease VP-polyprotein protease RdRp-polyprotein protease RdRp-polyprotein pet28a pet3cd T7 promoter IPTG 93 kda ptsv3c HinD III bp EcoR I bp Nde I Sac I bp Sma I Pst I bp Fig

46 (Fig. 31) ptsvvp pet3cd in vitro anti-vp-polyprotein in vitro ptsvvp ptsv3c kda VP1 VP2 VP3 protease RdRp-polyprotein pet3cd ptsvvp VP-ployprotein protease RdRp-polyprotein anti-vp-polyprotein protease RdRp-polyprotein VP-ployprotein E.coli T7 S30 Extract system T7 RNA polymrease pet3cd ptsvvp T7 promoter VP-ployprotein protease RdRp-polyprotein SDS-PAGE anti-vp-polyprotein Fig 32 pet3cd ptsvvp VP-ployprotein 36

47 RT-PCR RT-PCR PCR Tsai 2002 RT-PCR PCR RNA RT-PCR Mari 2002 NCBI NCBI, NC_ protease Mari 2002 HAV Poliovirus protease bp Bergmann et al., 1997 Cohen et al., 1987 clustal w Robles-Sikisaka 2001 NetPicoRNAV1.0 Blom et al.,

48 Altschul et al., 1997 picornavirus HAV Poliovirus dicistrovirus DCV CrPV bp protease bp TSV3CF1 TSV3CR1 RT-PCR protease RdRp-polyprotein bp TSV3CDF TSVorf1R2 RT-PCR ptsvvp pet28a T7 promoter T7 polymerase JM109 DE3 BL21 DE3 ptsvvp VP-polyprotein (Fig. 15.) (Bragard et al., 2000; Wizemann and Brunn, 1999) Hunke et al., 2003 VP-polyprotein detergent(1 triton-x M, 4 M, 6 M, 8 M 38

49 urea 6 M Gn-HCl) VP-polyprotein 8 M urea 6 M Gn-HCl superdex 200 VP-polyprotein detergent(1 triton-x M urea 1 -mercaptoethanol 6 M Guanidine-HCl) 8 M urea, Fig. 19, Fig. 20 VP-polyprotein superdex 200 Sosnovtsev (1998) Skerra 1991 VP-polyprotein N 6X-His tag phis-vp 6X-His tag (Fusion Protein) Ni 2+ 6X-His tag VP-polyprotein pqe40 mouse DHFR 26 kda DHFR 6X-His tag VP-polyprotein 6X-His tag Ni 2+ 39

50 anti-6x-his antibody 6X-His tag VP-polyprotein 6X-His tag VP-polyprotein 110 kda anti-6x-his antibody 6X-His tag VP-polyprotein 110 kda smear VP-polyprotein proteinase 6X-His tag VP-polyprotein 6X-His tag VP-polyprotein VP-ployprotein protease ptsvvp3c VP-ployprotein protease T5 promoter lacl q Bujard et al., 1987 VP-ployprotein T7 promoter DE3 VP-ployprotein protease ptsvvp3c JM109(DE3) VP-ployprotein protease(fig. 26) VP-polyprotein 110 kda protease VP-polyprotein kda VP1 VP2 VP3 inclusion body 40

51 Rudolph et al., 1996 protease VP-polyprotein Anti-VP-polyprotein VP-polyprotein VP-polyprotein VP-polyprotein SDS-PAGE ISCO electroeluter SDS-PAGE commassie blue CuCl 2 Lee et al., 1987 gel destain solution 0.25 M EDTA ph8.0, 0.25 M Tris ph9.0 commassie blue electroelute 120 V 2.5 Centricon YM-30 electroelute buffer PBS Bradford protein assay VP-polyprotein 100 µg/ml PBS Fig

52 ptsvvp ptsvvp3c VP-polyprotein VP-polyprotein Fig kda VP-polyprotein 110 kda smear anti-6x-his antibody PintÓ (2002) HAV HAV 3C protease HAV polyprotein protease protease HAV 3C protease HAV polyprotein TSV VP-polyprotein protease in vitro VP-ployprotein protease precursor protease VP-ployprotein protease in vitro VP-ployprotein protease E. coli T7 S30 Extract system E. coli endoproteinase ion protease Nevin et al., 1991 ptsvvp3c 42

53 E.coli T7 S30 Extract system T7 polymerase T7 promoter Studier et al., 1986 VP-polyprotein E. coli RNA polymerase protease ptsvvp3c T5 promoter protease anti-vp-polyprotein antibody kda VP1 VP2 VP3 94 kda 78 kda anti-vp-polyprotein antibody in vitro ptsv3c ptsvvp VP-ployprotein VP-ployprotein protease RdRp-polyprotein anti-vp-polyprotein 94 kda 78 kda anti-vp-polyprotein antibody in vitro pet3cd ptsvvp VP-ployprotein picornavirus protease RdRp-polyprotein P3 precursor P3 region PintÓ et al., 2002, Bedard Semler, 2004 Harris et al.,1994 Probst 1998 HAV 3C pro -containing proteins (P3, 3ABC, 3BCD, 3CD, 3BC, and 3C) COS7 cells. P1-2A precursor capsid protein precursor P3 43

54 3C pro -containing proteins in vitro VP-ployprotein protease protease RdRp-polyprotein VP2+VP1 VP1+VP3 precursor protein VP-polyprotein 44

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65 Fig. 1. Schematic diagram of the genome organization of TSV. Numbers indicate nucleotide positions. ORFs 1 and 2 are shown as open boxes and UTRs as a single line. The approximate positions of the BIR-like sequence (BIR), helicase (H), protease (P) and RNA-dependent RNA polymerase (RdRp) are indicated. Arrows represent the N termini of the capsid proteins.(mari, et al.,

66 TSVcoatR1 TSVcoatF1 B 3.2 kb Bam HI, Nco I Fig.2. ptsvvp TSVcoatF1 TSVcoatR1 3.2 kb VP-polyprotein BamH I pet28a Nco I Klenow BamH I pet28a PCR T4 ligase coat polyprotein ptsvvp Multiple cloning sites B Bam H I 56

67 M Fig. 3. Restriction enzymes mapping profile of ptsvvp vector. Lane 1 is ptsvvp plasmid. Lane 2 is EcoR I restriction enzyme mapping profiles of ptsvvp vector. Lane 3 is Sac I and Xho I and restriction enzyme mapping profiles of ptsvvp vector. M: DNA marker. 57

68 TSV3CF1 K TSV3CR1 S 1 kb Kpn I Sal I Fig. 4. ptsv3c TSV3CF1 TSV3CR1 1 kb TSV protease Kpn I Sal I pqe30 PCR T4 ligase poly His protease ptsv3c Multiple cloning sites S Sal I K Kpn I 58

69 M Fig. 5. Restriction enzymes mapping profile of ptsv3c vector. Lane 1 is ptsv3c plasmid. Lane 2 is BamH I restriction enzyme mapping profile of ptsv3c vector. Lane 3 is Kpn I and Sal I restriction enzyme mapping profile of ptsv3c vector. M: DNA marker. 59

70 Bam H I/Klenow Xho I/Nhe I/Klenow T5 promoter T7 promoter Fig. 6. ptsvvp3c ptsv3c Xho I Nhe I 1.2 kb protease ptsvvp BamH I protease Klenow T4 ligase protease ptsvvp3c 60

71 M Fig. 7. Restriction enzymes mapping profile of ptsvvp3c vector. Lane 1 is ptsvvp3c plasmid. Lane 2 is Xho I restriction enzyme mapping profile of ptsvvp3c vector. Lane 3 is EcoR I restriction enzyme mapping profile of ptsvvp3c vector. M: DNA marker. 61

72 TSVHisVPF1 TSVcoatR1 B His-VP 3.2 kb B Bam HI Fig. 8. phis-vp TSVHisVPF1 TSVcoatR1 3.2 kb VP-polyprotein pqe30 BamH I T4 ligase 6X-His tag VP-polyprotein phis-vp Multiple cloning sites B Bam H I 62

73 M Fig. 9. Restriction enzymes mapping profile of phis-vp vector. Lane 1 is phis-vp plasmid. Lane 2 is EcoR I restriction enzyme mapping profiles of phis-vp vector. Lane 3 is Sac I and Xho I and restriction enzyme mapping profiles of phis-vp vector. Lane 4 is HinD III restriction enzyme mapping profiles of phis-vp vector. M: DNA marker. 63

74 TSV3CDF N TSVorf1R2 S protease RdRp-polyprotein 2.5kb N S Fig. 10. pet3cd TSV3CDF TSVorf1R2 protease RdRp-polyprotein 2.5kb Nhe I Sal I pet28a PCR T4 ligase 6X-His tag protease RdRp pet3cd Multiple cloning sites S Sal I N Nhe I 64

75 M Fig. 11. Restriction enzymes mapping profile of pet-3cd vector. Lane 1 is pet-3cd plasmid. Lane 2 is EcoR I restriction enzyme mapping profiles of pet-3cd vector. Lane 3 is HinD III restriction enzyme mapping profiles of pet-3cd vector. Lane 4 is Sac I and Nde I and restriction enzyme mapping profiles of pet-3cd vector. Lane 5 is Sma I and Pst I and restriction enzyme mapping profiles of pet-3cd vector. M: DNA marker. 65

76 Fig.12. TSVorf1F2 orf-1-2r orf-1-2f TSVorf-BR1 TSVorf1F2 TSVorf-BR1 TSV3CDF TSVorf1R2 66

77 M bps Fig. 13. Detection of virus-infected samples by using RT-PCR. The total RNAs extracted from normal shrimp (lane 1) or virus-infected shrimp (lane 2), were used as template in the RT-PCR with primer TSVF1 and TSVR1. The RT-PCR was carried at the condition mentioned in section Material and Methods. M: DNA marker. 67

78 M Fig. 14. The SDS-PAGE analysis of protein from the clone ptsvvp. The ptsvvp bearing E.coli BL21(DE3) cells was incubated in 37. When cell density reach to OD , 0.84 mm IPTG was added. Lane 1 was the ptsvvp bearing E.coli BL21(DE3) cells without induction. After 4 hours induction, collected debris and analysis by SDS-PAGE (lane 2). M: protein marker. The arrow indicates the VP-poltprotein. 68

79 M Fig. 15. The SDS-PAGE analysis of ptsvvp VP-polyprotein solubility. The E.coli BL21(DE3) cell bearing ptsvvp was incubated in 37 C. When cell density reached to OD , 0.84 mm IPTG was added. After 4 hours induction, collected debris and analysis by SDS-PAGE. The lane 1 was E.coli BL21(DE3) cell bearing ptsvvp with induction.the collected cell was broken by French press and centrifuge under 39,400 g for 20 mins. The supernatant(lane 2) and debris(lane 3) were analysis by SDS-PAGE. M: protein marker. 69

80 M Fig. 16. The effects of oxygen concentration to VP-polyprotein expression. The cell incubated in 50 ml (lane 1), 75 ml (lane 2), 100 ml (lane 3) 150 ml (lane 4)broth volume at 170 rpm. M: protein marker. The arrow indicates the VP-poltprotein. 70

81 M Fig. 17. SDS-PAGE analysis the VP-polyprotein expressed at 30 C or 37 C. The lane 1 was E.coli BL21(DE3) cell bearing ptsvvp without induction. The E.coli BL21(DE3) cell bearing ptsvvp was incubated in 30 C lane 4 and lane 5 or 37 C lane 2 and lane 3. When cell density reached to OD , 0.84 mm IPTG was added. After 4 hours induction, collect debris and analysis by SDS-PAGE. The collected cell was broken by French press and centrifuge under 39,400 g for 20 mins. The supernatant(lane 2 and lane 4) and debris(lane 3 and lane 5) were analysis by SDS-PAGE. M: protein marker. The arrow indicates the VP-poltprotein. 71

82 M Fig. 18. SDS-PAGE analysis of different harvest fraction from superdex 200 dialysised ptsvvp induced production. The lane were fraction M: protein marker. The arrow indicates the VP-poltprotein. 72

83 M Fig. 19. SDS-PAGE analysis of different harvest fraction from superdex 200 dialysised ptsvvp induced production. The lane were fraction M: protein marker. The arrow indicates the VP-poltprotein. 73

84 M Fig. 20. The SDS-PAGE analysis of protein from the clone ptsv3c. The ptsv3c bearing E.coli JM109 cells was incubated in 37 C. The lane 1 was E.coli bearing ptsv3c without induction. When cell density reach to OD , 0.84 mm IPTG was added. After 4 hours induction, collected debris and analysis by SDS-PAGE (lane 2). M: protein marker. The arrow indicates the putive protease. 74

85 M Fig. 21. The SDS-PAGE analysis of protein from the clone pqe30 purified by Ni 2+ spin column. The lane 1 was E.coli bearing ptsv3c with induction. The lane 3 and lane 5 were sample washed and collected by wash buffer. The lane 6 was the finally eluted out sample by elution buffer. M: protein marker. The arrow indicates the putive protease. 75

86 M Fig. 22. Western blotting analysis of TSV3C using anti-6x-his antibody The lane 1 was E.coli JM109 with induction. Lane 2 was the ptsv3c bearing E.coli JM109 cells without induction. The ptsv3c (lane 3) bearing E.coli JM109 cells was incubated in 37 C. When cell density reach to OD , 0.84 mm IPTG was added. After 4 hours induction, collect debris and analysis by SDS-PAGE. The samples were fractionated on a 12% SDS-PAGE. The gel was then transferred onto the nitrocellulose filter for Western blotting analysis using anti-6x-his antibody and goat-anti-mouse IgG. M represents a protein molecule marker. 76

87 M Fig. 23. The SDS-PAGE analysis of protein from the clone phis-vp. The lane 1 was the phis-vp bearing E.coli JM109 cells without induction.the phis-vp bearing E.coli JM109 cells was incubated in 37 C. When cell density reach to OD , 0.84 mm IPTG was added. After 4 hours induction, collect debris and analysis by SDS-PAGE (lane 2). M: protein marker. The arrow indicates the 6X His-tag VP-poltprotein. 77

88 M Fig. 24. The SDS-PAGE analysis of protein from the clone phis-vp purified by Ni 2+ spin column. The phis-vp bearing E.coli JM109 cells was incubated in 37. When cell density reach to OD , 0.84 mm IPTG was added. The lane 1 was E.coli bearing phis-vp without induction. After 4 hours induction, collected debris than lyesed by lyses buffer and purified by Ni 2+ spin column. The supernatant(lane 2) and debris(lane 3) were analysis by SDS-PAGE. The lane 4 was eluted out sample. M: protein marker. The arrow indicates the 6X His-tag VP-poltprotein. 78

89 M Fig. 25. Western blotting analysis of His tag VP-polyprotein using anti-6x-his antibody The phis-vp (lane 3) bearing E.coli Jm109 cells was incubated in 37. When cell density reach to OD , 0.84 mm IPTG was added. After 4 hours induction, collected debris and analysis by SDS-PAGE. The lane 1 was E.coli JM109 with induction. Lane 2 was the phis-vp bearing E.coli JM109 cells induction. The samples were fractionated on a 12% SDS-PAGE. The gel was then transferred onto the nitrocellulose filter for Western blotting analysis using anti-6x-his antibody and goat-anti-mouse IgG. M represents a protein molecule marker. 79

90 M Fig. 26. The SDS-PAGE analysis of protein from the clone ptsvvp3c. The ptsvvp3c bearing E.coli JM109 cells and E.coli JM109 (DE3) cells was incubated in 37. Lane 1 was the ptsvvp3c bearing E.coli JM109 cells without induction. When cell density reach to OD , 0.84 mm IPTG was added. After 4 hours induction, collect debris and analysis by SDS-PAGE (lane 2, lane 3). M: protein marker. The black arrow indicates the VP-poltprotein. The red arrow indicates the protease. 80

91 116 M Fig. 27. The SDS-PAGE analysis of VP-polyprotein from electroelution. Lane 1 VP-polyprotein was recycled from eletroelution 2.5h. Lane 2 VP-polyprotein was recycled from eletroelution 3h. M: protein marker. The arrow indicates the VP-poltprotein. 81

92 No.4 No.5 Fig. 28. Dot Blotting test of immobilized VP-polyprotein in nitrocellulose filter. VP-polyprotein was immobilized onto the nitrocellulose filter at room temperature for 1h, followed by washing once with PBS. After those washing steps, followed by blocking with non-fat milk for 1h. Prior to carry out immuno-blotting, the samples were washed with PBS-Tween and PBS and blotted with No.4 and No.5 poly-anti-vp-polyprotein-antibody and goat-anti-mouse IgG as described in the section of Materials and Methods. 82

93 M Fig. 29. Western blotting analysis of VP-polyprotein. The lane 1 was negative control E.coli BL21(DE3) with induction. The ptsvvp (lane 2) or ptsvvp3c (lane 3) bearing E.coli BL21(DE3) cells was incubated in 37. When cell density reach to OD , 0.84 mm IPTG was added. After 4 hours induction, collected debris and analysis by SDS-PAGE. The ptsvvp (lane 4) or ptsvvp3c (lane 5) bearing E.coli BL21(DE3) cells was negstive control without induction. The lane 6 was VP-polyprotein recovered from electroeultion. The samples were fractionated on a 12% SDS-PAGE. The gel was then transferred onto the nitrocellulose filter for Western blotting analysis using poly-anti-vp-polyprotein-antibody and goat-anti-mouse IgG. M represents a protein molecule marker. 83

94 VP-polyprotein 94(VP1+VP3) 78(VP2+VP1) 55(VP1) 40 (VP2) 25(VP3) Fig. 30. Western blotting analysis of vp-polyprotein and putative protease from in vitro transcription and translation. The vectors, pet28a (lane 3), ptsv3c (lane 4), and ptsvvp3c (lane 6) were in vitro transcripted and translated. The lane 1 was positive control E.coli bearing phis-vp with induction. The lane 2 was negative control E.coli BL21(DE3) with induction. The lane 5 was vp-polyprotein recovered from electroeultion. The samples were fractionated on a 12% SDS-PAGE. The gel was then transferred onto the nitrocellulose filter for Western blotting analysis using poly-anti-vp-polyprotein-antibody and goat-anti-mouse IgG. M represents a protein molecule marker. 84

95 116- M Fig. 31. The SDS-PAGE analysis of protein from the clone pet3cd. The pet3cd bearing E.coli BL21(DE3) cells was incubated in 37. When cell density reach to OD , 0.84 mm IPTG was added. Lane 1 was the pet3cd bearing E.coli BL21(DE3) cells without induction. After 4 hours induction, collected debris and analysis by SDS-PAGE (lane 2). M: protein marker. The arrow indicates the protease-rdrp-polyprotein.. 85

96 118- M Fig. 32. Western blotting analysis of vp-polyprotein and putative protease RdRp-polyprotein from in vitro transcription and translation. The lane 1 was E.coli BL21 (DE3) with induction. The vectors, pet28a (lane 2), pet3cd (lane 3), and pet3cd(lane 5) with ptsvvp were in vitro transcripted and translated. The lane 4 was vp-polyprotein recovered from electroeultion. The lane 6 was positive control E.coli bearing phis-vp with induction. The samples were fractionated on a 12% SDS-PAGE. The gel was then transferred onto the nitrocellulose filter for Western blotting analysis using poly-anti-vp-polyprotein-antibody and goat-anti-mouse IgG. M represents a protein molecule marker. 86

97 Table.1 Comparison of the putative cleavage sites of the capsid proteins encoded by ORF2 between TSV and the CrPV-like viruses

98 Table.2 TSVcoatF1 5 -CCTGCTAACCCAGTTGAAATTGAT-3 TSVcoatR1 TSV3CF1 TSV3CR1 5 -GAATTCGGATCCCACCTGAAGGGTGTTAAATTA-3 5 -AAGACTGGTACCGTGGAATCCCATCAGGACTTTAA-3 5 -AGTAATGTCGACTTTTGTTGGATTGGGTGCTTCT-3 TSVHisVPF1 5 -GAATTCGGATCCATGCCTGCTAACCCAGTTGAA-3 TSV3CDF 5 - AAGACTGCTAGCGTGGAATCCCATCAGGAC-3' TSVorf1F2 5'- ATAAGCCTGCAGACTGCTAGCTATACTATGGCCTCT- 3' TSVorf1R2 5 - CGATCGGGTGGTCGACTTACTTTAAGTCCAATAGCTG -3' orf-1-2f 5 - CCCATAATTCAGGGTATATCTAGTTATGCTCTTCG- 3' TSVorf-B-R1 5'- TTAAAGTCCTGATGGGATTCCACCACAGCAGTCTT- 3' TSVR1 5 -AAGTAGACAGCCGCGCTT-3 TSVF1 5 -TACATGAGAGCTTGGTCC-3 TSVpolyT 5 -TTTTTTTTTTAGGAATCCGCCGCTT-3 88

99 Table.3 1. Condition A 40 1 hour min 95 2 min sec sec for 30 cycles sec min 2. Condition B 40 1 hour 95 2 min sec sec for 30 cycles sec min 3. Condition C 40 1 hour min 89

100 4. Condition D 95 2 min sec sec for 30 cycles sec min 90

101 . A pet28 91

102 . B pqe30 92

103 . C ptsvvp 93

104 94

105 . D ptsv3c 95

106 96

107 . E phis-vp G 97

108 98