NIER-SP 신속한노로바이러스진단을위한검사방법연구 Study for Rapid Diagnostics Methods of Norovirus
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1 NIER-SP 신속한노로바이러스진단을위한검사방법연구 Study for Rapid Diagnostics Methods of Norovirus
2 제출문 국립환경과학원장귀하
3 요약문 G.4 type.., rvlp (Recombination Virus-Like Particle). rvlp (hybridization) probe,.
4 VP1 region rvlp size chromatography. rvlp, (1) ELISA test test, (2) RT-PCR ELISA test, (3),. probe Dot-hybridization (sensitivity) (specificity), Dot blot. rvlp,.
5 I
6 <Table 1> Immunization schedule 23 <Table 2> 1 st Bleeding ELISA test 24 <Table 3> 2 nd Bleeding ELISA test 25 <Table 4> Norovirus G, G Probe design 32
7 <Figure 1> Organization of the Norovirus 2 <Figure 2> Norovirus structure protein 3 <Figure 3> Food poisoning occurrence during 2002~2013 in South Korea 5 <Figure 4> Baculovirus Expression Vector System 8 <Figure 5> Verification of VP1(ORF2) gene expression by Western Blotting 9 <Figure 6> Probe design 9 <Figure 7> Membrane blotting diagram 11 <Figure 8> Comparison of sensitivity to detect of Norovirus RNA by RT-PCR and Real time PCR 12 <Figure 9> BEVS-EGFP expression system 15 <Figure 10> Confirmation of the BEVS-EGFP expression system by EGFP 16 <Figure 11> Sequences of Norovirus VP1 and VP2 regions 17 <Figure 12> Verification of VP1 and VP2 gene in shuttle vector by PCR 18 <Figure 13> Schematic diagram for recombinant Baculovirus propagation containing Norovirus VP1 and VP2 genes 18 <Figure 14> Schematic diagram for rvlp propagation using recombinant Baculovirus expression system and confirmation of VP1 and VP2 proteins expression by Western Blotting method 20 <Figure 15> Expression of ORF2, verification by Western Blotting 21 <Figure 16> Verification by Coomassie blue staining and Western Blotting 22 <Figure 17> Verification of concentrated rvlp by coomassie blue staining and Western Blotting 22 <Figure 18> 1 st Bleeding ELISA test 24 <Figure 19> 2 nd Bleeding ELISA test 25 <Figure 20> Anti rvlp antibody sensitivity test 26 <Figure 21> Human Norovirus detection by RT-PCR 27 <Figure 22> Anti rvlp antibody ELISA test with human Norovirus stool sample 28 <Figure 23> Commercial Norovirus detection kit test 29 <Figure 24> Norovirus G sequence analysis using Meg-align program 30 <Figure 25> Norovirus G sequence analysis using Meg-align program 31
8 <Figure 26> Probe design using Oligo 6 program 32 <Figure 27> G, G probe specificity test 33 <Figure 28> G, G probe sensitivity test 34 <Figure 29> Human norovirus G, G hybridization test with designed probe 35
9 I.
10 Fig 1. Organization of the Norovirus
11 Fig 2. Norovirus structure protein
12
13 Fig 3. Food poisoning occurrence during 2002~2013 in South Korea ( - )
14 . 1.. rvlp 1) capside rvlp 2) rvlp 3) rvlp, His- rvlp 4) *rvlp (Recombination Virus-like particle: ). (hybridization) probe 1) genotype probe 2) Hybridization dot blot. 1) RT-PCR 2) rvlp ELISA 3) 2.. rvlp capsid ORF2 (VP1 region) epitope (Baculovirus Expression Vector System; BEVS). Autographa californica nuclear polyhedrosis virus (AcNPV)
15 (Fig 4).,. (Adeno- Retrovirus ) 17,18. AcNPV 130 kb DNA (12 kb ). BEVS. ORF2(VP1) ORF3(VP2). ORF2 capsid. VP1 shell domain S-domain protruding domain P-domain. P-domain P1 P2 sub-domain. P2 domain strain conserved region. P2-domain receptor 8,9. C- 6 His. ORF2(VP1) ORF3(VP2) ( ), VP1 VP2.
16 Fig 4. Baculovirus Expression Vector System ( - : Baculovirus ) VP1 ganciclovir negative selection VP1. rvlp size exclusion, Western blotting capsid protein (Fig 5). rvlp.
17 Fig 5. Verification of VP1(ORF2) gene expression by Western Blotting. (hybridization) probe NCBI, DDBJ, EMBL GenBank. DNAStar Meg-align program probe, Oligio6 melting temperature secondary structure (Fig 6). Fig 6. Probe design Hybridization probe test
18 probe target primer PCR cloning transformation. transformation competent cell (E.coli DH5a). 5 ml LB 12, 50 ml LB ,000 rpm, mm CaCl 15 ml. 10 7,000 rpm, mm CaCl MgCl 10 ml 10 7,000 rpm, mm CaCl 2 ml % glycerol ml tube -70. Transformation competent cell 200 ligation DNA 30. Competent cell ligation DNA , 2. LB (Ampicillin) X-gal (20 /ml) 40 IPTG (200 /ml) 4 spreading colony. white colony (Ampicillin) LB. 1.5 ml tube 13,000 rpm 1. Pellet (resuspension) pellet RNase PD (lysis) PD (neutralization) PD ,000 rpm 3. collection tube column 13,000 rpm 30. Column collection tube W ,000 rpm % 600 column 13,000 rpm. Column collection tube 13,000 rpm ml tube column (elution) Elution ,000 rpm 2. probe Dot blot hybridization. Dot blot hybridization. Hybridization membrane blotting (Fig 7).
19 Fig 7. Membrane blotting diagram Nitrocellulose membrane DNA 1 spot, membrane 80 2 baking. membrane prehybridizaion solution (5X Denhardt's, 5X SSC, 0.1% SDS, 0.1mg/mL denatured salmon sperm DNA) 65, 15 prehybridizaion. Biotin-labeled probe hybridization solution 65 1 hybridization. Nitrocellulose membrane 2X wash buffer (2X SSC, 0.1% SDS) 2, 0.5X wash buffer (0.5X SSC, 0.1% SDS) 2. Wash nitrocellulose membrane blocking solution 30, blocking solution streptavidin-ap conjugate 75 mu/ml (1:5000) solution membrane 30, 15 wash buffer 2. Membrane detection buffer 10 BCIP/NBT substrate solution 30. probe, Dot-blot.. RT-PCR rvlp., 500 ~ 1,000 L 1 MDS (Cuno) 1.5%
20 Beef extract. 20 ~ 30 ml membrane 140 Qiagen viral RNA mini kit viral RNA 80 Viral RNA 70., stool stool 50 mg 1X PBS vortex micro centrifuge 10,000 rpm Qiagen viral RNA mini kit viral RNA 80 Viral RNA 70. RNA 10 0 ~ RT-PCR Real-time PCR. RT-PCR reverse transcriptase reverse transcriptase , 55 30, cycle final extension 72 5 (Fig 8). Fig 8. Comparison of sensitivity to detect of Norovirus RNA by RT-PCR and Real time PCR test ELISA probe Dot blot hybridization.
21 3. rvlp Hybridization rvlp rvlp probe Hybridization
22 15% 5% 19% rvlp probe probe probe 19% 19% 19% 19% 19% Dot blot 19% Dot blot 19%
23 . 1. rvlp. reporter gene EGFP (Enhanced Green Fluorescence Protein) (2013 ) 4 4, EGFP. reporter gene EGFP. ( ), 4 4. reporter gene EGFP (Fig 9,10). Fig 9. BEVS-EGFP expression system EGFP PCR western blot (Fig 10).
24 Fig 10. Confirmation of the BEVS-EGFP expression system by EGFP. VP1, VP2 region epitope (2013 ) Caliciviridae 7.6 kb single stranded RNA 3 ORF. 5 genogroup (G ~G ). Human capsid RNA polymerase 2 genogroup G and G G 14 G 17 genotypes. ORF2 capsid. VP1 S-domain P-domain P-domain P1 P2 sub-domain. P2 domain strain conserved region. P2-domain receptor.
25 Fig 11. Sequences of Norovirus VP1 and VP2 regions VP1, VP2 region (Fig 11). VP1 (ORF2), VP2 (ORF3), VP1 + VP2 shuttle vector. shuttle vector clone (Fig 12).
26 Fig 12. Verification of VP1 and VP2 gene in shuttle vector by PCR 1) VP1 (ORF2) 2) VP2 (ORF3) 3) VP1 + VP2 (1) Recombination Shuttle vector entry clone recombinase DNA (Fig 13). Fig 13. Schematic diagram for recombinant Baculovirus propagation containing Norovirus VP1 and VP2 genes
27 (2) 1 st Passage Recombinant DNA Sf9 Sf21 Liposome. wild-type (gangcyclovir). (3) 2 nd Passage Recombinant 1 st passage recombinant PCR. 1 st passage recombinant 4 70 stock, Sf9 2 nd passage recombinant. (4) rvlp rvlp. 2 nd passage. C- 6 His rvlp 6x-His Western Blotting. 3 (VP1, VP2 VP1+VP2) rvlp (Fig 14).
28 Fig 14. Schematic diagram for rvlp propagation using recombinant Baculovirus expression system and confirmation of VP1 and VP2 proteins expression by Western Blotting method VP1, VP2 region capside protein.. Major capside protein coding VP1(ORF2), rvlp (2014 ) VP1, VP2 region major capside protein coding VP1(ORF2) rvlp rvlp. VP1(ORF2) capside protein major protein coding
29 ORF2 insect cell plant cell self-assembly rvlp. (1) rvlp Sf9 recombinant rvlp. 5~7 cell cell rvlp. VP1(ORF2) C- 6 His rvlp 6x-His Western Blotting. cell lysis supernatant cell pellet Western Blotting ORF2 protein size supernatant pellet Human rvlp (Fig 15). Fig 15. Expression of ORF2, verification by Western Blotting (2) rvlp size exclusion chromatography. FPLC cell lysate supernatant column(ge Healthcare, HiPrep26/60 Sephacryl S-400 High Resolution) loading. rvlp 180 molecules 90 dimer cell protein elution elution 1 2 fraction
30 rvlp. fraction coomassie blue staining Western Blotting (Fig 16). Fig 16. Verification by Coomassie blue staining and Western Blotting (3) rvlp. 1 2 fraction rvlp fraction elution solution Millipore Centricon,. rvlp coomassie blue staining Western Blotting (Fig 17), BCA Protein Assay(Thermo #23227) BSA gel coomassie blue staining (Data not shown). Fig 17. Verification of concentrated rvlp by coomassie blue staining and Western Blotting
31 (4), rvlp (AB frontier). Mouse BALB/C Female, 6 3 mouse polyclonal antibody. 3 1 st Boosting, 5 1 st Bleeding ELISA test 6 2 nd Boodting 8 2 nd Bleeding ELISA test titer 9 Heart Puncture mouse antisera (Table 1). ELISA test indirect ELISA serum 1:100 1:100,000, O.D. 1 st Bleeding ELISA test 250 ng mouse antiserum titer mouse 3 2 O.D 1: , ELISA titer (Table 2) (Fig 18). 2 nd Bleeding ELISA test 250 ng mouse antiserum titer mouse 3 1: , ELISA titer (Table 3) (Fig 19). Time Week 0 Description Injection Week 3 1 st Boosting Week 5 1 st Bleeding ELISA Week 6 2 nd Boosting Week 8 2 nd Bleeding ELISA Week 9 Week 10 Heart Puncture Sample Table 1. Immunization schedule
32 ELISA test 1 st Bleeding Antibody coating 250 ng/well Measurement filter 450 nm Mouse IgG HRP 1:10,000dilution TMB 5 min Mouse Serum dilution factor PBS # # # : :1, :5, :10, :50, :100, Table 2. 1 st Bleeding ELISA test Fig st Bleeding ELISA test
33 ELISA test 2 nd Bleeding Antibody coating 250 ng/well Measurement filter 450 nm Mouse IgG HRP 1:10,000dilution TMB 5 min Mouse #1 Serum #2 #3 dilution factor PBS : :1, :5, :10, :50, :100, Table 3. 2 nd Bleeding ELISA test Fig nd Bleeding ELISA test
34 (5) test ELISA. rvlp 1 ng/ml 0.1 pg/ml serial dilution ELISA plate 4 over night incubation. Blocking step PBS 5% non fat milk 3. antibody 1:1000 PBS 2% non fat milk coating rvlp 1. secondary antibody anti mouse IgG labeled HRP (abcam #ab97051) 1:10,000 PBS 2% non fat milk 1. ELISA TMB substrate solution ~15 stop solution. Absorbance 450. rvlp ELISA (Fig 20, a) (Fig 20, b). 0.1 ng/ml negtive (blank). Fig 20. Anti rvlp antibody sensitivity test
35 (6) RT-PCR G type (Fig 21, a). RT-PCR DNA band sequencing blast G type 10 (Fig 21, b). Fig 21. Human Norovirus detection by RT-PCR (7) RT-PCR G type 10 ELISA 2 (Fig 22). ELISA test coating buffer resuspension ~ 1.3 negative(blank) (Fig 22, a) (Fig 22, b) DENKA QuickNavi-Norovirus 50% (8,29,37,38,47 ) (Fig 23, a) SD SD Norovirus 50% (8,29,37,38,47 ) (Fig 23, b).
36 Fig 22. Anti rvlp antibody ELISA test with human Norovirus stool sample
37 Fig 23. Commercial Norovirus detection kit test
38 2. (hybridization) probe. Probe Probe NCBI, DDBI, EMBL GenBank Genogroup (G -1~8), Genogroup (G -1~17). (7.6Kb) VP1 region(conserved region) target specific (Fig 24, 25). Fig 24. Norovirus G sequence analysis using Meg-align program
39 Fig 25. Norovirus G sequence analysis using Meg-align program Specific probe DNAStar Meg-alingn program Oligo6 program melting temperature, secondary structure (Fig 26), GC content, GC clamp, 3 end stablity, cross homology probe (Table 4). Genogroup 4 probe Cosmo Genetech 5 end biotin modification. probe probe 8,, Dot-blot.
40 Fig 26. Probe design using Oligo 6 program Probe Sequence(5-3 ) Size,GC,Tem GIBiotin.1 GIBiotin.2 GIBiotin.3 GIBiotin.4 GIIBiotin.1 GIIBiotin.2 GIIBiotin.3 GIIBiotin.4 GGA CAG GAG ATC GCA ATC TCC TGC CCG AAT TYG TAA ATG ATG ATG GCG TCT AAG GAC GGC AGG CCA TGT TCC GCT GGA TGC GNT TCC ATG AYC TBG GDY TDT GGA CAG GRG A GGK GAT GAT GAR ATH GTN TCA ACT GAY ATA GAN TTT GAC CCA NCH MGV TTV ACM CAR RT CCH TCY GAR ATG GAY GTK GGB GAC TAY RTN ATW AGR GTB AAR GAA GGN CTH CCC TCA GG GTG AAT GAA GAT GGC GTC GAR TGA CGC YRM YCC ATC KRM TGR KKS YGC HGC C CCC ATC TGA TGG GTC CRC AGC CAA CCT CGT CCC AGA GGT CAA CAA TGA GGT TAT GGC CTA TGT TCC CCC AYA TAA TAG TRG ATG TTA GGC AAY TGG AAC CTG TGT TGA TCC CCT TAC C GGT TAT GCA GGT GGT TTT GAA GTG CAG GTR ATY CTC GCG GGG AAC GCG TTC ACC G Table 4. Norovirus G, G Probe design 57mer,50%,74 55mer,55%,79 59mer,38%,75 59mer,45%,79 55mer,50%,77 57mer,56, mer,45%,73 55mer,56%,76
41 (1) ( ), genogroup cross reaction Dot-blot. G specific 4 probe G spot G Negative spot. G specific 4 probe G spot G Negative spot probe (Fig 27). Fig 27. G, G probe specificity test (2) ( ) Plasmid DNA Dot-blot. probe 0.5 probe 1, probe 3,4 probe (Fig 28).
42 Fig 28. G, G probe sensitivity test
43 . hybridization probe hybridization G, G sample Dot hybridization. probe G Biotin1 G Biotin1. RNA preparation G ng/ul, G ng/ul serial dilution. G 10-2 G 10-3 Dot hybridization (Fig 29). Dot blot. Fig 29. Human norovirus G, G hybridization test with designed probe
44 . 1. rvlp...,. CDC 50% (ELISA ICT). RT-PCR. (1) EGFP - EGFP reporter gene. - DNA EGFP. rvlp. -,.. -
45 EGFP. (2) capside rvlp - capsid epitope. - Human capsid ORF2(VP1) capsid. capsid (immunogenicity) (infectivity). - capsid ORF2(VP1) rvlp genotype. (3) rvlp rvlp - VP1 region -. - Size chromatography. (4) - rvlp. - ELISA test test RT-PCR ELISA test %.
46 2. (hybridization) probe (1) genotype probe - Probe NCBI, DDBI, EMBL GenBank Genogroup (G -1~8), Genogroup (G -1~17). (7.6Kb) VP1 region(conserved region) target specific. - Hybridization membrane spotting probe molecules target molecule molecule, post-hybridization(washing) probe, hybridization Bioin avidin. Biotin probe labelling detection avidin-enzyme complex. (2) Hybridization dot blot - Genogroup, biotin-probe, dot-blot, probe. - probe spot. - G Biotin1, G Biotin2, G Biotin3 0.1 DNA, G Biotin DNA. G G Biotin1, G Biotin2, G Biotin3 0.1, G Biotin4 0.5 DNA. - probe hybridization G, G RNA preparation Dot hybridization. - Dot blot genogroup,.
47 3. ELISA test.. sensitivity specificity probe hybridization G, G RNA Dot hybridization. Dot blot, genogroup,.
48 .
49 1. M e d ic i, M.C., e t a l. N o v e l re c o m b in a n t G II.P 1 6 _ G II.1 3 a n d G II.P 1 6 _ G II.3 n o ro v iru s stra in s in Ita ly. V iru s re se a rc h 1 8 8, ( ). 2. W o n, Y.J., e t a l. F u ll-g e n o m ic a n a ly s is o f a h u m a n n o ro v iru s re c o m b in a n t G II.1 2 / 1 3 n o v e l s tra in iso la te d fro m S o u th K o re a. P lo S o n e 8, e ( ). 3. A d le r, J.L. & Z ic k l, R. W in te r v o m itin g d is e a s e. T h e Jo u rn a l o f in fe c tio u s d ise a s e s 1 1 9, ( ). 4. D e b b in k, K., L in d e sm ith, L.C. & B a ric, R.S. T h e s ta te o f n o ro v iru s v a c c in e s. C lin ic a l in fe c tio u s d is e a se s : a n o ffic ia l p u b lic a tio n o f th e In fe c tio u s D is e a s e s S o c ie ty o f A m e ric a 5 8, ( ). 5. A tm a r, R.L., e t a l. N o ro v iru s v a c c in e a g a in s t e x p e rim e n ta l h u m a n N o rw a lk V iru s illn e s s. T h e N e w E n g la n d jo u rn a l o f m e d ic in e 3 6 5, ( ). 6. W h ite, P.A. E v o lu tio n o f n o ro v iru s. C lin ic a l m ic ro b io lo g y a n d in fe c tio n : th e o ffic ia l p u b lic a tio n o f th e E u ro p e a n S o c ie ty o f C lin ic a l M ic ro b io lo g y a n d In fe c tio u s D ise a s e s 2 0, ( ). 7. M e sq u ita, J.R., B a rc la y, L., N a sc im e n to, M.S. & V in je, J. N o v e l n o ro v iru s in d o g s w ith d ia rrh e a. E m e rg in g in fe c tio u s d is e a s e s 1 6, ( ). 8. T h o rn e, L.G. & G o o d fe llo w, I.G. N o ro v iru s g e n e e x p re s s io n a n d re p lic a tio n. T h e Jo u rn a l o f g e n e ra l v iro lo g y 9 5, ( ). 9. H a rd y, M.E. N o ro v iru s p ro te in s tru c tu re a n d fu n c tio n. F E M S m ic ro b io lo g y le tte rs 2 5 3, 1-8 ( ) F a n k h a u s e r, R.L., e t a l. E p id e m io lo g ic a n d m o le c u la r tre n d s o f "N o rw a lk -lik e v iru se s " a s s o c ia te d w ith o u tb re a k s o f g a s tro e n te ritis in th e U n ite d S ta te s. T h e Jo u rn a l o f in fe c tio u s d ise a s e s 1 8 6, 1-7 ( ) T u a n Z a in a z o r, C., e t a l. T h e s c e n a rio o f n o ro v iru s c o n ta m in a tio n in fo o d a n d fo o d h a n d le rs. Jo u rn a l o f m ic ro b io lo g y a n d b io te c h n o lo g y 2 0, ( ) W id d o w s o n, M.A., e t a l. N o ro v iru s a n d fo o d b o rn e d ise a s e, U n ite d S ta te s, E m e rg in g in fe c tio u s d is e a se s 1 1, ( ) L e n n o n, G., e t a l. A c o m p a riso n o f th e e ffic ie n c y o f E L IS A a n d se le c te d p rim e r s e ts to d e te c t N o ro v iru s is o la te s in so u th e rn Ire la n d o v e r a fo u r- y e a r p e rio d ( ): v a ria tio n in d e te c tio n ra te s a n d e v id e n c e fo r c o n tin u in g p re d o m in a n c e o f N o V G II.4 g e n o ty p e. A rc h iv e s o f v iro lo g y 1 5 9, ( ) H a n, T.H., K im, C.H., C h u n g, J.Y., P a rk, S.H. & H w a n g, E.S. E m e rg e n c e o f n o ro v iru s G II-4 / v a ria n t a n d re c o m b in a n t s tra in s in S e o u l, K o re a. A rc h iv e s o f v iro lo g y 1 5 6, ( ) L a R o s a, G., P o u rs h a b a n, M., Ia c o n e lli, M. & M u s c illo, M. D e te c tio n o f g e n o g ro u p IV n o ro v iru s e s in e n v iro n m e n ta l a n d c lin ic a l sa m p le s a n d p a rtia l se q u e n c in g th ro u g h ra p id a m p lific a tio n o f c D N A e n d s. A rc h iv e s o f v iro lo g y 1 5 3, ( ).
50 1 6. L a R o s a, G., P o u rs h a b a n, M., Ia c o n e lli, M., V e n n a ru c c i, V.S. & M u sc illo, M. M o le c u la r d e te c tio n o f h e p a titis E v iru s in se w a g e s a m p le s. A p p lie d a n d e n v iro n m e n ta l m ic ro b io lo g y 7 6, ( ) v a n O e rs, M.M. O p p o rtu n itie s a n d c h a lle n g e s fo r th e b a c u lo v iru s e x p re s sio n s y s te m. Jo u rn a l o f in v e rte b ra te p a th o lo g y S u p p l, S ( ) L iu, F., W u, X., L i, L., L iu, Z. & W a n g, Z. U s e o f b a c u lo v iru s e x p re s sio n s y s te m fo r g e n e ra tio n o f v iru s - lik e p a rtic le s: s u c c e ss e s a n d c h a lle n g e s. P ro te in e x p re ss io n a n d p u rific a tio n 9 0, ( ) 김현수, 김경희, 권혜원, 강태영, 허미나, 김한성, 김재석, 송원근, 강희정, 이규만. 노로바이러스감염진단을위한신속항원검사법의평가 : 효소면역검사법 (E L IS A ) 및실시간정량역전사중합효소연쇄반응검사법과의비교. L a b M e d O n lin e ; V o l. 1, N o. 4 : 이나리. 노로바이러스와식중독. 한국식품연구원식품안전성연구본부식품위해평가팀. S a fe F o o d ; 2 : 식품의약품안정청. 노로바이러스식중독예방을위한지침서
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'' - 1 - - 2 - - 3 - - 4 - - 5 - ' - 6 - - 7 - - 8 - - 9 - - 10 - - 11 - Super-Bio Co., Ltd. KWON, Suk-Tae Thermostable DNA Polymerase-encoding Gene from Thermus sp. X-1 an d Amino Acid Sequence
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