WO2013031956A1 - 遺伝子型3aのC型肝炎ウイルスゲノム由来の核酸を含む核酸構築物 - Google Patents
遺伝子型3aのC型肝炎ウイルスゲノム由来の核酸を含む核酸構築物 Download PDFInfo
- Publication number
- WO2013031956A1 WO2013031956A1 PCT/JP2012/072179 JP2012072179W WO2013031956A1 WO 2013031956 A1 WO2013031956 A1 WO 2013031956A1 JP 2012072179 W JP2012072179 W JP 2012072179W WO 2013031956 A1 WO2013031956 A1 WO 2013031956A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- hcv
- seq
- base
- amino acid
- Prior art date
Links
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 179
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 179
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 179
- 101800001554 RNA-directed RNA polymerase Proteins 0.000 claims abstract description 60
- 101710144111 Non-structural protein 3 Proteins 0.000 claims abstract description 52
- 101800001014 Non-structural protein 5A Proteins 0.000 claims abstract description 45
- 101800001020 Non-structural protein 4A Proteins 0.000 claims abstract description 41
- 101800001019 Non-structural protein 4B Proteins 0.000 claims abstract description 41
- 241000711549 Hepacivirus C Species 0.000 claims description 668
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 412
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 193
- 108090000623 proteins and genes Proteins 0.000 claims description 162
- 230000035772 mutation Effects 0.000 claims description 140
- 239000002245 particle Substances 0.000 claims description 93
- 235000001014 amino acid Nutrition 0.000 claims description 92
- 238000006467 substitution reaction Methods 0.000 claims description 88
- 235000018102 proteins Nutrition 0.000 claims description 79
- 102000004169 proteins and genes Human genes 0.000 claims description 79
- 238000000034 method Methods 0.000 claims description 71
- 150000001413 amino acids Chemical group 0.000 claims description 70
- 101710118188 DNA-binding protein HU-alpha Proteins 0.000 claims description 68
- 101710144128 Non-structural protein 2 Proteins 0.000 claims description 68
- 101710199667 Nuclear export protein Proteins 0.000 claims description 68
- 229940024606 amino acid Drugs 0.000 claims description 50
- 101710132601 Capsid protein Proteins 0.000 claims description 49
- 108091026898 Leader sequence (mRNA) Proteins 0.000 claims description 47
- 239000002773 nucleotide Substances 0.000 claims description 43
- 125000003729 nucleotide group Chemical group 0.000 claims description 43
- 101710125507 Integrase/recombinase Proteins 0.000 claims description 40
- 101710185720 Putative ethidium bromide resistance protein Proteins 0.000 claims description 39
- 239000000126 substance Substances 0.000 claims description 30
- 108091036066 Three prime untranslated region Proteins 0.000 claims description 28
- 229960005486 vaccine Drugs 0.000 claims description 27
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 25
- 239000004475 Arginine Substances 0.000 claims description 24
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 24
- 229960000310 isoleucine Drugs 0.000 claims description 23
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 23
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 22
- 239000004473 Threonine Substances 0.000 claims description 22
- 230000000694 effects Effects 0.000 claims description 21
- 238000012360 testing method Methods 0.000 claims description 21
- 241000700605 Viruses Species 0.000 claims description 17
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 16
- 238000007792 addition Methods 0.000 claims description 15
- 238000012217 deletion Methods 0.000 claims description 15
- 241001493065 dsRNA viruses Species 0.000 claims description 15
- 230000037430 deletion Effects 0.000 claims description 14
- 208000005176 Hepatitis C Diseases 0.000 claims description 10
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 10
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 10
- 239000004472 Lysine Substances 0.000 claims description 9
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 9
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 9
- 230000003612 virological effect Effects 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 7
- 239000000427 antigen Substances 0.000 claims description 7
- 108091007433 antigens Proteins 0.000 claims description 7
- 102000036639 antigens Human genes 0.000 claims description 7
- 239000004471 Glycine Substances 0.000 claims description 6
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 6
- 235000004279 alanine Nutrition 0.000 claims description 6
- 238000011002 quantification Methods 0.000 claims description 6
- 229940113082 thymine Drugs 0.000 claims description 5
- 229940035893 uracil Drugs 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 113
- 210000004027 cell Anatomy 0.000 description 150
- 108091026890 Coding region Proteins 0.000 description 123
- 230000010076 replication Effects 0.000 description 88
- 239000000047 product Substances 0.000 description 83
- 108020004414 DNA Proteins 0.000 description 79
- 239000013604 expression vector Substances 0.000 description 55
- 239000002299 complementary DNA Substances 0.000 description 54
- 239000012634 fragment Substances 0.000 description 43
- 210000004748 cultured cell Anatomy 0.000 description 27
- 108020003589 5' Untranslated Regions Proteins 0.000 description 26
- 108020005345 3' Untranslated Regions Proteins 0.000 description 25
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 24
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 24
- 108091008146 restriction endonucleases Proteins 0.000 description 20
- 208000015181 infectious disease Diseases 0.000 description 19
- 206010059866 Drug resistance Diseases 0.000 description 18
- 108700008625 Reporter Genes Proteins 0.000 description 17
- 230000003044 adaptive effect Effects 0.000 description 17
- 239000012228 culture supernatant Substances 0.000 description 17
- 108060001084 Luciferase Proteins 0.000 description 15
- 239000002243 precursor Substances 0.000 description 15
- 239000003814 drug Substances 0.000 description 14
- 230000037429 base substitution Effects 0.000 description 13
- 229940079593 drug Drugs 0.000 description 13
- 238000004520 electroporation Methods 0.000 description 13
- 241000710188 Encephalomyocarditis virus Species 0.000 description 12
- 229930193140 Neomycin Natural products 0.000 description 12
- 229960004927 neomycin Drugs 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 239000002671 adjuvant Substances 0.000 description 11
- 108020004705 Codon Proteins 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 238000000246 agarose gel electrophoresis Methods 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 8
- 108700039791 Hepatitis C virus nucleocapsid Proteins 0.000 description 8
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 238000003259 recombinant expression Methods 0.000 description 8
- 230000003362 replicative effect Effects 0.000 description 8
- 101800001838 Serine protease/helicase NS3 Proteins 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 7
- 238000007796 conventional method Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 230000002458 infectious effect Effects 0.000 description 7
- 230000007246 mechanism Effects 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 102000006992 Interferon-alpha Human genes 0.000 description 6
- 108010047761 Interferon-alpha Proteins 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- 230000005757 colony formation Effects 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 6
- 238000003757 reverse transcription PCR Methods 0.000 description 6
- GWCTUYUNVJTNJR-UHFFFAOYSA-N 1-cyclohexyl-2-(4-phenylmethoxyphenyl)benzimidazole-5-carboxylic acid Chemical compound C=1C=C(OCC=2C=CC=CC=2)C=CC=1C1=NC2=CC(C(=O)O)=CC=C2N1C1CCCCC1 GWCTUYUNVJTNJR-UHFFFAOYSA-N 0.000 description 5
- 108010086093 Mung Bean Nuclease Proteins 0.000 description 5
- 108010067390 Viral Proteins Proteins 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 230000001332 colony forming effect Effects 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 4
- NYPIRLYMDJMKGW-VPCXQMTMSA-N 4-amino-1-[(2r,3r,4r,5r)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyloxolan-2-yl]pyrimidin-2-one Chemical compound C[C@@]1(F)[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C=C1 NYPIRLYMDJMKGW-VPCXQMTMSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 238000012300 Sequence Analysis Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000001638 lipofection Methods 0.000 description 4
- 238000004020 luminiscence type Methods 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 108700026220 vif Genes Proteins 0.000 description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- 206010065051 Acute hepatitis C Diseases 0.000 description 3
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 3
- 102000014150 Interferons Human genes 0.000 description 3
- 108010050904 Interferons Proteins 0.000 description 3
- 101150095629 NS2 gene Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 229940123066 Polymerase inhibitor Drugs 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 3
- 230000006819 RNA synthesis Effects 0.000 description 3
- 101710172711 Structural protein Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 208000037621 acute hepatitis C virus infection Diseases 0.000 description 3
- NWMHDZMRVUOQGL-CZEIJOLGSA-N almurtide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)CO[C@@H]([C@H](O)[C@H](O)CO)[C@@H](NC(C)=O)C=O NWMHDZMRVUOQGL-CZEIJOLGSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- PJZPDFUUXKKDNB-KNINVFKUSA-N ciluprevir Chemical compound N([C@@H]1C(=O)N2[C@H](C(N[C@@]3(C[C@H]3\C=C/CCCCC1)C(O)=O)=O)C[C@H](C2)OC=1C2=CC=C(C=C2N=C(C=1)C=1N=C(NC(C)C)SC=1)OC)C(=O)OC1CCCC1 PJZPDFUUXKKDNB-KNINVFKUSA-N 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 229940079322 interferon Drugs 0.000 description 3
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 239000000829 suppository Substances 0.000 description 3
- 229960002935 telaprevir Drugs 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 2
- 235000002198 Annona diversifolia Nutrition 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 241000282828 Camelus bactrianus Species 0.000 description 2
- 241000282836 Camelus dromedarius Species 0.000 description 2
- 102000009016 Cholera Toxin Human genes 0.000 description 2
- 108010049048 Cholera Toxin Proteins 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108090000331 Firefly luciferases Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108700015872 N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108010076039 Polyproteins Proteins 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 102100038313 Transcription factor E2-alpha Human genes 0.000 description 2
- 229930003427 Vitamin E Natural products 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 210000000628 antibody-producing cell Anatomy 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
- 150000001483 arginine derivatives Chemical group 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 238000000975 co-precipitation Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000000415 inactivating effect Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 238000013081 phylogenetic analysis Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 229930182490 saponin Natural products 0.000 description 2
- 150000007949 saponins Chemical class 0.000 description 2
- 235000017709 saponins Nutrition 0.000 description 2
- 239000006152 selective media Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- 235000019165 vitamin E Nutrition 0.000 description 2
- 229940046009 vitamin E Drugs 0.000 description 2
- 239000011709 vitamin E Substances 0.000 description 2
- CXNPLSGKWMLZPZ-GIFSMMMISA-N (2r,3r,6s)-3-[[(3s)-3-amino-5-[carbamimidoyl(methyl)amino]pentanoyl]amino]-6-(4-amino-2-oxopyrimidin-1-yl)-3,6-dihydro-2h-pyran-2-carboxylic acid Chemical compound O1[C@@H](C(O)=O)[C@H](NC(=O)C[C@@H](N)CCN(C)C(N)=N)C=C[C@H]1N1C(=O)N=C(N)C=C1 CXNPLSGKWMLZPZ-GIFSMMMISA-N 0.000 description 1
- YHQZWWDVLJPRIF-JLHRHDQISA-N (4R)-4-[[(2S,3R)-2-[acetyl-[(3R,4R,5S,6R)-3-amino-4-[(1R)-1-carboxyethoxy]-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]amino]-3-hydroxybutanoyl]amino]-5-amino-5-oxopentanoic acid Chemical compound C(C)(=O)N([C@@H]([C@H](O)C)C(=O)N[C@H](CCC(=O)O)C(N)=O)C1[C@H](N)[C@@H](O[C@@H](C(=O)O)C)[C@H](O)[C@H](O1)CO YHQZWWDVLJPRIF-JLHRHDQISA-N 0.000 description 1
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 1
- GUWSQVZFXHIGLN-UHFFFAOYSA-M 1-(4-amino-2-methylpyrimidin-5-ylmethyl)-3-(2-hydroxyethyl)-2-methylpyridinium bromide Chemical compound [Br-].NC1=NC(C)=NC=C1C[N+]1=CC=CC(CCO)=C1C GUWSQVZFXHIGLN-UHFFFAOYSA-M 0.000 description 1
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 description 1
- 108010000239 Aequorin Proteins 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 102100024321 Alkaline phosphatase, placental type Human genes 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 108010039224 Amidophosphoribosyltransferase Proteins 0.000 description 1
- 241000272517 Anseriformes Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 101150045282 CD81 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 108010039939 Cell Wall Skeleton Proteins 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- 108090000600 Claudin-1 Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000710781 Flaviviridae Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000282838 Lama Species 0.000 description 1
- 241000282842 Lama glama Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108700020354 N-acetylmuramyl-threonyl-isoglutamine Proteins 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 241001045988 Neogene Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010010677 Phosphodiesterase I Proteins 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- 241000242583 Scyphozoa Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 241000271567 Struthioniformes Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 1
- 241000839309 Thesea Species 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 108700022715 Viral Proteases Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- VEZXCJBBBCKRPI-UHFFFAOYSA-N beta-propiolactone Chemical compound O=C1CCO1 VEZXCJBBBCKRPI-UHFFFAOYSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- CXNPLSGKWMLZPZ-UHFFFAOYSA-N blasticidin-S Natural products O1C(C(O)=O)C(NC(=O)CC(N)CCN(C)C(N)=N)C=CC1N1C(=O)N=C(N)C=C1 CXNPLSGKWMLZPZ-UHFFFAOYSA-N 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000004520 cell wall skeleton Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- QTTMOCOWZLSYSV-QWAPEVOJSA-M equilin sodium sulfate Chemical compound [Na+].[O-]S(=O)(=O)OC1=CC=C2[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4C3=CCC2=C1 QTTMOCOWZLSYSV-QWAPEVOJSA-M 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000003505 heat denaturation Methods 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229960001388 interferon-beta Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- JMUHBNWAORSSBD-WKYWBUFDSA-N mifamurtide Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCCCCCCCCCC)COP(O)(=O)OCCNC(=O)[C@H](C)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)OC(O)[C@@H]1NC(C)=O JMUHBNWAORSSBD-WKYWBUFDSA-N 0.000 description 1
- 229960005225 mifamurtide Drugs 0.000 description 1
- 108700007621 mifamurtide Proteins 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 101150091879 neo gene Proteins 0.000 description 1
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 108010031345 placental alkaline phosphatase Proteins 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001515 polyalkylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000955 prescription drug Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 229960000380 propiolactone Drugs 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000020978 protein processing Effects 0.000 description 1
- 150000003834 purine nucleoside derivatives Chemical class 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
- 229960000329 ribavirin Drugs 0.000 description 1
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 150000003355 serines Chemical group 0.000 description 1
- 229960002091 simeprevir Drugs 0.000 description 1
- JTZZSQYMACOLNN-VDWJNHBNSA-N simeprevir Chemical compound O=C([C@@]12C[C@H]1\C=C/CCCCN(C)C(=O)[C@H]1[C@H](C(N2)=O)C[C@H](C1)OC=1C2=CC=C(C(=C2N=C(C=1)C=1SC=C(N=1)C(C)C)C)OC)NS(=O)(=O)C1CC1 JTZZSQYMACOLNN-VDWJNHBNSA-N 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 229940031439 squalene Drugs 0.000 description 1
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000019635 sulfation Effects 0.000 description 1
- 238000005670 sulfation reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940021747 therapeutic vaccine Drugs 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- XETCRXVKJHBPMK-MJSODCSWSA-N trehalose 6,6'-dimycolate Chemical compound C([C@@H]1[C@H]([C@H](O)[C@@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](COC(=O)C(CCCCCCCCCCC3C(C3)CCCCCCCCCCCCCCCCCC)C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)O2)O)O1)O)OC(=O)C(C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)CCCCCCCCCCC1CC1CCCCCCCCCCCCCCCCCC XETCRXVKJHBPMK-MJSODCSWSA-N 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/186—Hepatitis C; Hepatitis NANB
Definitions
- the present invention relates to a nucleic acid derived from the genotype 3a hepatitis C virus genome, a nucleic acid construct containing the nucleic acid, and a method for screening an anti-hepatitis C virus substance.
- HCV hepatitis C virus
- HCV hepatitis C virus
- development research of anti-HCV drugs Specifically, a system for amplifying HCV in cultured cells and a system for evaluating the proliferation of HCV in cultured cells are necessary, and if these systems can be constructed, the above research is considered to progress dramatically.
- HCV is a virus whose genome is a single-stranded (+) strand sense RNA belonging to the Flaviviridae family, and is known to cause hepatitis C. HCV is classified into a number of types according to genotype or serotype, and according to the phylogenetic analysis method by Simmonds et al. Using the base sequence of the HCV strain, the HCV genotype is classified into genotypes 1 to 6, and They are classified into subtypes (Non-Patent Document 1). At present, the full-length genome sequences of a plurality of HCV genotypes have been determined (Non-Patent Documents 2 to 5).
- Non-Patent Documents 6 and 7 are characterized by a large proportion of genotype 3 HCV-infected patients.
- the HCV subgenomic replicon RNA is an RNA that includes a part of the HCV genome, and has no ability to produce infectious HCV particles, but is an RNA that can autonomously replicate RNA derived from the HCV genome when introduced into cultured cells. That is.
- the full genome replicon RNA of HCV is the RNA containing the full length of the HCV genome, that is, the 5 ′ untranslated region, the structural gene, the nonstructural gene, and the 3 ′ untranslated region. It is RNA that can autonomously replicate genome-derived RNA.
- RNA having the ability to produce infectious HCV particles in an in vitro system using cultured cells is RNA derived from the JFH-1 strain of genotype 2a, in order to obtain a raw material for HCV vaccine.
- the ability to prepare a large amount of HCV particles in an in vitro system is limited to HCV of JFH-1 strain or full genome replicon derived from JFH-1 strain.
- Main treatment for hepatitis C is performed by monotherapy of interferon- ⁇ and interferon- ⁇ and combination therapy of interferon- ⁇ and purine-nucleoside derivative ribavirin.
- the therapeutic effect of interferon is related to the HCV genotype, and is known to be less effective for genotype 1b and more effective for genotypes 2a and 3a (Non-Patent Literature). 14). The reason why the therapeutic effect of interferon differs depending on the genotype of HCV has not yet been clarified, but one of the causes is thought to be due to differences in the replication mechanism and replication efficiency of HCV.
- HCV subgenomic replicon RNAs are limited to several types derived from HCV strains of genotypes 1a, 1b and 2a, and for full genome replicon RNAs that produce infectious HCV particles, Only those derived from a genome consisting of a chimera of a structural gene derived from the genome of the JFH-1 strain of genotype 2a or a non-structural gene derived from a strain different from the JFH-1 strain. is there. Therefore, it is difficult to elucidate the relationship between HCV genotype and HCV replication mechanism or replication efficiency, and the types of HCV particles that can be artificially prepared as raw materials for HCV vaccines are also limited to genotype 2a. It is the current situation.
- an object of the present invention is to provide a replicon RNA having an autonomous replication ability derived from a novel genotype 3a HCV strain and a novel genotype 3a HCV strain.
- the present invention includes the following.
- the amino acid sequence of the NS4A protein of amino acid numbers 1664 to 1717, the amino acid sequence of the NS4B protein of amino acid numbers 1718 to 1978, the amino acid sequence of the NS5A protein of amino acid numbers 1979 to 2430, and the amino acid sequence of the NS5B protein of amino acid numbers 2431 to 3021 A nucleic acid comprising a coding base sequence and a 3 ′ untranslated region comprising the base sequence of nucleotide numbers 9407 to 9655 of SEQ ID NO: 1 in this order (however, if the nucleic acid is RNA, thymine ( t) is read as uracil (u)).
- the base sequence encoding the amino acid sequence of the NS3 protein includes the base sequence of base numbers 3437 to 5329 of SEQ ID NO: 1
- the base sequence encoding the amino acid sequence of the NS4A protein includes the base sequence of base numbers 5330 to 5491 of SEQ ID NO: 1
- the base sequence encoding the amino acid sequence of the NS4B protein includes the base sequence of base numbers 5492 to 6274 of SEQ ID NO: 1
- the base sequence encoding the amino acid sequence of the NS5A protein includes the base sequence of base numbers 6275 to 7630 of SEQ ID NO: 1
- the base sequence encoding the amino acid sequence of the NS5B protein comprises the base sequence of base numbers 7631 to 9406 of SEQ ID NO: 1, The nucleic acid according to [1] above.
- the base sequence encoding the Core protein, the base sequence encoding the E1 protein, the base sequence encoding the E2 protein, the base sequence encoding the p7 protein, and the base sequence encoding the NS2 protein of the hepatitis C virus genome The nucleic acid according to any one of [1] to [4] above, further comprising:
- the base sequence encoding the Core protein is a base sequence encoding the amino acid sequence of the Core protein of amino acid numbers 1 to 191 of SEQ ID NO: 14,
- the base sequence encoding the E1 protein is a base sequence encoding the amino acid sequence of the E1 protein of amino acid numbers 192 to 383 of SEQ ID NO: 14,
- the base sequence encoding the E2 protein is a base sequence encoding the amino acid sequence of the E2 protein of amino acid numbers 384 to 752 of SEQ ID NO: 14
- the base sequence encoding the p7 protein is a base sequence encoding the amino acid sequence of the p7 protein of amino acid numbers 753 to 815 of SEQ ID NO: 14
- the base sequence encoding NS2 protein is a base sequence encoding the amino acid sequence of NS2 protein of amino acid numbers 816 to 1032 of SEQ ID NO: 14.
- nucleic acids described in [1] to [8] above may further contain a foreign gene and / or an IRES sequence.
- a hepatitis C virus subgenomic replicon RNA comprising the nucleic acid according to any one of [1] to [4] and [8] above.
- a hepatitis C virus full-genome replicon RNA comprising the nucleic acid according to any one of [5] to [7] above.
- nucleic acids described in [1] to [8] above may be DNA.
- An expression vector containing the nucleic acid described in [1] to [8] above, particularly such DNA is also a preferred embodiment of the present invention.
- the cells according to [12] above or the mixture of hepatitis C virus particles and hepatitis C virus sensitive cells according to [11] above are cultured both in the presence and absence of a test substance.
- the amounts of subgenomic replicon RNA, full-genome replicon RNA or hepatitis C virus particles quantified in the culture cultured in the presence of the test substance are cultured in the absence of the test substance, respectively.
- test substance Evaluating the test substance to have anti-hepatitis C virus activity when less than the amount of subgenomic replicon RNA, full genome replicon RNA or hepatitis C virus particles quantified in the cultured culture; A method for screening for an anti-hepatitis C virus substance.
- E1 A base sequence encoding a protein, a base sequence encoding an E2 protein, and a base sequence encoding a p7 protein;
- a base sequence encoding the NS2 protein comprising the sequence of base numbers 2786 to 3436 of SEQ ID NO: 1, a base sequence encoding the NS2 protein of the hepatitis C virus genome other than the hepatitis C virus genome of SEQ ID NO: 1, or a sequence number Part of the base sequence encoding the NS2 protein consisting of the base sequence of base numbers 2786 to 3436 of 1 and part of the base sequence encoding the NS2 protein of the hepatitis C virus genome other than the hepatitis C virus genome
- a hepatitis C virus chimeric full-genome replicon RNA comprising the nucleic acid according to [16] to [18] above.
- a genotype 3a HCV replicon RNA having autonomous replication ability in cultured cells can be provided.
- the quantification result of the copy number of HCV subgenomic replicon RNA contained in a S310A subgenomic replicon replicating cell is shown. It is a figure which shows the sensitivity with respect to the interferon of replicon RNA replication in a S310A subgenomic replicon replication cell (clone). It is a figure which shows the sensitivity with respect to NS3 protease inhibitor VX-950 of the replicon RNA replication in a S310A subgenomic replicon replication cell (clone).
- FIG. 1 is a view showing the structure of a chimeric HCV genome including a structural gene derived from -1 strain or TH strain.
- S310A R2198H, S310A S2210I, or S310A R2895K which are HCV full-genome replicon RNA (variant full-length genomic RNA) of the mutant S310A strain, contain the mutations R2198H, S2210I, or R2895K, respectively, and the HCV genome shown at the top The positions of these mutations are collectively shown on the structure (in fact, each mutation is included alone).
- Hepatitis C virus is a virus whose genome is a single-stranded (+) strand sense RNA.
- the genome of HCV has a 5 ′ untranslated region (5′UTR) and bases encoding Core protein, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein It consists of a sequence (viral protein coding region) and a 3 ′ untranslated region (3′UTR).
- HCV genome (HCV full length genome) is 5′UTR in order from 5 ′ side to 3 ′ side, Core protein, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protein, NS4A protein, NS4B protein, NS5A It is an RNA comprising a base sequence encoding a protein and NS5B protein and a 3′UTR.
- HCV full length genome In order to distinguish from a nucleic acid consisting of a part of the HCV genome, the HCV genome consisting of the full length is referred to as “HCV full length genome”, “full length HCV genome”, “HCV full length genomic RNA”, “full length HCV genomic RNA” or “full length. Also referred to as “genomic RNA”.
- HCV virus particles contain an HCV genome inside a virus shell composed of HCV structural proteins.
- HCV Core protein, E1 protein, E2 protein, and p7 protein are “structural proteins” that form HCV particles, and nucleic acids that encode these structural proteins are referred to as “structural genes”. Sequences on the HCV genome that contain these structural genes are also called “structural regions”. NS2 protein, NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein of HCV are “nonstructural proteins” that do not form HCV particles, and a nucleic acid encoding this nonstructural protein is referred to as “nonstructural gene”. . Sequences on the HCV genome that contain these nonstructural genes are also referred to as “nonstructural regions”. Nonstructural proteins have functions involved in HCV genome replication, HCV protein processing, and the like.
- HCV 5 'untranslated region (5' UTR) provides an internal ribosome entry site (hereinafter IRES) for protein translation and elements necessary for replication.
- IRES internal ribosome entry site
- the 5'UTR of HCV is a region of about 360 nucleotides from the 5 'end of the genome.
- HCV 3 'untranslated region (3'UTR) assists HCV replication.
- the 3'UTR of HCV includes a variable region, a poly U region, and an additional region of about 100 nucleotides.
- HCV 10 types of viral proteins (Core protein, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein) are linked together in this order. (Polyprotein), and then 10 types of mature viral proteins (Core protein, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protein, NS4A protein, NS4B by intracellular and viral proteases, respectively. Protein, NS5A protein and NS5B protein).
- HCV Various genotypes are known for HCV, and it is known that HCV genomes of various genotypes have similar gene structures.
- the “genotype” of HCV means a genotype classified according to the international classification by Simmonds et al.
- the nucleic acid consisting of the base sequence of SEQ ID NO: 1 in the sequence listing is the genome of the S310A strain, which is a novel genotype 3a HCV isolated from an acute severe hepatitis C patient.
- the sequence shown in SEQ ID NO: 1 is the cDNA sequence of the full-length genomic RNA of the S310A strain, and the RNA sequence is obtained by replacing thymine (t) in the base sequence with uracil (u).
- t thymine
- u uracil
- the 5 ′ untranslated region (SEQ ID NO: 1) of the above S310A strain
- the 5 ′ untranslated region (SEQ ID NO: 1)
- the Core protein coding sequence is The E1 protein coding sequence consists of the sequence of base numbers 914 to 1489 of SEQ ID NO: 1
- the E2 protein coding sequence consists of the sequence of base numbers 1490 to 2596 of SEQ ID NO: 1.
- the p7 protein coding sequence consists of the sequence of base numbers 2597 to 2785 of SEQ ID NO: 1
- the NS2 protein coding sequence consists of the sequence of base numbers 2786 to 3436 of SEQ ID NO: 1
- the NS3 protein coding sequence It consists of the sequence of base numbers 3437-5329 of number 1
- NS4A protein coding sequence Is composed of the sequences of base numbers 5330 to 5491 of SEQ ID NO: 1
- the NS4B protein coding sequence is composed of the sequences of base numbers 5492 to 6274 of SEQ ID NO: 1
- the NS5A protein coding sequence is from base numbers 6275 to 5 of SEQ ID NO: 1.
- the NS5B protein coding sequence consists of the sequence of base numbers 7631 to 9406 of SEQ ID NO: 1, and the 3 ′ untranslated region (3′UTR) consists of the sequence of base numbers 9407 to 9655 of SEQ ID NO: 1. Become. The structure of the full-length HCV genome of this S310A strain is shown in FIG. 1A.
- the 5 ′ untranslated region (5′UTR) of the full-length HCV genome (SEQ ID NO: 1) of the S310A strain consists of the base sequence shown in SEQ ID NO: 2, and the Core protein coding sequence is the base sequence shown in SEQ ID NO: 3.
- the E1 protein coding sequence consists of the base sequence shown in SEQ ID NO: 4, the E2 protein coding sequence consists of the base sequence shown in SEQ ID NO: 5, the p7 protein coding sequence consists of the base sequence shown in SEQ ID NO: 6, and the NS2 protein
- the coding sequence consists of the base sequence shown in SEQ ID NO: 7, the NS3 protein coding sequence consists of the base sequence shown in SEQ ID NO: 8, the NS4A protein coding sequence consists of the base sequence shown in SEQ ID NO: 9, and the NS4B protein coding sequence is SEQ ID NO:
- the NS5A protein coding sequence is represented by SEQ ID NO: 11. Consists to nucleotide sequence, NS5B protein coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 12, 3 'untranslated region (3'UTR) has a base sequence shown in SEQ ID NO: 13.
- the amino acid sequence of the precursor protein of S310A strain is shown in SEQ ID NO: 14.
- the amino acid sequence of the precursor protein of the S310A strain shown in SEQ ID NO: 14 consists of the sequences of base numbers 341 to 9406 (including the stop codon) of the cDNA sequence of the full-length genomic RNA of the wild type S310A strain shown in SEQ ID NO: 1. It is encoded by the base sequence of the part.
- the present invention provides the genome of S310A strain, which is the above-mentioned novel genotype 3a HCV, and replicon RNA or nucleic acid thereof having autonomous replication ability derived from this S310A strain.
- Nucleic acid includes RNA and DNA.
- protein coding region protein-encoding base sequence
- protein-encoding sequence protein-encoding sequence
- protein-encoding sequence in this specification are base sequences encoding the amino acid sequence of a given protein. The start and stop codons may or may not be included.
- nucleic acid is RNA and a base sequence or base of RNA is specified with reference to a sequence number in the sequence listing, thymine (t) in the base sequence shown in the sequence number is uracil ( It shall be read as u).
- the present invention relates to a 5 ′ untranslated region comprising the base sequence of base numbers 1 to 340 of SEQ ID NO: 1, of the genome of genotype 3a hepatitis C virus (for example, S310A strain or a variant thereof) And the amino acid sequence of the NS3 protein of amino acid numbers 1033 to 1663, the amino acid sequence of the NS4A protein of amino acid numbers 1664 to 1717, and the amino acid number 1718 to SEQ ID NO: 14 (precursor protein encoded by the base sequence of SEQ ID NO: 1) A base sequence encoding the amino acid sequence of NS4B protein of 1978, the amino acid sequence of NS5A protein of amino acid numbers 1979 to 2430, and the amino acid sequence of NS5B protein of amino acid numbers 2431 to 3021 and the bases of base numbers 9407 to 9655 of SEQ ID NO: 1 Including array 3 'and a non-translated region in this order, providing a nucleic acid.
- the 5 ′ untranslated region of the nucleic acid contains one or more (eg, 2 to 20, preferably 2 to 5) nucleotide sequences of nucleotide numbers 1 to 340 of SEQ ID NO: 1. Base deletions, substitutions or additions may be included.
- the 5 ′ untranslated region of the nucleic acid is 95% or more, preferably 97% or more, more preferably 99% or more, for example, 99.5% or more of the nucleotide sequence identical to the nucleotide sequence of nucleotide numbers 1 to 340 of SEQ ID NO: 1. It is preferable to have properties.
- the nucleic acid of the present invention encodes an amino acid sequence of NS3 protein consisting of a 5 ′ untranslated region containing the nucleotide sequence of nucleotide numbers 1 to 340 of SEQ ID NO: 1 and nucleotide numbers 3437 to 5329 of SEQ ID NO: 1.
- a base sequence encoding the amino acid sequence of NS4A protein of base numbers 5330 to 5491 of SEQ ID NO: 1 and a base sequence encoding the amino acid sequence of NS4B protein of base numbers 5492 to 6274 of SEQ ID NO: 1.
- the 5 ′ untranslated region of the nucleic acid contains one or more (eg, 2 to 20, preferably 2 to 5) nucleotide sequences of nucleotide numbers 1 to 340 of SEQ ID NO: 1. Base deletions, substitutions or additions may be included.
- the 5 ′ untranslated region of the nucleic acid is 95% or more, preferably 97% or more, more preferably 99% or more, for example, 99.5% or more of the nucleotide sequence identical to the nucleotide sequence of nucleotide numbers 1 to 340 of SEQ ID NO: 1. It is preferable to have properties.
- the nucleic acid of the present invention encodes the base sequence encoding the Core protein, the base sequence encoding the E1 protein, the base sequence encoding the E2 protein, the base sequence encoding the p7 protein, and the NS2 protein of the hepatitis C virus genome. It is not necessary to include the base sequence to be.
- Such nucleic acids of the invention can encode HCV subgenomic replicon RNA.
- the nucleic acid of the present invention encodes the base sequence encoding the Core protein, the base sequence encoding the E1 protein, the base sequence encoding the E2 protein, the base sequence encoding the p7 protein, and the NS2 protein of the hepatitis C virus genome. It may further include a base sequence that Such nucleic acids of the invention can encode HCV full genome replicon RNA.
- the base sequence encoding the Core protein, the base sequence encoding the E1 protein, the base sequence encoding the E2 protein, the base sequence encoding the p7 protein, and the base sequence encoding the NS2 protein are C of genotype 3a. It may be derived from the genome of hepatitis B virus (for example, S310A strain or a variant thereof).
- the base sequence encoding the Core protein is preferably a base sequence encoding the amino acid sequence of the Core protein of amino acid numbers 1 to 191 of SEQ ID NO: 14.
- the base sequence encoding the E1 protein is preferably a base sequence encoding the amino acid sequence of the E1 protein of amino acid numbers 192 to 383 of SEQ ID NO: 14.
- the base sequence encoding the E2 protein is preferably a base sequence encoding the amino acid sequence of the E2 protein of amino acid numbers 384 to 752 of SEQ ID NO: 14.
- the base sequence encoding the p7 protein is preferably a base sequence encoding the amino acid sequence of the p7 protein of amino acid numbers 753 to 815 of SEQ ID NO: 14.
- the base sequence encoding the NS2 protein is preferably a base sequence encoding the amino acid sequence of the NS2 protein of amino acid numbers 816 to 1032 of SEQ ID NO: 14.
- the base sequence encoding Core protein, the base sequence encoding E1 protein, the base sequence encoding E2 protein, the base sequence encoding p7 protein, and the base sequence encoding NS2 protein are also genotypes other than genotype 3a.
- genotype 3a For example, 1a, 1b, 2a, 2b, 2c, 3b, 4, 5a, or 6a
- 1a, 1b, 2a, 2b, 2c, 3b, 4, 5a, or 6a may be derived from the genome of HCV (for example, an existing strain of HCV).
- the nucleic acid of the present invention may be a nucleic acid containing one or more (preferably 2 to 50, for example, 2 to 10) base mutations in the base sequence of the nucleic acid.
- the base mutation is not limited, but is preferably a base deletion, substitution or addition.
- the base mutation may be a synonymous substitution that does not cause an amino acid substitution, or may be a non-synonymous substitution that causes an amino acid substitution as long as the autonomous replication ability is not lost. As long as the autonomous replication ability is not lost, the amino acid substitution may be a conservative substitution or a non-conservative substitution.
- the nucleic acid of the present invention can also be substituted with a 5 ′ untranslated region containing the nucleotide sequence of nucleotide numbers 1 to 340 of SEQ ID NO: 1 with a genotype other than genotype 3a (eg, 1a, 1b, 2a, 2b, 2c, 3b, 5a, or 6a) of the HCV (eg, existing strains of HCV) may be included.
- a genotype other than genotype 3a eg, 1a, 1b, 2a, 2b, 2c, 3b, 5a, or 6a
- HCV existing strains of HCV
- the nucleic acid of the present invention may also contain a foreign gene (such as a drug resistance gene or a reporter gene) and an IRES sequence.
- the nucleic acid of the present invention may be HCV replicon RNA such as HCV subgenomic replicon RNA or HCV full genome replicon RNA or a nucleic acid encoding the same.
- the nucleic acid of the present invention may be, for example, an expression cassette containing a base sequence encoding HCV replicon RNA.
- the nucleic acid of the present invention may be DNA, RNA, a chimera of DNA and RNA, or the like, and may contain a modified base or the like.
- replicon RNA refers to RNA having the ability to replicate autonomously in cultured cells (typically HCV sensitive cells). Since the replicon RNA introduced into the cell replicates autonomously and its RNA copy is distributed to the daughter cells after cell division, the replicon RNA can be stably introduced into the cell.
- HCV replicon RNA refers to RNA that autonomously replicates, including part or the entire length of HCV genomic RNA.
- An autonomously replicating RNA containing a part of the HCV genomic RNA is called an HCV subgenomic replicon RNA
- an autonomously replicating RNA containing the full length of the HCV genomic RNA is called an HCV full genomic replicon RNA.
- HCV replicon RNA encompasses both HCV subgenomic replicon RNA and HCV full genomic replicon RNA.
- the “HCV subgenomic replicon RNA” in the present invention includes a 5 ′ untranslated region (5′UTR) of HCV, a nucleotide sequence encoding NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein, and 3 ′ non-translated region.
- the translation region (3′UTR) is preferably included in this order from the 5 ′ side to the 3 ′ side.
- the HCV subgenomic replicon RNA preferably contains a foreign gene (for example, drug resistance gene or reporter gene) and an IRES sequence for detection thereof. In such a case, the foreign gene (drug resistance gene or reporter gene) ) And IRES sequences are preferably included 5 ′ of the NS3 protein coding region of the HCV subgenomic replicon RNA.
- the “HCV subgenomic replicon RNA” of the present invention preferably contains the nucleic acid of the present invention.
- the “HCV subgenomic replicon RNA” of the present invention is preferably expressed from the nucleic acid of the present invention.
- HCV subgenomic replicon RNA in the present invention is a 5 ′ untranslated region (5′UTR) of HCV, a sequence of 57 nucleotides from the 5 ′ end of the base sequence encoding the Core protein, A base sequence encoding a foreign gene (drug resistance gene or reporter gene), an IRES sequence, NS3 protein, NS4A protein, NS4B protein, NS5A protein, and NS5B protein, and a 3 ′ untranslated region (3′UTR), It is included in this order from the 5 ′ side to the 3 ′ side.
- 5′UTR 5 ′ untranslated region
- HCV full genome replicon RNA in the present invention includes 5 ′ untranslated region (5′UTR) of HCV, Core protein, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protein, NS4A protein, NS4B protein, A base sequence encoding NS5A protein and NS5B protein and a 3 ′ untranslated region (3′UTR) are preferably arranged in this order from the 5 ′ side to the 3 ′ side.
- the HCV full genome replicon RNA may further comprise a foreign gene (drug resistance gene or reporter gene) and an IRES sequence. In such a case, it is preferable that the foreign gene (drug resistance gene or reporter gene) and the IRES sequence are included 5 'to the base sequence encoding the Core protein of the HCV full genome replicon RNA.
- HCV full-length genomic nucleic acid When the HCV full-length genomic nucleic acid has autonomous replication ability, the genome is a replicon RNA.
- a replicon RNA containing an HCV full-length genomic nucleic acid is referred to as an HCV full genome replicon RNA.
- HCV full genome replicon RNA When RNA consisting of the full-length HCV genomic sequence (HCV full-length genomic RNA) has autonomous replication ability, it is an HCV full-genome replicon RNA.
- HCV replicon RNA HCV full-genome replicon RNA and HCV subgenomic replicon RNA
- examples of drug resistance genes that the HCV replicon RNA (HCV full-genome replicon RNA and HCV subgenomic replicon RNA) and the nucleic acid of the present invention can contain include, for example, neomycin resistance gene, hygromycin resistance gene, thymidine kinase gene, kanamycin resistance gene , A pyrithiamine resistance gene, an adenylyltransferase gene, a zeocin resistance gene, a puromycin resistance gene, a blasticidin S resistance gene, and the like.
- a neomycin resistance gene and a hygromycin resistance gene are preferable, and a neomycin resistance gene is more preferable.
- reporter genes that can be included in the HCV replicon RNA and the nucleic acid of the present invention include, for example, structural genes of enzymes that catalyze luminescence reactions and color reactions.
- preferable reporter genes include transposon Tn9-derived chloramphenicol acetyltransferase gene, E. coli-derived ⁇ -glucuronidase or ⁇ -galactosidase gene, luciferase gene, green fluorescent protein gene, jellyfish-derived aequorin gene, secretory placental alkaline phosphatase ( SEAP) gene and the like.
- the drug resistance gene and the reporter gene may be contained in the HCV replicon RNA or the nucleic acid of the present invention.
- One drug resistance gene or reporter gene may be contained in the HCV replicon RNA or the nucleic acid of the present invention, or may be contained in two or more. When two or more drug resistance genes or reporter genes are included, each gene may be linked to the virus-derived 2A peptide gene so as to be in the correct reading frame (in frame).
- 2A peptides As 2A peptides, 2A peptide derived from Thesea assigna virus (T2A), 2A peptide derived from Foot-and-mouth disease virus (F2A), 2A peptide derived from Equin rhinitis A virus (E2A2 peptide) (E2A peptide from E2A), Porcin (P2A) (Kim et al., PoLos One., 2011, Vol. 6 (4), e18556).
- T2A virus
- F2A Foot-and-mouth disease virus
- E2A2 peptide Equin rhinitis A virus
- P2A Porcin
- the IRES sequence that can be contained in the HCV replicon RNA and the nucleic acid of the present invention is the same as described above, and means, for example, an internal ribosome binding site capable of binding a ribosome inside RNA and initiating translation.
- Preferred examples of the IRES sequence include EMCV IRES (internal ribosome binding site of encephalomyocarditis virus), FMDV IRES, HCV IRES, etc., EMCV IRES and HCV IRES are more preferred, and EMCV IRES is most preferred.
- the drug resistance gene and / or the reporter gene are linked so that they are translated from the HCV replicon RNA in the correct reading frame (in frame).
- the proteins encoded by the HCV replicon RNA or the nucleic acid of the present invention are translated and expressed as a series of polypeptides, then cleaved into individual proteins by proteases and released from each other via protease cleavage sites or the like. It is preferable to connect.
- HCV-sensitive cells refer to cells that permit HCV particle infection and HCV replicon RNA replication in cell culture systems.
- cells in which the CD81 gene and / or Claudin1 gene is expressed in Huh7 cells, HepG2 cells, IMY-N9 cells, HeLa cells or 293 cells can also be mentioned (Lindenbach et al., Science, 2005, Vol. 309, p. 309). 623-626; Evans et al., Nature, 2007, 446, 801-805; Akawawa et al., J. Virol. 2007, 81, pp. 5036-5045).
- Huh7 cells or Huh7 cell derivatives are particularly preferably used.
- the “derived strain” refers to a strain derived from the cell.
- the nucleic acid and HCV replicon RNA of the present invention may also contain adaptive mutations.
- the adaptive mutation that improves the replication ability of the HCV subgenomic replicon RNA of the S310A strain described above is based on the amino acid sequence shown in SEQ ID NO: 14 (the full-length amino acid sequence of the precursor protein of the S310A strain).
- T1286I (the 1286th threonine (T) is mutated to isoleucine (I)
- T2188A (the 2188th threonine (T) is mutated to alanine (A)
- R2198H (the 2198th arginine (R) is Mutated to histidine (H)
- S2210I (2210th serine (S) mutated to isoleucine (I)
- T2496I (2496th threonine (T) mutated to isoleucine (I)
- R2895G (2895)
- Th arginine (R) is mutated to glycine (G))
- R2895K second 95 arginine (R) mutation
- K lysine
- nucleic acid of the present invention and the HCV replicon RNA, for example, the HCV genome of the S310A strain described above, thereby improving the replication ability of the HCV subgenomic replicon RNA or HCV full-genome replicon RNA.
- nucleic acids encoding them can be obtained.
- more preferred base mutations of the nucleic acid and HCV replicon RNA of the present invention are amino acid sequence T1286I mutation, R2198H mutation, S2210I mutation or R2895K mutation, and further preferred base mutations.
- the mutation is a mutation that causes an R2198H mutation, an S2210I mutation, or an R2895K mutation in the amino acid sequence.
- adaptive mutations described in known documents can be introduced alone or in combination with the above mutations.
- the introduced mutation may be any mutation that improves the replication ability of the HCV replicon RNA derived from the S310A strain.
- T1286I is a NS3 protein
- T2188A, R2198H and S2210I mutations are NS5A proteins
- T2496I, R2985G and R2985K are mutations in NS5B proteins.
- a PCR method or a commercially available mutation introduction kit for example, KOD-Plus-Mutageness Kit manufactured by Toyobo Co., Ltd.
- a PCR method for example, PCR using a vector obtained by cloning a cDNA of genotype 3a wild type HCV genomic RNA as a template and using forward and reverse primers containing a mutation to be introduced designed from the sequence of the cDNA
- the target sequence portion can be amplified.
- a plurality of different PCR products having overlapping sequences with each other are synthesized, these PCR products are mixed, and using this as a template, a forward primer containing the 5 ′ end of the target nucleic acid and the complementary strand of the nucleic acid
- the target nucleic acid can be amplified by performing PCR using a reverse primer containing a 5 ′ end.
- Each end of the synthesized nucleic acid is cleaved with a restriction enzyme, and the cDNA of wild-type HCV genomic RNA cleaved with the same enzyme is ligated to a cloned vector.
- Basic techniques of such operation are described in, for example, International Publication No. 04/104198, International Publication No. 06/022422, Wakita et al., 2005, Nature Medicine, No. 11, p. 791-796, and Lindenbach et al., 2005, Science, 309, p. 623-626.
- nucleic acid and HCV subgenomic replicon RNA of the present invention includes a 5 ′ untranslated region (5′UTR) (SEQ ID NO: 2) in the full-length HCV genome (SEQ ID NO: 1) of the S310A strain described above, NS3 protein coding sequence (SEQ ID NO: 8), NS4A protein coding sequence (SEQ ID NO: 9), NS4B protein coding sequence (SEQ ID NO: 10), NS5A protein coding sequence (SEQ ID NO: 11), NS5B protein coding sequence (SEQ ID NO: 12), Examples include a nucleic acid containing a 3 ′ untranslated region (3′UTR) (SEQ ID NO: 13) in this order from the 5 ′ side to the 3 ′ side.
- 5′UTR 5 ′ untranslated region
- a 5 ′ untranslated region (SEQ ID NO: 2), NS3 protein in the full-length HCV genome (SEQ ID NO: 1) of S310A strain Coding sequence (SEQ ID NO: 8), NS4A protein coding sequence (SEQ ID NO: 9), NS4B protein coding sequence (SEQ ID NO: 10), NS5A protein coding sequence (SEQ ID NO: 11), NS5B protein coding sequence (SEQ ID NO: 12), 3 ′
- the base sequence comprising the untranslated region (3′UTR) (SEQ ID NO: 13) in this order from the 5 ′ side to the 3 ′ side, selected from the group consisting of T1286I, T2188A, R2198H, S2210I, T2496I, R2895G and R2895K Or a nucleic acid containing at least one mutation.
- nucleic acid and HCV subgenomic replicon RNA of the present invention 5 ′ untranslated region (5′UTR), NS3 protein coding sequence, NS4A protein coding sequence, NS4B protein coding sequence in the full-length HCV genome of S310A strain, NS5A protein coding sequence, NS5B protein coding sequence, a nucleic acid comprising a 3 ′ untranslated region (3′UTR) in this order from the 5 ′ side to the 3 ′ side, and comprising a mutation of T1286I, R2198H or R2895K
- nucleic acids also included are nucleic acids.
- a 5 ′ untranslated region 5′UTR
- NS3 protein coding sequence NS4A protein coding sequence
- NS4B protein coding sequence in the full-length HCV genome of S310A strain
- a nucleic acid comprising NS5A protein coding sequence, NS5B protein coding sequence, 3 ′ untranslated region (3′UTR) in this order from 5 ′ side to 3 ′ side, and a nucleic acid comprising a mutation of R2198H or R2895K Can be mentioned.
- the HCV subgenomic replicon RNA may further contain a drug resistance gene and / or a reporter gene and an IRES sequence. In this case, it is preferable to insert a drug resistance gene or / and a reporter gene downstream of the 5 'UTR and further insert an IRES sequence downstream thereof.
- HCV subgenomic replicon RNA of the present invention a nucleic acid consisting of the base sequence represented by SEQ ID NO: 16 (wild-type S310A strain HCV subgenomic replicon RNA, FIG. 1C) can be mentioned.
- a mutation selected from the group consisting of T1286I, T2188A, R2198H, S2210I, T2496I, R2895G, and R2895K is introduced into the nucleotide sequence shown in SEQ ID NO: 16 More preferably, RNA into which a mutation of T1286I, R2198H or R2895K has been introduced may be mentioned.
- SEQ ID NO: 17 sequence containing the T1286I mutation in the HCV subgenomic replicon RNA of the wild type S310A strain
- SEQ ID NO: 18 sequence containing the R2198H mutation in the HCV subgenomic replicon RNA of the wild type S310A strain
- a nucleic acid comprising the base sequence shown in SEQ ID NO: 19 sequence containing R2895K mutation in the wild-type S310A HCV subgenomic replicon RNA
- the nucleic acid constituting the HCV subgenomic replicon RNA of the present invention further has other mutations in bases other than the bases causing the above mutations, but the nucleic acid that provides the same replication ability as the nucleic acid containing the above mutations It may be.
- Examples of such other mutation include substitution of one or a plurality of bases, and a nucleic acid having the other mutation is 90% or more, preferably 95% or more, more preferably 97%, of the base sequence of the original nucleic acid. It is preferable to have the above base sequence identity.
- such other mutations include deletion or addition of one or more bases.
- the nucleic acid having the other mutation has 90% or more, preferably 95% or more, more preferably 97% or more of the base sequence of the original nucleic acid, and is within the sequence encoding the protein.
- deletion or addition of it is preferable that the reading frame translated into the amino acid sequence of the protein does not shift.
- other mutations include deletion, substitution or addition of one or more bases in the 5 ′ untranslated region or 3 ′ untranslated region of the HCV genome, and having such other mutations.
- the nucleic acid preferably has 90% or more, preferably 95% or more, more preferably 97% or more of the nucleotide sequence identity with the base sequence of the original nucleic acid.
- such other mutations include deletion, substitution or addition of one or more bases in the base sequence (viral protein coding region) encoding the HCV protein of the HCV genome.
- amino acids or amino acid residues are described by the one-letter code or three-letter code of amino acids commonly used in the field of biology (Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition, 1989), and so on.
- the amino acids described in the above include amino acids that have undergone post-translational modifications such as hydroxylation, glycosylation, and sulfation.
- the amino acid at a specific position in the amino acid sequence represented by SEQ ID NO: “the amino acid sequence represented by the sequence number“ X ”” is the “Y” th (amino acid) ”. It may be specified by the expression.
- “the“ Y ”-th (amino acid) when the amino acid sequence of the precursor protein of the S310A strain shown in SEQ ID NO: 14 is used as a reference” means that the amino acid is S310A of HCV shown in SEQ ID NO: 14. This means that the amino acid residue is located at the “Y” -th position when the first amino acid (methionine) at the N-terminus is defined as the first position in the amino acid sequence of the precursor protein of the strain.
- amino acid designated by using the expression “Yth (amino acid)” shown in SEQ ID NO: “X” when the amino acid sequence is used as a reference is the “Y” -th amino acid of SEQ ID NO: “X”.
- it may be located at the “Y” position in the sequence variant of the sequence number “X” (such as a truncated sequence), but it is located at the “Y” position. You don't have to.
- the 2496th threonine is substituted with isoleucine, the NS3 protein of the S310A strain, the NS4A protein,
- the N-terminal truncation of the amino acid sequence of SEQ ID NO: 14 is positioned at position 2496 in SEQ ID NO: 14.
- the threonine whose position from the N-terminal on the truncation sequence is not the 2496th position is replaced with isoleucine in the protein.
- the notation such as R2895K indicates substitution of an amino acid at a specific position, but also means a base mutation that causes the amino acid substitution depending on the context.
- the base mutation may be referred to as R2895K substitution (mutation).
- a nucleic acid encoding an amino acid sequence having the mutation may be referred to as a nucleic acid containing R2895K substitution (mutation) or a nucleic acid into which R2895K substitution (mutation) has been introduced.
- such a mutation may be referred to as a base mutation that causes R2895K substitution (mutation) in the amino acid sequence or a mutation that causes R2895K substitution (mutation) in the amino acid sequence.
- an HCV replicon RNA introduced with an R2895K substitution (mutation) may be referred to as an R2895K mutant HCV replicon RNA.
- it may be expressed as “including substitution (mutation) of T2496I / R2895K”.
- “Amino acid substitution” may be expressed as “amino acid mutation”.
- the base mutation that causes a specific amino acid substitution can be selected based on a well-known genetic code table.
- the mutation that causes the R2895K substitution can occur from codons “CGU”, “CGC”, “CGA”, “CGG”, “AGA” or “AGG” encoding arginine, from codons “AAA” or “AAG” encoding lysine.
- the base mutation that causes R2895K substitution is a mutation that changes the codon “AGA” (positions 9023 to 9025 of SEQ ID NO: 1) to “AAG” or “AAA”. It is.
- the 9024th to 9025th base sequence of SEQ ID NO: 1 is mutated from 5′-GA-3 ′ to 5′-AG-3 ′, or the 9024th base is changed from G to A. It is to be mutated.
- the amino acid substitution of the 2198th arginine to histidine is expressed as R2198H.
- the base mutation that causes the R2198H substitution is the codon “CGU” that encodes arginine (6932 to 6934 of SEQ ID NO: 1) and the codon that encodes histidine “ It is a mutation that changes to “CAU” or “CAC”.
- the amino acid substitution of the 1286th threonine to isoleucine is expressed as T1286I.
- the base mutation that causes the T1286I substitution is the codon “ACU” (4196th to 4198th in SEQ ID NO: 1) encoding threonine, and the codon encoding isoleucine “ It is a mutation that changes to “AUU”, “AUC”, or “AUA”.
- the base number of the base sequence shown in SEQ ID NO. Is based on the base sequence shown in SEQ ID NO. With the 5′-terminal first base as the first base number.
- HCV subgenomic replicon RNA can be obtained by transcription (expression) from an expression vector.
- Basic techniques for the construction of HCV subgenomic replicon RNA expression vectors are described in Lohmann et al., Science, 1999, 285, p. 110-113 and Kato et al., Gastroenterology, 2003, vol. 125, p. 1808-1817.
- 5 ′ untranslated region 5′UTR
- NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein and the 3 ′ untranslated region (3′UTR) in the downstream of the T7 promoter HCV subgenomic replicon RNA expression vectors can be constructed.
- the linking site may contain an additional sequence such as a restriction enzyme site.
- the above HCV subgenomic replicon RNA can be synthesized by a polymerase from the constructed HCV subgenomic replicon RNA expression vector.
- synthesis by MEGAscript T7 kit (Ambion) can be used as a method for producing RNA in vitro using a nucleic acid obtained by cloning HCV cDNA under the control of the T7 promoter as a template.
- HCV subgenomic replicon RNA transcribed from this vector replicates autonomously when introduced into a cell.
- the present invention includes cells into which HCV subgenomic replicon RNA has been introduced.
- any promoter such as SP6 promoter, T3 promoter, T5 promoter and the like can be used as well as T7 promoter, but T7 promoter is preferable.
- pUC19 (TaKaRa), pBR322 (TaKaRa), pGEM-T, pGEM-T Easy, pGEM-3Z (all Promega), pSP72 (Promega), pCRII (Invitrogen), pT7Blue (Novagen)
- pUC19 (TaKaRa)
- pBR322 TiKaRa
- pGEM-T As vectors, pUC19 (TaKaRa), pBR322 (TaKaRa), pGEM-T, pGEM-T Easy, pGEM-3Z (all Promega), pSP72 (Promega), pCRII (Invitrogen), pT7Blue (Novagen)
- pT7Blue (Novagen)
- Various vectors such as the company) can be used.
- the cell into which the HCV subgenomic replicon RNA is introduced may be any cell that allows replication of the HCV subgenomic replicon RNA, and examples thereof include the HCV sensitive cells described above. Huh7 cells or derivatives of Huh7 cells are particularly preferably used.
- Any known method can be used as a method for introducing HCV subgenomic replicon RNA into cells.
- Examples thereof include calcium phosphate coprecipitation method, DEAE dextran method, lipofection method, microinjection method, and electroporation method, preferably lipofection method and electroporation method, more preferably electroporation method. .
- the method for evaluating the replication ability of the introduced HCV subgenomic replicon RNA includes measuring the function of a foreign gene linked to the HCV subgenomic replicon RNA, that is, the function expressed by the expression of the gene.
- the replication ability of the HCV subgenomic replicon RNA can be evaluated by measuring the number of cells or the number of cell colonies that grow on the selective medium containing the drug. In this case, it can be evaluated that the greater the number of cells or the number of cell colonies, the higher the replication ability.
- the foreign gene is an enzyme
- the replication activity of the HCV subgenomic replicon RNA can be evaluated by measuring the enzyme activity. In this case, it can be evaluated that the higher the enzyme activity, the higher the replication ability.
- the replication ability of HCV subgenomic replicon RNA can also be directly evaluated by quantifying the amount of RNA replicated by quantitative RT-PCR.
- HCV full-genome replicon RNA of the present invention includes HCV full-length genomic RNA itself, and can be prepared by the same procedure as the above-described HCV subgenomic replicon RNA.
- the HCV full-genome replicon RNA of the present invention has an adaptive mutation that improves the replication ability of the HCV subgenomic replicon RNA, as in the case of the HCV subgenomic replicon RNA, such as the wild-type S310A strain of genotype 3a.
- HCV full genome replicon RNA may be produced by introducing it into full-length genomic RNA.
- the HCV genome in which the above-mentioned adaptive mutation is introduced into the HCV full-length genomic RNA of the wild-type S310A strain is referred to as S310A strain mutant or mutant S310A strain.
- the mutation may be introduced into the full-length HCV genome of the wild-type S310A strain using the above method, and the structural gene portion of the wild-type HCV genome is linked to the subgenomic replicon mutant. Mutation may be introduced.
- HCV full genome replicon RNA is the full length genomic RNA of S310A strain (SEQ ID NO: 1), ie, 5 ′ untranslated region (5′UTR) (SEQ ID NO: 2), Core protein coding sequence (sequence No. 3), E1 protein coding sequence (SEQ ID NO: 4), E2 protein coding sequence (SEQ ID NO: 5), p7 protein coding sequence (SEQ ID NO: 6), NS2 protein coding sequence (SEQ ID NO: 7), NS3 protein coding sequence (sequence) No.
- NS4A protein coding sequence SEQ ID NO: 9
- NS4B protein coding sequence SEQ ID NO: 10
- NS5A protein coding sequence SEQ ID NO: 11
- NS5B protein coding sequence SEQ ID NO: 12
- 3′ untranslated region 3′UTR
- HCV full genome replicon RNA is a nucleic acid in which the above-mentioned adaptive mutation is introduced into the full-length genomic RNA of the S310 strain.
- the full-length genomic RNA (SEQ ID NO: 1) of the above-mentioned S310A strain that is, 5 ′ untranslated region (5′UTR) (SEQ ID NO: 2), Core protein coding sequence ( SEQ ID NO: 3), E1 protein coding sequence (SEQ ID NO: 4), E2 protein coding sequence (SEQ ID NO: 5), p7 protein coding sequence (SEQ ID NO: 6), NS2 protein coding sequence (SEQ ID NO: 7), NS3 protein coding sequence ( SEQ ID NO: 8), NS4A protein coding sequence (SEQ ID NO: 9), NS4B protein coding sequence (SEQ ID NO: 10), NS5A protein coding sequence (SEQ ID NO: 11), NS5B
- the full-length genomic RNA of S310A strain (SEQ ID NO: 1), that is, 5 ′ untranslated region (5′UTR), Core protein coding sequence, E1 protein coding sequence, E2 protein coding sequence, p7 protein coding sequence, NS2 protein
- a nucleic acid containing a mutation of R2198H, S2210I or R2895K can be mentioned.
- HCV full genome replicon RNA may further contain a drug resistance gene and / or a reporter gene, and an IRES sequence. At this time, it is preferable to insert a drug resistance gene or / and a reporter gene downstream of the 5 'untranslated region (5' UTR) and an IRES sequence downstream thereof.
- SEQ ID NO: 49 full genome base sequence including mutation of S2210I
- SEQ ID NO: 50 full genome base sequence including mutation of R2198H
- SEQ ID NO: 51 mutant of R2895K
- RNA containing the base sequence shown in Full genome base sequence containing Specifically, a nucleic acid comprising a base sequence having a mutation that causes substitution of serine at position 2210 with isoleucine shown in SEQ ID NO: 49, substitution of arginine at position 2198 with histidine shown in SEQ ID NO: 50 Or a nucleic acid comprising a base sequence having a mutation that causes substitution of arginine at position 2895 to lysine, as shown in SEQ ID NO: 51.
- the nucleic acid constituting the HCV full-genome replicon RNA of the present invention further has other mutations in bases other than the bases that cause the mutations described above, but a nucleic acid that provides the same replication ability as a nucleic acid containing the mutations described above. It may be.
- examples of such other mutations include deletion, substitution or addition of one or more bases, and the nucleic acid having the other mutations is 90% or more, preferably 95% or more of the base sequence of the original nucleic acid, More preferably, it has a base sequence identity of 97% or more.
- such other mutations include deletion, substitution or addition of one or more bases in the 5 ′ untranslated region or 3 ′ untranslated region of the HCV genome, and having such other mutations.
- the nucleic acid preferably has 90% or more, preferably 95% or more, more preferably 97% or more of the nucleotide sequence identity with the base sequence of the original nucleic acid. Further, such other mutations include deletion, substitution or addition of one or more bases in the nucleotide sequence (viral protein coding region) encoding the HCV protein of the HCV genome.
- the nucleic acid has 90% or more, preferably 95% or more, more preferably 97% or more of the nucleotide sequence identity of the original nucleic acid, and in the case of deletion or addition, the amino acid sequence of the HCV protein It is preferable that the translated reading frame does not shift.
- the expression vector used for the production of the above HCV full genome replicon RNA can be produced by using the technique described in International Publication No. 05/080575. Specifically, cDNA corresponding to the full-length genomic RNA of HCV is reconstructed by a conventional method and inserted downstream of the promoter to prepare a DNA clone.
- the promoter is preferably contained in a plasmid clone. As the promoter, T7 promoter, SP6 promoter, T3 promoter, T5 promoter and the like can be used, but T7 promoter is preferable.
- pUC19 (TaKaRa), pBR322 (TaKaRa), pGEM-T, pGEM-T Easy, pGEM-3Z (all Promega), pSP72 (Promega), pCRII (Invitrogen), pT7Blue (Novagen) Etc.) can be used.
- RNA is synthesized by a polymerase using the prepared DNA clone as a template.
- RNA is produced in vitro using a nucleic acid obtained by cloning HCV cDNA under the control of the T7 promoter as a template, it can be synthesized with MEGAscript T7 kit (Ambion). RNA synthesis can be initiated from the 5'UTR by conventional methods.
- the DNA clone is a plasmid clone
- RNA can also be synthesized using a DNA fragment excised from the plasmid clone by a restriction enzyme as a template.
- the HCV full-genome replicon RNA or nucleic acid thereof is autonomously replicated when introduced into cultured cells (typically HCV-sensitive cells) to produce HCV particles (hepatitis C virus). Infecting cultured cells (typically HCV-sensitive cells) with HCV particles containing HCV full-genome replicon RNA or its nucleic acid as a viral genome will produce HCV particles. That is, a cultured cell into which the above HCV full genome replicon RNA or a nucleic acid thereof has been introduced, or a cultured cell infected with HCV particles containing the above HCV full genome replicon RNA or a nucleic acid thereof as a viral genome, It can be used for mass production.
- the cultured cells into which the HCV full genome replicon RNA or the nucleic acid thereof has been introduced, or the HCV full genome replicon RNA or the nucleic acid thereof as a viral genome.
- HCV particles produced from cultured cells typically HCV sensitive cells
- HCV particles hepatitis C virus
- HCV genomic RNA is replicated and packaged in cells, allowing HCV particles to be produced repeatedly.
- Infection of cultured cells with HCV particles can be performed, for example, by adding a culture supernatant of cultured cells into which HCV full-genome replicon RNA or a nucleic acid thereof has been introduced to HCV-sensitive cells (for example, Huh7 cells).
- HCV-sensitive cells for example, Huh7 cells
- the cells to which the above HCV full genome replicon RNA or its nucleic acid is introduced or the cells to be infected with the above HCV particles are preferably cultured cells, which allow HCV replicon RNA replication and HCV particle formation. And the above-mentioned HCV sensitive cells. Huh7 cells or derivatives of Huh7 cells are particularly preferably used.
- Any known method can be used as a method for introducing HCV full genome replicon RNA into cells.
- Examples thereof include calcium phosphate coprecipitation method, DEAE dextran method, lipofection method, microinjection method, and electroporation method, preferably lipofection method and electroporation method, more preferably electroporation method. .
- the replication ability of the HCV full-genome replicon RNA As a method for evaluating the replication ability of the introduced HCV full-genome replicon RNA, there is a method of measuring the function of a foreign gene linked to the HCV full-genome replicon RNA, that is, the function expressed by the expression of the gene.
- the foreign gene is a drug resistance gene
- the replication ability of the HCV full-genome replicon RNA can be evaluated by measuring the number of cells or the number of cell colonies growing in a selective medium containing the drug. In this case, it can be evaluated that the greater the number of cells or the number of cell colonies, the higher the replication ability.
- the foreign gene is an enzyme
- the replication activity of HCV full genome replicon RNA can be evaluated by measuring the enzyme activity. In this case, it can be evaluated that the higher the enzyme activity, the higher the replication ability.
- the replication ability of HCV full genome replicon RNA can also be directly evaluated by quantifying the amount of RNA replicate
- the present invention also includes a viral genome containing the nucleic acid and a hepatitis C virus particle (hepatitis C virus) containing the nucleic acid as a viral genome.
- a viral genome containing the nucleic acid
- a hepatitis C virus particle hepatitis C virus
- the above HCV full genome replicon RNA or nucleic acid thereof has the ability to produce HCV particles in cultured cells.
- the RNA is introduced into cells and the presence of HCV particles in the cell culture supernatant is measured. That's fine.
- the ability of cells to produce HCV particles can be detected using a protein that constitutes the HCV particles released into the culture supernatant, for example, an antibody against Core protein, E1 protein, or E2 protein.
- a protein that constitutes the HCV particles released into the culture supernatant for example, an antibody against Core protein, E1 protein, or E2 protein.
- the presence of HCV particles may be indirectly detected by amplifying and detecting HCV full-genome replicon RNA contained in HCV particles in the culture supernatant by RT-PCR using specific primers. it can.
- the culture supernatant of cells into which HCV full-genome replicon RNA or its nucleic acid has been introduced is treated with HCV-sensitive cells (eg, Huh7 cells).
- HCV-sensitive cells eg, Huh7 cells
- the cells are immunostained with an anti-Core antibody to count the number of infected cells, or the cell extract is electrophoresed on SDS-polyacrylamide gel and the Core protein is detected by Western blot. Can be judged.
- the present invention also provides chimeric nucleic acids derived from the genomes of two or more hepatitis C virus strains, including genotype 3a HCV (eg, S310A strain).
- genotype 3a HCV eg, S310A strain
- a genome sequence derived from the S310A strain of genotype 3a and an HCV genome other than the HCV genome (SEQ ID NO: 1) of the S310A strain such as various genotypes (1a, 1b, 2a, 2b, 3a, 3b, etc.) Chimera type HCV genome (chimera HCV genome), chimera type HCV subgenomic replicon RNA (chimeric HCV subgenomic replicon RNA), chimera, including existing HCV strains and genotype HCV genomic sequences other than genotype 3a HCV full-genome replicon RNA (chimeric HCV full-genome replicon RNA), chimera-type HCV particles (chimeric HCV particles), and the like are provided.
- the chimeric HCV genome and the chimeric HCV full genome replicon RNA refer to an HCV genome and an HCV full genome replicon RNA containing HCV genome sequences of two or more different strains, respectively, and these chimeric HCV genome or chimeric HCV full genome.
- HCV particles produced from replicon RNA are referred to as chimeric HCV particles.
- Such a chimeric HCV genome is also included in the nucleic acid of the present invention.
- the above-mentioned chimeric HCV genome comprises a non-structural gene of S310A strain or S310A strain mutant (S310A strain introduced with adaptive mutation) and a structural gene of other HCV strain (other than S310A strain and S310A strain mutant). It may be a chimeric genome of HCV.
- the above chimeric HCV genome may have a mutation such as an adaptive mutation, or may be prepared using a S310A strain mutant.
- the S310A strain mutant used for the above chimeric HCV genome specifically includes a mutation of T1286I, T2188A, R2198H, S2210I, T2496I, R2895G or R2895K in the S310A strain, and preferably includes a mutation of R2198H, S2210I or R2895K.
- the S310A strain mutant includes SEQ ID NO: 49 (full genome base sequence including a mutation of S2210I), SEQ ID NO: 50 (full genome base sequence including a mutation of R2198H), or SEQ ID NO: 51 (mutation of R2895K). It may be a nucleic acid comprising the base sequence shown in (full genome base sequence).
- the chimeric HCV genome includes, for example, a base sequence encoding the core protein of hepatitis C virus, a base sequence encoding the E1 protein, a base sequence encoding the E2 protein, and a base sequence encoding the p7 protein.
- the base sequence encoding the NS3 protein consisting of the sequence of base numbers 3437-5329, the base sequence encoding the NS4A protein consisting of the sequence of base numbers 5330-5491, and the NS4B protein consisting of the sequence of base numbers 5492-6274 in SEQ ID NO: 1.
- the chimeric HCV genome of the present invention also encodes a Core protein of, for example, a hepatitis C virus genome other than the S310A strain (for example, an existing strain of hepatitis C virus or an HCV genome of a genotype other than genotype 3a).
- a part of the base sequence encoding the NS2 protein consisting of the sequence of base numbers 2786 to 3436 of SEQ ID NO: 1 in the sequence listing, and other than the S310A strain A chimeric NS2 in which a part of the base sequence encoding the NS2 protein of the genome of hepatitis C virus (for example, the genome of an existing strain of hepatitis C virus or the genotype of HCV other than genotype 3a) is linked
- the chimeric HCV genome of the present invention may contain a 5 ′ untranslated region of the HCV genome of genotype 3a, for example, a 5 ′ untranslated region containing the nucleotide sequence of nucleotide numbers 1 to 340 of SEQ ID NO: 1 at the 5 ′ end.
- the HCV genome other than the HCV genome of S310A strain (SEQ ID NO: 1), for example, the genome of HCV existing strains of various genotypes or the 5 ′ untranslated region of the hepatitis C virus genome of genotypes other than genotype 3a May be included at the 5 ′ end.
- FIG. 16 An example of such a chimeric HCV genome is shown in FIG. 16, for example, the 5 ′ untranslated region of J6CF strain, JFH-1 strain or TH strain, the structural gene (Core to p7) and the N-terminus of its NS2 gene.
- Part of the sequence for example, the sequence encoding the 1st to 16th amino acid sequences from the N-terminus of the NS2 protein
- the sequence of the C-terminal side of the NS2 gene of the S310A strain for example, the sequence from the N-terminus of the NS2 protein
- NS3 to NS5B a non-structural gene other than NS2 gene
- These may include mutations of T1286I, T2188A, R2198H, S2210I, T2496I, R2895G or R2895K.
- the present invention also provides a chimeric full genome replicon RNA containing these chimeric HCV genomes.
- the chimeric HCV genome is prepared, for example, by recombining the coding sequences of Core protein, E1 protein, E2 protein and p7 protein, which are structural genes of the genome of the S310A strain mutant, with the structural genes of other HCV strains. be able to.
- This basic technique is described in, for example, Wakita et al., Nature Medicine, 2005, Vol. 11, p. 791-796; Lindenbach et al., Science, 2005, Vol. 309, p. 623-626; Pietschmann et al., Proc. Natl. Acad. Sci. U. S. A. 2007, volume 103, p. 7408-7413.
- HCV is classified into 6 types from genotype 1 to genotype 6, and each type is further classified into several subtypes.
- an existing strain of HCV that is already known, specifically, as a genotype 1a HCV strain, the H77 strain (GenBank accession number AF011751), and as a genotype 1b HCV strain, the J1 strain (GenBank accession number D89815), Con1 strain (GenBank accession number AJ238799, Con-1 strain, sometimes referred to as con1 strain), TH strain (Wakita et al., The Journal of Biological Chemistry, 1994, Vol. 269).
- Genotype 2a HCV strains include JFH-1 strain (GenBank accession number AB047639, sometimes referred to as JFH1 strain), J6CF strain (GenBank accession number AF177036), JCH-1 strain (GenBank accession number AB047640).
- JCH-2 strain (GenBank accession number AB047641), JCH-3 strain (GenBank accession number AB047642), JCH-4 strain (GenBank accession number AB047643), JCH-5 strain (GenBank accession number AB047644) and JCH -6 strains (GenBank accession number AB047645) are known.
- HC-J8 strain (GenBank accession number D01221) and the like as HCV strains of genotype 2b
- NZL1 strain GenBank accession number D17763
- K3a / 650 strain (GenBank accession) as HCV strains of genotype 3a No.
- the chimeric HCV genome described above is an NS2 derived from an HCV strain other than the Core protein, E1 protein, E2 protein, and p7 protein derived from an HCV strain other than the S310A strain variant, or an S310A strain variant or an S310A strain variant.
- the NS2 protein may be a chimeric NS2 protein (chimeric NS2 protein) composed of the NS2 protein of the S310A strain variant and the NS2 protein derived from the S310A strain and HCV strains other than the variant.
- the HCV strain other than the HCV strain S310A and other than the S310A strain mutant is preferably an existing strain of the HCV described above.
- NS2 protein consisting of the full-length amino acid sequence.
- the NS2 protein may be derived from the S310A strain variant, or a part of the NS2 protein derived from the HCV strain other than the S310A strain variant and the S310A strain variant. It may be a chimeric NS2 protein consisting of the rest of the NS2 protein.
- the chimeric NS2 protein has the same function as the non-chimeric NS2 protein.
- a part of the NS2 protein derived from the HCV strain other than the S310A strain mutant consists of a base sequence encoding from the N-terminal amino acid residue to the 16th amino acid residue of the NS2 protein
- the S310A strain and its The rest of the NS2 protein derived from the mutant consists of a base sequence encoding from the 17th amino acid residue to the C-terminal amino acid residue from the N-terminus.
- the nucleic acid of the chimeric HCV genome preferably further contains a 5 'UTR on the 5' side of the base sequence encoding the Core protein and a 3 'UTR on the 3' side of the region encoding the NS5B protein.
- the 5 ′ UTR and / or 3 ′ UTR may be a sequence derived from any HCV strain, but preferably a 5 ′ UTR derived from an HCV strain other than the S310A strain mutant and a 3 ′ UTR derived from the S310A strain mutant. is there.
- the HCV strain other than the S310A strain mutant that is, the existing strain of HCV is preferably a strain belonging to genotype 1a, 1b or 2a.
- Strains belonging to genotype 1a include, for example, the H77 strain.
- Strains belonging to genotype 1b include, for example, TH strain, Con1 strain, J1 strain and derivatives thereof.
- Strains belonging to genotype 2a include, for example, JFH-1 strain, J6CF strain and the like. Preferred strains are JFH-1 strain, J6CF strain or TH strain, and particularly preferred is JFH-1 strain.
- the genome base sequence information of HCV strains other than the S310A strain and its mutants can be obtained from the above literature or GenBank.
- the nucleic acid of the above chimeric HCV genome is, for example, a chimeric nucleic acid derived from the J6CF strain and the S310A strain, and contains at least one mutation selected from the group consisting of T1286I, S2210I, R2198H, and R2895K.
- the nucleic acid of the chimeric HCV genome is, for example, a chimeric nucleic acid derived from the JFH-1 strain and the S310A strain, and contains at least one mutation selected from the group consisting of T1286I, S2210I, R2198H, and R2895K. Yes.
- the nucleic acid of the above chimeric HCV genome is, for example, a chimeric nucleic acid derived from the TH strain and the S310A strain, and contains at least one mutation selected from the group consisting of T1286I, S2210I, R2198H, and R2895K.
- FIG. 16 shows the structure of the HCV genome of S310A strain mutant (S310A strain introduced with adaptive mutation of R2198H, S2210I or R2895K), J6CF strain, JFH-1 strain and TH strain, and examples of chimeric nucleic acids.
- a chimeric HCV genome comprising a non-structural gene of S310A strain mutant (S310A strain introduced with adaptive mutation of R2198H, S2210I or R2895K) and a structural gene of J6CF strain, JFH-1 strain or TH strain (respectively J6CF / S310A, JFH-1 / S310A, TH / S310A).
- the 5 ′ untranslated region is derived from the same HCV strain as the structural region (J6CF strain, JFH-1 strain, or TH strain, respectively).
- the 3 ′ untranslated region is derived from the same S310A strain as the nonstructural region
- the NS2 coding sequence is derived from the NS2 sequence derived from the same HCV strain as the structural region (J6CF strain, JFH-1 strain or TH strain, respectively) and the S310A strain. Chimera with the NS2 sequence.
- These chimeric nucleic acids have an adaptive mutation of R2198H, S2210I or R2895K.
- the present invention provides an HCV virus genome containing the above-described chimeric HCV genome nucleic acid, a hepatitis C virus containing the above chimeric HCV genome nucleic acid as a virus genome, an HCV full genome replicon RNA, an expression vector, or a chimeric HCV particle.
- the chimeric HCV particles can be produced with high efficiency in a cell culture system and have a high infectivity.
- a chimeric HCV gene can be prepared by performing PCR using a vector obtained by cloning cDNA of each HCV genomic RNA as a template, using synthetic DNA as a primer, amplifying and ligating necessary regions of each HCV gene. it can.
- the chimeric HCV gene cDNA can be ligated to an appropriate restriction enzyme site downstream of a promoter such as T7 promoter to produce an expression vector for synthesizing HCV genomic RNA.
- a promoter such as T7 promoter
- RNA transcribed from this expression vector is introduced into HCV-sensitive cells (eg, Huh7 cells), viral replication and packaging occur, and infectious HCV particles can be produced.
- the HCV full genome replicon RNA containing the chimeric HCV gene has the ability to replicate in cells, the ability to produce HCV particles, and the infectivity of the produced HCV particles can be confirmed by the above method.
- the present invention also provides a method for screening an anti-hepatitis C virus substance using the above HCV subgenomic replicon RNA, HCV full genome replicon RNA or hepatitis C virus particles.
- the cultured cells into which the above HCV subgenomic replicon RNA has been introduced can be used for screening for compounds that inhibit the replication of the HCV subgenomic replicon RNA. That is, an anti-HCV substance can be screened by culturing cultured cells into which the above HCV subgenomic replicon RNA is introduced in the presence of a test substance and detecting the replicon RNA in the obtained culture.
- the culture includes a culture supernatant and a cell lysate. In this case, when the replicon RNA is not present in the culture or is less than in the absence of the test substance, it can be determined that the test substance can inhibit the replication of the HCV subgenomic replicon RNA.
- 57 nucleotides of the 5 ′ untranslated region (5′UTR) (SEQ ID NO: 2) and the Core protein coding sequence (SEQ ID NO: 3) of the S310A strain or a variant thereof , Luciferase gene, EMCV IRES sequence, NS3 protein coding sequence (SEQ ID NO: 8), NS4A protein coding sequence (SEQ ID NO: 9), NS4B protein coding sequence (SEQ ID NO: 10), NS5A protein coding sequence (SEQ ID NO: 11), NS5B protein HCV subgenomic replicon RNA consisting of RNA linked in the order of coding sequence (SEQ ID NO: 12) and 3 ′ untranslated region (3′UTR) (SEQ ID NO: 13) is introduced into Huh7 cells, followed by addition of a test substance The luciferase activity is measured 48 to 72 hours later. It can be determined that a test substance that can suppress
- HCV particles obtained by introducing the HCV full-genome replicon RNA or its nucleic acid into cultured cells (typically HCV-sensitive cells) are neutralizing antibodies and compounds that inhibit HCV infection.
- HCV-sensitive cells typically HCV-sensitive cells
- HCV particles obtained by introducing the HCV full-genome replicon RNA or its nucleic acid into cultured cells (typically HCV-sensitive cells) are neutralizing antibodies and compounds that inhibit HCV infection.
- it can also be suitably used for vaccines and antigens for producing anti-HCV antibodies.
- HCV particles In order to use the HCV particles for screening for substances that inhibit HCV infection or replication, cells that produce the HCV particles are cultured in the presence or absence of a test substance, or the HCV particles are used. Culturing with HCV-sensitive cells, that is, culturing a mixture of HCV particles and HCV-sensitive cells, or culturing HCV-infected cells infected with the HCV particles, and detecting HCV replicon RNA or HCV particles in the resulting culture A method is mentioned.
- detection refers to quantifying the amount of HCV replicon RNA or HCV particles in the culture. In this case, when the HCV replicon RNA and HCV particles are not present in the culture or are less than in the absence of the test substance, it can be evaluated that the test substance can inhibit HCV infection and replication. .
- HCV sensitive cells are cultured with the HCV particles, and HCV replicon RNA or HCV particles in the obtained culture are detected.
- Anti-HCV substances can be screened by determining whether to suppress replication of replicon RNA or formation of HCV particles.
- Detection of HCV replicon RNA in the culture includes measuring the function of a foreign gene linked to the HCV replicon RNA, that is, the function expressed by the expression of the gene.
- HCV replicon RNA can be detected by measuring the enzyme activity.
- HCV replicon RNA can also be detected by quantifying the amount of RNA replicated by quantitative RT-PCR.
- HCV particles In order to detect the presence of HCV particles in the culture, detection using an antibody against a protein (for example, Core protein, E1 protein or E2 protein) constituting HCV particles released in the culture supernatant, or infection Detection of the presence of nonstructural proteins in cells by immunostaining with antibodies to nonstructural proteins, or RT-PCR method using specific primers for HCV genomic RNA contained in HCV particles in culture supernatant
- a protein for example, Core protein, E1 protein or E2 protein constituting HCV particles released in the culture supernatant
- infection Detection of the presence of nonstructural proteins in cells by immunostaining with antibodies to nonstructural proteins, or RT-PCR method using specific primers for HCV genomic RNA contained in HCV particles in culture supernatant
- the presence of HCV particles can also be indirectly detected by amplifying and detecting by.
- HCV full genome replicon RNA used for screening contains a foreign gene, specifically, for example, HCV 5′UTR of S310A mutant, 57 nucleotides of Core protein coding sequence, luciferase gene, EMCV IRES sequence Core protein coding sequence, E1 protein coding sequence, E2 protein coding sequence, p7 protein coding sequence, NS2 protein coding sequence, NS3 protein coding sequence, NS4A protein coding sequence, NS4B protein coding sequence, NS5A protein coding sequence, NS5B protein coding sequence HCV full-genome replicon RNA consisting of RNA in which 3′UTRs are sequentially linked in the direction from 5 ′ to 3 ′.
- HCV full-genome replicon RNA consisting of RNA in which 3′UTRs are sequentially linked in the direction from 5 ′ to 3 ′.
- the linking site may contain an additional sequence such as a restriction enzyme site.
- the HCV full-genome replicon RNA is introduced into Huh7 cells to obtain HCV particles, the HCV particles are infected with HCV-sensitive cells, and at the same time, a test substance is added, and luciferase activity is measured 48 to 72 hours later. It can be determined that a substance that suppresses luciferase activity compared to the absence of a test substance has an action of suppressing HCV infection.
- the anti-HCV substance is selected as one capable of suppressing viral infection or replication.
- a viral genome containing the HCV full genome replicon RNA or a nucleic acid thereof and a hepatitis C virus containing the HCV full genome replicon RNA or a nucleic acid thereof as a viral genome can also be used.
- the present invention provides a hepatitis C virus vaccine (HCV vaccine) containing the above hepatitis C virus particles (HCV particles).
- HCV vaccine hepatitis C virus vaccine
- the HCV particles or a part thereof can be used as a vaccine as it is, but it is preferably used after being attenuated or inactivated by a method known in the art.
- Inactivation of the virus can be achieved by, for example, adding an inactivating agent such as formalin, ⁇ -propiolactone, glutardialdehyde, etc. to the virus suspension and reacting with the virus (Appiahgari, M. et al. B. & Vrati, S., Vaccine, 2004, Vol. 22, pp. 3669-3675).
- the HCV vaccine can be prepared for administration as either a solution or a suspension.
- it can be prepared in the form of a solid (eg, a lyophilized preparation) suitable for dissolution or suspension in a liquid so that it can be reconstituted immediately prior to use.
- a solid eg, a lyophilized preparation
- Such solids or preparations may be emulsified or encapsulated in liposomes.
- Active immunogenic ingredients such as HCV particles are pharmaceutically acceptable and excipients compatible with the active ingredients can often be mixed.
- Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol and the like, or mixtures thereof.
- the HCV vaccine may contain small amounts of adjuvants (eg, humidifiers or emulsifiers), pH buffering agents, and / or adjuvants that enhance vaccine efficacy.
- adjuvants eg, humidifiers or emulsifiers
- Adjuvant is a non-specific stimulator of the immune system. They enhance the host immune response to the HCV vaccine. Accordingly, in a preferred form, the HCV vaccine includes an adjuvant. The efficacy of an adjuvant can be determined by measuring the amount of antibody produced by administering a vaccine composed of HCV particles.
- adjuvants examples include, but are not limited to: Aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (referred to as CGP11637, nor-MDP) N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, MTP-PE and And RIBI).
- RIBI contains three components extracted from bacteria: monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (HPL + TDM + CWS) in a 2% squalene / Tween® 80 emulsion.
- one or more compounds having adjuvant activity can be added to the HCV vaccine.
- adjuvants known in the art include Freund's complete and incomplete adjuvants, vitamin E, nonionic block polymers, muramyl dipeptides, saponins, mineral oils, vegetable oils and Carbopol.
- Adjuvants particularly suitable for mucosal application include, for example, E. coli heat-labile toxin (LT) or cholera toxin (CT).
- Other suitable adjuvants include, for example, aluminum hydroxide, aluminum phosphate or aluminum oxide, oily emulsions (eg Bayol® or Marcol 52®), saponins or vitamin E solubilizate.
- the HCV vaccine is usually administered parenterally, for example, by injection, such as subcutaneous injection or intramuscular injection.
- Alternative formulations suitable for other modes of administration include suppositories and in some cases oral formulations.
- HCV vaccine and pharmaceutically acceptable carriers or diluents include stabilizers, carbohydrates (for example, sorbitol, mannitol, starch, Sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing substances such as bovine serum or skim milk, and buffers (eg, phosphate buffer).
- carbohydrates for example, sorbitol, mannitol, starch, Sucrose, glucose, dextran
- proteins such as albumin or casein
- protein-containing substances such as bovine serum or skim milk
- buffers eg, phosphate buffer
- binders and carriers used for suppositories can include, for example, polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 50%, preferably 1% -20%.
- Oral prescription drugs contain commonly used excipients. Examples of the excipient include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like.
- the HCV vaccine takes the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders, 10% -95%, preferably 25% -70% active ingredient (HCV Particles or a part thereof).
- the HCV vaccine is administered in a manner suitable for the dosage form and in such an amount as to have a prophylactic and / or therapeutic effect.
- the amount of antigen to be administered usually ranges from 0.01 ⁇ g to 100,000 ⁇ g per dose, which depends on the patient being administered, the ability to synthesize antibodies in the patient's immune system, and the degree of protection desired. Depends on. It also depends on the route of administration such as oral, subcutaneous, intradermal, intramuscular and intravenous routes.
- the HCV vaccine can be given on a single dose schedule or a combined dose schedule.
- it is a combined dosing schedule.
- 1 to 10 individual doses are given at the start of the inoculation, followed by time intervals required to maintain and / or enhance the immune response, for example 1 to 2 as the second dose.
- Another dose can be given after 4 months. If necessary, subsequent administrations can be given after several months.
- the dosage regimen will also be determined, at least in part, by the individual's needs and will depend on the judgment of the physician.
- There is a method in which the HCV vaccine is administered to a healthy person induces an immune response against HCV in the healthy person, and is used prophylactically against a new HCV infection.
- a method of use as a therapeutic vaccine that eliminates HCV by administering to a patient infected with HCV and inducing a strong immune response against HCV in vivo.
- the HCV particles are useful as an antigen for producing anti-HCV antibodies.
- Antibodies can be produced by administering the HCV particles to mammals or birds. Examples of mammals include mice, rats, rabbits, goats, sheep, horses, cows, guinea pigs, dromedaries, bactrian camels, and llamas. Dromedary, Bactrian camel and llama are suitable for preparing an antibody consisting only of H chain. Examples of avian animals include chickens, geese, and ostriches. Sera of animals administered with the HCV particles can be collected, and antibodies can be obtained according to known methods.
- the present invention also provides these anti-HCV antibodies.
- the anti-HCV antibodies can be used as neutralizing antibodies capable of inactivating HCV.
- a hybridoma that produces monoclonal antibody-producing cells can be produced using the cells of an animal immunized with the HCV particles.
- a method for producing a hybridoma is well known, and a method described in Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, 1988) can be used.
- Monoclonal antibody-producing cells may be prepared by cell fusion, or may be prepared by other methods such as immortalization of B lymphocytes by introduction of oncogene DNA or infection with Epstein-Barr virus.
- Monoclonal antibodies and polyclonal antibodies obtained by these methods are useful for diagnosis, treatment, and prevention of HCV.
- the antibody produced using the HCV particles as an antigen can be administered as a pharmaceutical together with a pharmaceutically acceptable solubilizer, additive, stabilizer, buffer, and the like.
- the administration route may be any administration route, but is preferably subcutaneous, intradermal, or intramuscular administration, and more preferably intravenous administration.
- the present invention also provides a method for treating or preventing HCV comprising administering the vaccine or antibody of the present invention as described above to a subject in need of treatment.
- the present invention also provides a pharmaceutical composition comprising the vaccine or antibody according to the present invention.
- the pharmaceutical composition may contain a pharmaceutically acceptable carrier such as a solubilizer, an additive, a stabilizer, and a buffer.
- Example 1 Construction of HCV subgenomic replicon RNA expression vector of wild-type S310A strain
- An HCV virus strain was isolated from a 71-year-old acute hepatitis C patient infected with genotype 3a HCV and named S310A strain. The patient was diagnosed as infected with genotype 3a HCV at the age of 59 and had undergone liver transplantation due to cirrhosis four years later. Specifically, RNA was extracted and purified from patient sera using Isogen-LS (Nippon Gene), and cDNA was synthesized with random hexamer primers.
- Isogen-LS Natural Gene
- PCR primers were designed based on the conserved sequences of four known genotype 3a HCV genomes (GenBank accession numbers AF046866, D28917, X76918, and D17763), and 9 fragments were synthesized using the synthesized cDNA.
- the cDNA was amplified separately.
- the 5 ′ end sequence for which it was difficult to obtain an amplification product was obtained using the 5 ′ RACE method.
- Each fragment was cloned into a sequencing cloning vector, pGEM-T EASY (Promega).
- the base sequences of these clones were analyzed by a conventional method, and the full-length genomic RNA sequence of the S310A strain was determined.
- a cDNA fragment corresponding to this full-length genomic RNA was synthesized by a conventional method.
- the cDNA sequence corresponding to the full-length genomic RNA sequence of S310A strain is shown in SEQ ID NO: 1.
- the base sequence of the full-length genome of the wild type S310A strain is shown in SEQ ID NO: 1. Further, the 5′UTR base sequence of the S310A strain is SEQ ID NO: 2, the Core protein coding sequence is SEQ ID NO: 3, the E1 protein coding sequence is SEQ ID NO: 4, the E2 protein coding sequence is SEQ ID NO: 5, and the p7 protein coding sequence is SEQ ID NO: 6, NS2 protein coding sequence is SEQ ID NO: 7, NS3 protein coding sequence is SEQ ID NO: 8, NS4A protein coding sequence is SEQ ID NO: 9, NS4B protein coding sequence is SEQ ID NO: 10, NS5A protein coding sequence is SEQ ID NO: 11, NS5B protein coding The sequence is shown in SEQ ID NO: 12, and the nucleotide sequence of 3′UTR is shown in SEQ ID NO: 13, respectively.
- the amino acid sequence of the HCV precursor protein (polyprotein) encoded by the base sequence of SEQ ID NO: 1 (the full-length genome sequence of the wild type S310A strain) is shown in SEQ ID NO: 14.
- the amino acid sequence shown in SEQ ID NO: 14 is encoded by base numbers 341 to 9406 (including a stop codon) of the base sequence of SEQ ID NO: 1.
- the amino acid sequence of the region from NS3 protein to NS5B protein in the precursor protein of S310A strain is shown in SEQ ID NO: 15. This amino acid sequence of SEQ ID NO: 15 (NS3 to NS5B region) corresponds to amino acids 1033 to 3021 of the amino acid sequence shown in SEQ ID NO: 14.
- HCV subgenomic replicon RNA expression vector pS310ASGR-Neo was performed based on the procedures shown in Kato et al. (Gastroenterology, 2003, Vol. 125, p. 1808-1817) and International Publication No. 04/104198. It was.
- cDNA of the full-length genomic RNA (FIG. 1A) of the wild type S310A strain was inserted under the control of the T7 promoter in the plasmid vector pUC19 to prepare a recombinant plasmid pS310A.
- Plasmid pS310ASGR-Neo was constructed by substitution with EMCV IRES (internal ribosome binding site of encephalomyocarditis virus). This was named HCV subgenomic replicon RNA expression vector pS310ASGR-Neo.
- FIG. 1B shows the structure of the HCV subgenomic replicon RNA expression vector pS310ASGR-Neo.
- the expression vector pS310ASGR-Neo 5'UTR, 57 nucleotides (HCV IRES) from the N-terminus of the Core protein coding sequence, NS3 to NS5B protein coding sequence and 3'UTR are sequences derived from the S310A strain.
- T7 means T7 promoter.
- the T7 promoter is a sequence element required for transcription of HCV subgenomic replicon RNA from each expression vector using T7 RNA polymerase.
- “Neo” indicates a neomycin resistance gene
- “EMCV IRES” indicates an internal ribosome binding site of encephalomyocarditis virus.
- C is the Core protein coding sequence
- E1 is the E1 protein coding sequence
- E2 is the E2 protein coding sequence
- p7 is the p7 protein coding sequence
- NS2 is the NS2 protein coding sequence
- NS3 is NS3
- “4A” represents the NS4A protein coding sequence
- “4B” represents the NS4B protein coding sequence
- “NS5A” represents the NS5A protein coding sequence
- NS5B represents the NS5B protein coding sequence.
- HCV subgenomic replicon RNA prepared from the expression vector pS310ASGR-Neo is RNA in which the region downstream from the T7 promoter is transcribed, as shown in FIG. 1C.
- EcoRI “EcoRI”, “PmeI”, “SnaBI” and “XbaI” in the figure indicate restriction enzyme sites. In FIG. 9, 10, 12, 14, and 16, the same notation is used.
- cDNA of S310A subgenomic replicon RNA is linked downstream of the T7 promoter.
- the cDNA base sequence of HCV subgenomic replicon RNA of S310A strain is shown in SEQ ID NO: 16.
- Example 2 Preparation of HCV subgenomic replicon RNA of wild type S310A strain
- the expression vector pS310ASGR-Neo constructed in Example 1 was cleaved with a restriction enzyme XbaI.
- Mung Bean Nuclease 20U total reaction volume: 50 ⁇ L was added to 10-20 ⁇ g of the XbaI cleaved fragment, and the mixture was incubated at 30 ° C. for 30 minutes.
- Mung Bean Nuclease is an enzyme that catalyzes a reaction that selectively degrades and smoothes a single-stranded portion in double-stranded DNA.
- RNA transcription by RNA polymerase is performed using the above XbaI cleaved fragment as a template DNA as it is, a replicon RNA in which 4 bases of CUAG, which is a part of the recognition sequence of XbaI, are added to the 3 ′ end is synthesized. End up. Therefore, in this example, the XbaI cleavage fragment was treated with Mung Bean Nuclease to remove 4 bases of CTAG from the XbaI cleavage fragment.
- the Mbaan Nuclease-treated solution containing the XbaI-cleaved fragment is subjected to protein removal treatment according to a conventional method to purify the XbaI-cleaved fragment from which 4 CTAG bases have been removed, and use this in the next reaction.
- Template DNA was used. From this template DNA, RNA was synthesized in vitro by a transcription reaction using T7 promoter using MEGAscript (registered trademark) of Ambion. Specifically, 20 ⁇ L of a reaction solution containing 0.5 to 1.0 ⁇ g of template DNA was prepared based on the manufacturer's instructions and reacted at 37 ° C. for 3 to 16 hours.
- RNA synthesis After completion of RNA synthesis, DNase I (2U) was added to the reaction solution and reacted at 37 ° C. for 15 minutes to remove the template DNA, and further RNA extraction with acidic phenol was performed to transcribe from pS310ASGR-Neo.
- the HCV subgenomic replicon RNA (FIG. 1C) (SEQ ID NO: 16) of the wild type S310A strain was obtained.
- Example 3 Establishment of HCV subgenomic replicon cell clone of S310A strain Huh7 cells by electroporation of HCV subgenomic replicon RNA (1 ⁇ g, 3 ⁇ g, 10 ⁇ g or 30 ⁇ g) of wild type S310A strain prepared in Example 2 Introduced.
- Huh7 cells (3 ⁇ 10 6 cells) that had been subjected to electroporation were seeded in a culture dish, cultured for 16 to 24 hours, and then G418 (neomycin) was added to the culture dish. Thereafter, the culture was continued while changing the culture medium twice a week.
- HCV subgenomic replicon RNA-introduced cells For the HCV subgenomic replicon RNA-introduced cells in which colony formation was observed, surviving cell colonies were further cloned from the culture dish after 21 days of culture and the culture was continued. By cloning such colonies, 10 cell clones could be established. These cell clones were named S310A subgenomic replicon-replicating cells, and each clone was assigned a clone number (number: 1-10). In the established cell clone, the introduced HCV subgenomic replicon RNA of the S310A strain is autonomously replicated.
- Example 4 Quantification of copy number of HCV subgenomic replicon RNA in S310A subgenomic replicon-replicating cells Using established 10 clones (clone numbers: 1 to 10) of S310A subgenomic replicon-replicating cells, The copy number of genomic replicon RNA was quantified. Quantification of the copy number of the HCV subgenomic replicon RNA is described in Takeuchi et al. (Gastroenterology, 1999, 116, 636-642) and Kato et al. (Gastroenterology, 2003, 125, p. 1808-). 1817).
- This method is a detection system using the TaqMan probe method (Parkin Elmer, Applied Biosystems). Specifically, first, total RNA was extracted from S310A subgenomic replicon-replicating cells by a conventional method. Next, rTth DNA polymerase was used to synthesize cDNA from total RNA, and using the synthesized cDNA as a template, primers 5′-CGGGAGAGCATCATGTGG-3 ′ (SEQ ID NO: 24) and 5′-AGTACCACAAGGCCCTTTC-3 ′ (SEQ ID NO: 25) was used for PCR amplification.
- HCV subgenomic replicon RNA expression vector of the JFH-1 strain (its structure is the 5 ′ untranslated region (5′UTR) of the HCV of the JFH-1 strain and the 57 nucleotide of the 5 ′ end of the Core protein coding region).
- HCV subgenomic replicon RNA Sequence, neomycin resistance gene, EMCV IRES sequence, base sequence encoding NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein, and 3 'untranslated region (3'UTR) on the 5' side
- HCV subgenomic replicon RNA is described in Kato et al. (Gastroenterology, 2003, 125, pp. 1808-1817) and International Publication No. 04 / This was carried out based on the method described in No. 104198. Introduction into Huh7 cells and copy number quantification were performed in the same manner as described above.
- Quantitative results are shown in FIG.
- the numbers on the horizontal axis in the figure indicate the clone numbers of S310A subgenomic replicon-replicating cell clones.
- the vertical axis represents the number of copies of HCV subgenomic replicon RNA in the cell per 1 ⁇ g of total RNA of the cell.
- the numerical value of the bar graph upper part shows the copy number of HCV subgenomic replicon RNA in each cell.
- the established 10 clones of S310A subgenomic replicon-replicating cells were detected with RNA copy numbers equal to or higher than those of JFH-1 subgenomic replicon-replicating cells. It was shown that the HCV subgenomic replicon RNA derived from the S310A strain has almost the same or higher replication capacity as the HCV subgenomic replicon RNA of the JFH-1 strain.
- Example 5 Effect of antiviral agent on replicon RNA replication in S310A subgenomic replicon-replicating cells The effect of antiviral agent on replicon RNA replication in established S310A subgenomic replicon-replicating cells (clone) was examined.
- S310A subgenomic replicon-replicating cells (clone) and JFH-1 subgenomic replicon-replicating cells were seeded in a 24-well plate at 5 ⁇ 10 4 per well, and the next day, interferon- ⁇ (IFN- ⁇ ), NS3 protease inhibitors VX-950 (teleprevir) (Lin et al., Journal of Biological Chemistry, 2004, 279, p. 17508-17514) and BILN-2061 (Daniel et al., Nature, 2003, 426) , P.186-189), and NS5B polymerase inhibitor JTK-109 (Hirashima et al., Journal of Medicinal Chemistry, 2006, Vol.
- the results are shown in FIGS.
- the horizontal axis of the figure indicates the concentration of the added inhibitor.
- the vertical axis shows the rate of change in the amount of HCV RNA, which is the amount when the amount of subgenomic replicon RNA per 1 ⁇ g of total RNA in cells without an inhibitor (0 IU or 0 M) is 100%.
- the ratio (%) of the subgenomic replicon RNA amount when an inhibitor is added is shown.
- the bar graph shows that for each inhibitor concentration, the leftmost bar (white) is the JFH-1 subgenomic replicon replicating cell, the second bar from the left (light gray) is the S310A subgenomic replicon replicating cell clone 6, the third from the left
- the bar black shows the results for the S310A subgenomic replicon replicating cell clone 9
- the rightmost bar shows the results for the S310A subgenomic replicon replicating cell clone 10.
- NS3 protease inhibitors VX-950 and BILN-2061 were found to inhibit replication of JFH-1 subgenomic replicon, but did not inhibit replication of S310A subgenomic replicon (FIGS. 5 and 6). . This suggests that genotype 3a NS3 protease is different from genotype 2a NS3 protease and is not inhibited by existing NS3 protease inhibitors.
- JFH-1 subgenomic replicon is not subject to RNA replication inhibition by JTK-109, but S310A subgenomic replicon is caused by JTK-109, respectively.
- Significant inhibition of RNA replication was observed (FIG. 7).
- PSI-6130 was allowed to act, both RNA replication of JFH-1 and S310A subgenomic replicon inhibited in a concentration-dependent manner (FIG. 8).
- the subgenomic replicon of genotype 3a of the present invention is suitable as a tool for evaluating a drug that inhibits replication of genotype 3a.
- the replication ability of the subgenomic replicon derived from the HCV strain of genotype 3a is affected by factors different from the replication ability of the subgenomic replicon derived from the HCV strain of genotype 2a.
- the structures of the polymerases encoded by the HCV genome differ between genotypes 3a and 2a, and as a result, the effects of the drug compounds may also differ.
- Example 6 Sequence analysis of HCV subgenomic replicon RNA in S310A subgenomic replicon-replicating cells Sequence analysis of HCV subgenomic replicon RNA existing in S310A subgenomic replicon-replicating cells (clone) established in Example 3 was performed. It was.
- HCV subgenomic replicon RNA contained therein was amplified using RT-PCR.
- PCR amplification using cDNA synthesized by reverse transcription from HCV subgenomic replicon RNA as a template 5′-TAATACGACTCACTATAG-3 ′ (SEQ ID NO: 27) and 5′-GCGGCTCACGGACCTTTCAC-3 ′ (SEQ ID NO: 28) are used as primers. did.
- the PCR amplification product was cloned into a cloning vector for sequencing, and sequence analysis was performed by a conventional method.
- Table 1 shows adaptive mutations identified from HCV subgenomic replicon RNA in S310A subgenomic replicon-replicating cells as a result of sequence analysis.
- one site (T1286I: 1286th threonine (T) is isoleucine (I) in the NS3 protein region which is a non-structural region.
- T2188A the 2188th threonine (T) is mutated to alanine (A)
- R2198H the 2198th arginine (R) is mutated to histidine (H)
- S2210I 2210th serine (S) mutated to isoleucine (I)
- NS5B protein region at 3 sites T2496I: 2496th threonine (T) mutated to isoleucine (I)
- R2895G 2895th arginine (R) is mutated to glycine (G)
- R2895K The 2895 arginine (R) are lysine (K) mutation) was found base substitutions causing amino acid substitutions.
- FIG. 9 schematically shows the positions of these amino acid substitutions on the structure of the expression vector pS310ASGR-Neo. The positions of these amino acid substitutions are described based on the full-length amino acid sequence (SEQ ID NO: 14) of the precursor protein of the S310A strain.
- Example 7 Mutation introduction of wild type S310A strain into HCV subgenomic replicon RNA and analysis of its influence on replicon replication ability Base substitution causing amino acid substitution found in Example 6, ie, base mutation is wild type Whether the HCV subgenomic replicon RNA of the S310A strain affects replication in cells was examined as follows.
- Base substitutions that cause amino acid substitutions within the NS3 protein region, T1286I, amino acid substitutions within the NS5A protein region, T2188A, R2198H, and S2210I, and amino acid substitutions within the NS5B protein region, T2496I, R2895G, and R2895K alone Each was introduced into the HCV subgenomic replicon RNA expression vector pS310ASGR-Neo of the S310A strain prepared in Example 1.
- base substitutions that cause T2296I and R2895K, which are amino acid substitutions in the NS5B protein region were simultaneously introduced into the expression vector pS310ASGR-Neo.
- FIG. 10 shows the structure of an HCV subgenomic replicon RNA expression vector into which each of these amino acid substitutions has been introduced.
- the expression vector pS310ASGR-Neo into which T1286I substitution was introduced alone was named “pS310ASGR-Neo T1286I”.
- Expression vectors into which other amino acid substitutions were introduced were also named in the same manner (see FIG. 10).
- the expression vector pS310ASGR-Neo into which T2496I and R2895K substitutions were simultaneously introduced was named “pS310ASGR-Neo T2496I / R2895K”.
- a base substitution was introduced into pS310ASGR-Neo by repeated PCR using a primer containing a base mutation that caused an amino acid substitution to be introduced.
- PCR conditions used here are as follows. First, PCR template DNA, Pyrobest (registered trademark) DNA Polymerase kit (Takara Bio Inc.) attached 10 ⁇ l buffer 5 ⁇ L, 2.5 mM dNTP mixed solution 4 ⁇ L, 100 ⁇ M primer (each forward and reverse) was added to a total volume of 49.75 ⁇ L. Thereafter, 0.25 ⁇ L of Pyrobest (registered trademark) DNA Polymerase (Takara Bio Inc.) was added, and PCR was performed.
- Pyrobest registered trademark
- DNA Polymerase Takara Bio Inc.
- PCR was performed at 98 ° C for 2 minutes after heat denaturation, followed by 25 cycles of 98 ° C for 20 seconds, 55 ° C for 30 seconds and 72 ° C for 3 minutes, followed by final extension at 72 ° C for 10 minutes. Reaction was performed.
- Neo-S4 (5′-TCCTCGTGCTTTACGGTATC-3 ′ (SEQ ID NO: 29)) and 1286R (5′-GTTCCAATGCGACGGTGG-3 ′ (SEQ ID NO: 30) ) was used as a primer and PCR was performed under the above PCR conditions.
- the obtained PCR product was designated as PCR product no. It was set to 1.
- PCR product no. 2 PCR product no. 2.
- PCR product was purified and dissolved in 15 ⁇ L H 2 O.
- Purified PCR product no. 1 and PCR product no. 2 ⁇ l of the DNA of 2 was mixed.
- PCR was performed under the above PCR conditions using Neo-S4 (5′-TCCTCGTGCTTTACGGTATC-3 ′ (SEQ ID NO: 29)) and 5546R (5′-TCCTTTGAACTGGTGGGCTATT-3 ′ (SEQ ID NO: 32)) as primers. It was.
- the obtained PCR product was designated as PCR product no. It was set to 3.
- the PCR product was purified and dissolved in 30 ⁇ L H 2 O.
- PS310ASGR-Neo and purified PCR product no. 3 were digested with restriction enzymes SnaBI and EcoT22I, and each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. These two DNA fragments and DNA Ligation Kit (Takara Bio Inc.) were mixed and the two DNA fragments were ligated.
- the recombinant expression vector thus obtained (having a base substitution that causes amino acid substitution T1286I) was named pS310SGR-Neo T1286I.
- the sequence of the HCV subgenomic replicon RNA synthesized from pS310ASGR-Neo T1286I is shown in SEQ ID NO: 17.
- PCR was performed under the above PCR conditions.
- the obtained PCR product was designated as PCR product no. It was set to 4.
- PCR product no was set to 5.
- PCR product no. 4 and PCR product no. 1 ⁇ L of 5 DNA was mixed.
- PCR was performed under the above PCR conditions using 5240F (5′-TGGGGCCCTGCCAAAAATGAA-3 ′ (SEQ ID NO: 33)) and 7601R (5′-ACTAACGGTGGACCAAAGGT-3 ′ (SEQ ID NO: 36)) as a template DNA.
- the obtained PCR product was designated as PCR product no. It was set to 6.
- the PCR product was purified and dissolved in 30 ⁇ L H 2 O.
- PS310ASGR-Neo and purified PCR product no. 6 were digested with restriction enzymes XhoI and BamHI, and each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. These two DNA fragments and DNA Ligation Kit (Takara Bio Inc.) were mixed and the two DNA fragments were ligated.
- the recombinant expression vector thus obtained (having a base substitution that causes amino acid substitution T2188A) was named pS310ASGR-Neo T2188A.
- the sequence of the HCV subgenomic replicon RNA synthesized from pS310SAGR-Neo T2188A is shown in SEQ ID NO: 21.
- PCR product no was designated as PCR product no. It was set to 7.
- PCR product no was set to 8.
- PCR product no. 7 and PCR product no. 1 ⁇ L of 8 DNAs were mixed.
- PCR was carried out under the above PCR conditions using 5240F (5′-TGGGGCCCTCCAAAAATGAA-3 ′ (SEQ ID NO: 33)) and 7601R (5′-ACTAACGGGGACCAAGAGT-3 ′ (SEQ ID NO: 36)) as a template DNA.
- the obtained PCR product was designated as PCR product no. It was set to 9.
- the PCR product was purified and dissolved in 30 ⁇ L H 2 O.
- PS310ASGR-Neo and purified PCR product no. 9 were digested with restriction enzymes XhoI and BamHI, and each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. These two DNA fragments and DNA Ligation Kit (Takara Bio Inc.) were mixed and the two DNA fragments were ligated.
- the recombinant expression vector thus obtained (having a base substitution that causes amino acid substitution R2198H) was named pS310ASGR-Neo R2198H.
- the sequence of the HCV subgenomic replicon RNA synthesized from pS310ASGR-Neo R2198H is shown in SEQ ID NO: 18.
- PCR product no was designated as PCR product no. It was set to 10.
- PCR product no was set to 11.
- PCR product no. 10 and PCR product no. 1 ⁇ L of 11 DNAs were mixed.
- PCR was performed under the above PCR conditions using 7276F (5′-GTACCACCAACTGTCCCATGGA-3 ′ (SEQ ID NO: 39)) and 8579R (5′-CCGCCAGACAAGAAAGTCCCGGGT-3 ′ (SEQ ID NO: 42)) as a template DNA.
- the obtained PCR product was designated as PCR product no. It was set to 12.
- the PCR product was purified and dissolved in 30 ⁇ L H 2 O.
- PS310ASGR-Neo and purified PCR product no. 12 were digested with restriction enzymes XhoI and EcoRV, and each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. These two DNA fragments and DNA Ligation Kit (Takara Bio Inc.) were mixed and the two DNA fragments were ligated.
- the recombinant expression vector thus obtained (having a base substitution that causes amino acid substitution T2496I) was named pS310ASGR-Neo T2496I.
- the sequence of the HCV subgenomic replicon RNA synthesized from pS310ASGR-Neo T2496I is shown in SEQ ID NO: 22.
- PCR product no was set to 13.
- PCR was performed under the above PCR conditions.
- the obtained PCR product was designated as PCR product no. It was set to 14.
- PCR product no. 13 and the PCR product no. 14 ⁇ l of each DNA was mixed.
- PCR was carried out under the above PCR conditions using this template DNA and 7988F (5′-GCTCCGTCTGGGAGGATGTTGC-3 ′ (SEQ ID NO: 43)) and 3X-54R-2a (5′-GCGGCTCACGACCCTTTCAC-3 ′ (SEQ ID NO: 46)) as primers. Went.
- the obtained PCR product was designated as PCR product no. It was set to 15.
- the PCR product was purified and dissolved in 30 ⁇ L H 2 O.
- PS310ASGR-Neo and purified PCR product no. 15 were digested with restriction enzymes EcoRV and MfeI, and each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. These two DNA fragments and DNA Ligation Kit (Takara Bio Inc.) were mixed and the two DNA fragments were ligated.
- the recombinant expression vector thus obtained (having a base substitution that causes amino acid substitution R2895G) was named pS310ASGR-Neo R2895G.
- the sequence of the HCV subgenomic replicon RNA synthesized from pS310ASGR-Neo R2895G is shown in SEQ ID NO: 23.
- PCR product no was set to 16.
- PCR was performed under the above PCR conditions.
- the obtained PCR product was designated as PCR product no. It was set to 17.
- PCR product no. 16 and PCR product no. 17 ⁇ l of DNA was mixed by 1 ⁇ L.
- PCR was carried out under the above PCR conditions using this template DNA and 7988F (5′-GCTCCGTCTGGGAGGATGTTGC-3 ′ (SEQ ID NO: 43)) and 3X-54R-2a (5′-GCGGCTCACGACCCTTTCAC-3 ′ (SEQ ID NO: 46)) as primers. Went.
- the obtained PCR product was designated as PCR product no. It was set to 18.
- the PCR product was purified and dissolved in 30 ⁇ L H 2 O.
- PS310ASGR-Neo and purified PCR product no. 18 were digested with restriction enzymes EcoRV and MfeI, and each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. These two DNA fragments and DNA Ligation Kit (Takara Bio Inc.) were mixed and the two DNA fragments were ligated.
- the recombinant expression vector thus obtained (having a base substitution that causes amino acid substitution R2895K) was named pS310ASGR-Neo R2895K.
- the sequence of the HCV subgenomic replicon RNA synthesized from pS310ASGR-Neo R2895K is shown in SEQ ID NO: 19.
- T2496I and R2895K two amino acid substitutions were introduced into the NS5B protein region.
- the pS310ASGR-Neo T2496I and the purified PCR product no. 18 were digested with restriction enzymes EcoRV and MfeI, and each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. These two DNA fragments and DNA Ligation Kit (Takara Bio Inc.) were mixed and the two DNA fragments were ligated.
- the recombinant expression vector thus obtained (having the base substitution that causes amino acid substitutions T2496I and R2895K) was named pS310ASGR-Neo T2496I / R2895K.
- the sequence of the HCV subgenomic replicon RNA synthesized from pS310ASGR-Neo T2496I / R2895K is shown in SEQ ID NO: 20.
- PCR was performed under the above PCR conditions.
- the obtained PCR product was designated as PCR product no. It was set to 19.
- PCR product no was set to 20.
- PCR product no. 19 Each PCR product was purified and dissolved in 15 ⁇ L H 2 O.
- PCR was carried out under the above PCR conditions using 5240F (5′-TGGGGCCCTCCAAAAATGAA-3 ′ (SEQ ID NO: 33)) and 7601R (5′-ACTAACGGGGACCAAGAGT-3 ′ (SEQ ID NO: 36)) as a template DNA.
- the obtained PCR product was designated as PCR product no. 21.
- the PCR product was purified and dissolved in 30 ⁇ L H 2 O.
- PS310ASGR-Neo and purified PCR product no. 21 was digested with restriction enzymes XhoI and BamHI, and each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. These two DNA fragments and DNA Ligation Kit (Takara Bio Inc.) were mixed and the two DNA fragments were ligated.
- the recombinant expression vector thus obtained (having a base substitution that causes amino acid substitution S2210I) was named pS310ASGR-Neo S2210I.
- the sequence of the HCV subgenomic replicon RNA synthesized from pS310ASGR-Neo S2210I is shown in SEQ ID NO: 54.
- the expression vectors pS310ASGR-Neo, pS310ASGR-Neo T1286I, pS310ASGR-Neo T2188A, pS310ASGR-Neo R2198H, pS310ASGR-NeoS2210I, pS310ASGR24SG95SG96RSGNSRGS HCV subgenomic replicon RNA was synthesized from R2895K and pS310ASGR-Neo T2496I / R2895K using MEGAscript (registered trademark) of Ambion.
- HCV subgenomic replicon RNA About the produced HCV subgenomic replicon RNA, 0.3 ⁇ g of each subgenomic replicon RNA was introduced into Huh7 cells by electroporation. However, 10 ⁇ g of the HCV subgenomic replicon RNA (expressed from pS310ASGR-Neo) of the wild type S310A strain and the T2496I mutant HCV subgenomic replicon RNA (expressed from pS310ASGR-Neo T2496I) were introduced. Huh7 cells subjected to electroporation treatment were seeded in a culture dish and cultured for 16 to 24 hours, and then G418 (neomycin) was added to the culture dish. Thereafter, the culture was continued while changing the culture medium twice a week. After culturing for 21 days from the time of seeding, viable cells were stained with crystal violet.
- wild type is pS310ASGR-Neo
- T1286I is pS310ASGR-Neo T1286I
- T2188A is pS310ASGR-Neo T2188A
- R2198H is pS310ASGR-Neo R2198H
- S2210SG is pS310ASG24S
- PS310ASGR-Neo T2296I “ R2895G ”is pS310ASGR-Neo R2895G
- R2895K is pS310ASGR-Neo R2895K
- T2296I / R2895K is a pS310ASGR-Neo T2496I / R2HCV
- T2296I / R2895K is a pS310ASGR-Neo T2496I / R2HCV
- T2496I has a colony-forming ability comparable to that of the wild type, and there is no difference between the colony-forming ability of “R2895K” and “T2496I / R2895K”, the amino acid substitution T2496I It is considered that the replication ability of genomic replicon RNA is not affected (FIG. 11).
- the autonomous replication ability is maintained or increased when the amino acid substitution (mutation) is introduced into the HCV subgenomic replicon RNA of the wild type S310A strain.
- the amino acid substitution T1286I, R2198H or R2895K was significantly increased.
- Example 8 Evaluation of HCV subgenomic replicon RNA replication efficiency of mutant S310A strain using luciferase gene
- the structures of HCV subgenomic replicon RNA expression vectors pS310ASGR-Luc and HCV subgenomic replicon RNA synthesized from pS310ASGR-Luc are shown in FIG.
- the construction of the expression vector pS310ASGR-Luc was performed based on the procedure shown in the literature of Kato et al. (Journal of Clinical Microbiology, 2005, Vol. 43, p.5679-5684). Specifically, the expression vector p310ASGR-Luc was prepared by replacing the neomycin resistance gene (neo) of the HCV subgenomic replicon expression vector pS310ASGR-Neo with the firefly luciferase gene (Luc) (FIG. 12).
- a neomycin resistance gene (neo) was replaced with a firefly luciferase gene (Luc) in the same manner to produce a mutant of p310ASGR-Luc.
- the mutants of p310ASGR-Luc are p310ASGR-Luc T1286I for the T1286I mutant, p310ASGR-Luc T2188A for the T2188A mutant, p310ASGR-Luc R2198H for the T2188A mutant, and the pS310TSGR22L mutant for the S2210I mutant of S2210I.
- the p310ASGR-Luc T2496I and R2895G mutants were named p310ASGR-Luc R2895G, the R2895K mutant was named p310ASGR-Luc R2895K, and the T2496I / R2895K mutant was named p310ASGR-Luc T2496I / R2895K.
- HCV subgenomic replicon RNA was prepared from wild-type and mutant p310ASGR-Luc using the same method as in Example 2, and 5 ⁇ g of the RNA was introduced into 2 ⁇ 10 6 Huh7 cells by electroporation. Well plates were seeded. The seeded cells were collected after 24 hours and 72 hours, and the luciferase activity was detected using Luciferase assay system (Promega).
- wild type is pS310ASGR-Luc
- T1286I is pS310ASGR-Luc T1286I
- T2188A is pS310ASGR-Luc T2188A
- R2198H is pS310ASGR-Luc R2198H
- S2210SG is pS310ASG-T "PS310ASGR-Luc T2296I”
- R2895G is pS310ASGR-Luc R2895G
- R2895K is pS310ASGR-Luc R2895K
- T2496I / R2895K is pS310ASGR-Luc.
- subgenomic replicon RNA JFH1 / GND in which aspartic acid (D), which is the 2760th amino acid residue in NS5B polymerase of JFH-1 strain, was substituted with asparagine (N) was used as a negative control (Kato). Et al., J. Clin. Microbiol. 2005, 43, p.5679-5684).
- This subgenomic replicon RNA (JFH1 / GND) does not show replication ability 72 hours after introduction due to mutation of NS5B polymerase.
- the vertical axis of the figure shows the relative luminescence intensity of luciferase, and the higher the luminescence intensity, the higher the replication ability.
- the expression of the luciferase gene after 72 hours shows a value higher than JFH1 / GND, it indicates that the gene is continuously amplified (has autonomous replication ability).
- Example 9 Construction of HCV full genome replicon RNA (full-length genomic RNA) expression vector of mutant S310A strain HCV full genome replicon RNA of S310A strain having each amino acid substitution found in Example 6 in cultured cells
- HCV full genome replicon RNA expression vector having a full length HCV genome sequence was constructed.
- the expression vector pS310A prepared in Example 1 (recombinant plasmid in which the cDNA of the full-length genomic RNA of the wild type S310A strain was inserted under the control of the T7 promoter in pUC19) and each pS310ASGR-Neo mutant prepared in Example 7 were used.
- the HCV full-genome replicon RNA expression vector was used.
- pS310A prepared in Example 1 and pS310SGR-Neo T1286I prepared in Example 7 were digested with restriction enzymes SnaBI and BamHI. Each HCV cDNA fragment was fractionated and purified by agarose gel electrophoresis. DNA Ligation Kit (Takara Bio Inc.) was added to and mixed with the DNA fragment of pS310A and each DNA fragment of each pS310ASGR-Neo mutant, and the two DNA fragments were ligated.
- the HCV full genome replicon RNA expression vector having an amino acid substitution thus obtained was named pS310A T1286I.
- HCV full genome replicon RNA expression vectors having amino acid substitutions were named pS310A, T2188A, pS310A, R2198H, pS310A, S2210I, pS310A, T2496I, pS310A, R2895G, and pS310A, R2895K, respectively.
- Example 10 Evaluation of HCV particle production ability of HCV full-genome replicon RNA-introduced cells of mutant S310A strain
- the pS310A prepared in Example 1 and each expression vector prepared in Example 9 were cleaved with the restriction enzyme XbaI, Phenol / chloroform extraction and ethanol precipitation were performed.
- the XbaI cleaved fragment was treated with Mung Bean Nuclease to remove 4 bases of the extra 3 ′ terminal CTAG derived from the XbaI recognition sequence from the XbaI cleaved fragment.
- RNA synthesis After completion of RNA synthesis, DNase (2U) was added to the reaction solution and reacted at 37 ° C. for 15 minutes to remove the template DNA, and further RNA extraction with acidic phenol was carried out to obtain wild type and mutant S310A strains. HCV full genome replicon RNA was obtained.
- HCV full-genome replicon RNAs obtained here have the same nucleotide sequences as the full-length genomic RNA of the wild-type and mutant S310A strains, respectively.
- HCV full genome replicon RNA (wild type S310A strain full length genomic RNA) of wild type S310A strain was introduced with amino acid substitution (mutation) of “S310A”, T1286I, T2188A, T2198H, S2210I, T2496I, R2895G or R2895K, respectively.
- the S310 strain HCV full-genome replicon RNA full-length genomic RNA variant of the S310A strain
- S310A T1286I full-length genomic RNA variant of the S310A strain
- S310A T2188A full-length genomic RNA variant of the S310A strain
- S310A R2198H full-length genomic RNA variant of the S310A strain
- S310A S2210I They are referred to as “S310A T2496I”, “S310A R2895G” and “S310A R2895K”.
- the structure of the HCV full genome replicon RNA (HCV full length genome) of these S310A strains is shown in FIG.
- HCV full genome replicon RNA (10 ⁇ g) of the obtained wild type and mutant S310A was introduced (transfected) into Huh7 cells by electroporation. After the electroporation treatment, Huh7 cells were seeded in a culture dish and cultured for 16 to 24 hours, and then G418 (neomycin) was added to the culture dish. Thereafter, the culture was continued while being passaged twice a week. HCV particle production was confirmed by quantifying the HCV Core protein contained in the culture supernatant over time using an HCV antigen ELISA test kit (Ortho).
- the horizontal axis of the graph represents the cell culture time after the HCV full-genome replicon RNA (full-length RNA) of the wild type and mutant S310A strain was introduced into the cells, and the vertical axis represents the HCV core protein concentration in the cell culture supernatant. .
- T1286I is S310A T1286I
- T2188A is S310A T2188A
- R2198H is S310A R2198H
- S2210I is S310A S2210I
- T2496I is S310A T2496I
- R310G R2895K is S310A R2895K Represents cells into which full-genome replicon RNA (full-length RNA) has been introduced.
- HCV full-genome replicon RNA full-length RNA
- Genotype 3a HCV subgenomic replicon RNA that can be amplified in cultured cells, HCV full-genome replicon RNA that produces genotype 3a infectious HCV particles, and genotype-independent anti-HCV drugs, particularly effects
- the present invention can be used for screening anti-HCV drugs against genotype 3a without a typical therapeutic drug, research for elucidating the replication mechanism or replication efficiency of HCV, and development of an HCV vaccine using HCV particles.
- SEQ ID NO: 1 cDNA sequence of full-length genomic RNA of wild type S310A strain.
- 1 to 340 is the 5 ′ untranslated region (5′UTR)
- 341 to 913 are the Core protein
- 914 to 1489 are the E1 protein
- 1490 to 2596 are the E2 protein
- 2597 to 2785 are the p7 protein
- 2786 -3434 is NS2 protein
- 3437-5329 is NS3 protein
- 5330-5491 is NS4A protein
- 5492-6274 is NS4B protein
- 6275-7630 is NS5A protein
- 7631-9406 is NS5B protein
- positions 9407-9655 are the 3 ′ untranslated region (3′UTR).
- SEQ ID NO: 2 cDNA sequence of 5 ′ untranslated region (5′UTR) of wild-type S310A strain genomic RNA
- SEQ ID NO: 3 cDNA sequence of Core protein coding sequence of wild-type S310A strain genomic RNA
- SEQ ID NO: 4 Wild-type S310A strain CDNA sequence of E1 protein coding sequence of genomic RNA
- SEQ ID NO: 5 cDNA sequence of E2 protein coding sequence of wild type S310A strain genomic RNA
- SEQ ID NO: 6 cDNA sequence of p7 protein coding sequence of wild type S310A strain genomic RNA
- SEQ ID NO: 7 CDNA sequence of NS2 protein coding sequence of wild type S310A strain genomic RNA
- SEQ ID NO: 8 cDNA sequence of NS3 protein coding sequence of wild type S310A strain genomic RNA
- SEQ ID NO: 9 cD of NS4A protein coding sequence of wild type S310A strain genomic RNA
- a sequence SEQ ID NO: 10 cDNA sequence
- SEQ ID NO: 15 amino acid sequence of the region from NS3 protein to NS5B protein in the precursor protein of wild type S310A strain
- SEQ ID NO: 16 cDNA sequence of HCV subgenomic replicon RNA of wild type S310A strain
- SEQ ID NO: 17 pS310ASGR-Neo T1286I CDNA sequence of mutant S310A T1286I HCV subgenomic replicon RNA synthesized from SEQ ID NO: 18: cDNA sequence of mutant S310A R2198H HCV subgenomic replicon RNA synthesized from pS310ASGR-Neo R2198H
- SEQ ID NO: 19 pS310ASGR-Neo R2895K CDNA sequence of mutant S310A R2895K HCV subgenomic replicon RNA synthesized from SEQ ID NO: 20: pS310ASGR- cDNA sequence of mutant S310A T2296I / R2895K HCV subgeno
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Communicable Diseases (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
Abstract
Description
上記NS4Aタンパク質のアミノ酸配列をコードする塩基配列が配列番号1の塩基番号5330~5491の塩基配列を含み、
上記NS4Bタンパク質のアミノ酸配列をコードする塩基配列が配列番号1の塩基番号5492~6274の塩基配列を含み、
上記NS5Aタンパク質のアミノ酸配列をコードする塩基配列が配列番号1の塩基番号6275~7630の塩基配列を含み、
上記NS5Bタンパク質のアミノ酸配列をコードする塩基配列が配列番号1の塩基番号7631~9406の塩基配列を含む、
上記[1]記載の核酸。
(a) 第1286番目のスレオニンのイソロイシンへの置換
(b) 第2188番目のスレオニンのアラニンへの置換
(c) 第2198番目のアルギニンのヒスチジンへの置換
(d) 第2210番目のセリンのイソロイシンへの置換
(e) 第2496番目のスレオニンのイソロイシンへの置換
(f) 第2895番目のアルギニンのグリシンへの置換
(g) 第2895番目のアルギニンのリジンへの置換
E1タンパク質をコードする塩基配列が、配列番号14のアミノ酸番号192~383のE1タンパク質のアミノ酸配列をコードする塩基配列であり、
E2タンパク質をコードする塩基配列が、配列番号14のアミノ酸番号384~752のE2タンパク質のアミノ酸配列をコードする塩基配列であり、
p7タンパク質をコードする塩基配列が、配列番号14のアミノ酸番号753~815のp7タンパク質のアミノ酸配列をコードする塩基配列であり、
NS2タンパク質をコードする塩基配列が、配列番号14のアミノ酸番号816~1032のNS2タンパク質のアミノ酸配列をコードする塩基配列である、
上記[5]記載の核酸。
上記培養ステップで培養物中に得られたサブゲノムレプリコンRNA、フルゲノムレプリコンRNA又はC型肝炎ウイルス粒子の量を定量する定量ステップと、
上記定量ステップの結果、被験物質の存在下で培養した培養物中で定量されたサブゲノムレプリコンRNA、フルゲノムレプリコンRNA又はC型肝炎ウイルス粒子の量が、それぞれ上記被験物質の非存在下で培養した培養物中で定量されたサブゲノムレプリコンRNA、フルゲノムレプリコンRNA又はC型肝炎ウイルス粒子の量より少ない場合に、上記被験物質は抗C型肝炎ウイルス活性を有すると評価する評価ステップと、
を含む、抗C型肝炎ウイルス物質をスクリーニングする方法。
配列番号1の塩基番号2786~3436の配列からなるNS2タンパク質をコードする塩基配列、配列番号1のC型肝炎ウイルスゲノム以外のC型肝炎ウイルスゲノムのNS2タンパク質をコードする塩基配列、又は、配列番号1の塩基番号2786~3436の塩基配列からなるNS2タンパク質をコードする塩基配列の一部と配列番号1のC型肝炎ウイルスゲノム以外のC型肝炎ウイルスゲノムのNS2タンパク質をコードする塩基配列の一部とが連結されたキメラ型のNS2タンパク質をコードする塩基配列と、
配列番号1における、塩基番号3437~5329の配列からなるNS3タンパク質をコードする塩基配列、塩基番号5330~5491の配列からなるNS4Aタンパク質をコードする塩基配列、塩基番号5492~6274の配列からなるNS4Bタンパク質をコードする塩基配列、塩基番号6275~7630の配列からなるNS5Aタンパク質をコードする塩基配列及び塩基番号7631~9406の配列からなるNS5Bタンパク質をコードする塩基配列とを、
5’側から3’側に向かって、この順で含む、上記[5]記載の核酸。
(a) 第1286番目のスレオニンのイソロイシンへの置換
(b) 第2188番目のスレオニンのアラニンへの置換
(c) 第2198番目のアルギニンのヒスチジンへの置換
(d) 第2210番目のセリンのイソロイシンへの置換
(e) 第2496番目のスレオニンのイソロイシンへの置換
(f) 第2895番目のアルギニンのグリシンへの置換
(g) 第2895番目のアルギニンのリジンへの置換
配列表の配列番号1の塩基番号2786~3436の配列からなるNS2タンパク質をコードする塩基配列、S310A株以外のC型肝炎ウイルスのゲノム(例えばC型肝炎ウイルスの既存株のゲノム又は遺伝子型3a以外の遺伝子型のHCVのゲノム)のNS2タンパク質をコードする塩基配列、又は、配列表の配列番号1の塩基番号2786~3436の配列からなるNS2タンパク質をコードする塩基配列の一部とS310A株以外のC型肝炎ウイルスのゲノム(例えばC型肝炎ウイルスの既存株のゲノム又は遺伝子型3a以外の遺伝子型のHCVのゲノム)のNS2タンパク質をコードする塩基配列の一部とが連結されたキメラ型のNS2タンパク質をコードする塩基配列と、
配列表の配列番号1における、塩基番号3437~5329の配列からなるNS3タンパク質をコードする塩基配列、塩基番号5330~5491の配列からなるNS4Aタンパク質をコードする塩基配列、塩基番号5492~6274の配列からなるNS4Bタンパク質をコードする塩基配列、塩基番号6275~7630の配列からなるNS5Aタンパク質をコードする塩基配列及び塩基番号7631~9406の配列からなるNS5Bタンパク質をコードする塩基配列とが、
5’側から3’側に向かって、Coreタンパク質、E1タンパク質、E2タンパク質、p7タンパク質、NS2タンパク質、NS3タンパク質、NS4Aタンパク質、NS4Bタンパク質、NS5Aタンパク質及びNS5Bタンパク質、をコードする塩基配列の順で含まれている、核酸であってもよい。
遺伝子型3aのHCVに感染した71歳の急性C型肝炎患者からHCVウイルス株を単離し、S310A株と名付けた。この患者は、59歳のときに遺伝子型3aのHCVに感染していると診断され、4年後に肝硬変により、肝臓移植を受けていた。具体的には、患者血清からIsogen-LS(Nippon Gene社)を用いてRNAを抽出精製し、ランダムヘキサマープライマーでcDNAを合成した。既知の4種類の遺伝子型3aのHCVゲノムの保存された配列(GenBankアクセッション番号AF046866、D28917、X76918、及びD17763)を元にPCRプライマーを設計し、合成したcDNAを用いて、9つのフラグメントに分けてcDNAを増幅した。増幅産物が得られにくい5’末端の配列は5’RACE法を用いて増幅産物を得た。それぞれのフラグメントはシークエンス用クローニングベクター、pGEM-T EASY(Promega社)中にクローン化した。常法により、これらのクローンの塩基配列を解析し、S310A株の全長ゲノムRNA配列を決定した。この全長ゲノムRNAに対応するcDNA断片を常法により合成した。S310A株の全長ゲノムRNA配列に対応するcDNA配列を配列番号1に示す。
実施例1で構築した発現ベクターpS310ASGR-Neoを、制限酵素XbaIで切断した。次いで、XbaI切断断片 10~20μgに、Mung Bean Nuclease 20U(トータル反応液量 50μL)を加えて30℃で30分間インキュベートした。Mung Bean Nucleaseは、二本鎖DNA中の一本鎖部分を選択的に分解して平滑化する反応を触媒する酵素である。通常、上記XbaI切断断片をそのまま鋳型DNAとして用いてRNAポリメラーゼによるRNA転写を行うと、XbaIの認識配列の一部であるCUAGの4塩基が3’末端に余分に付加されたレプリコンRNAが合成されてしまう。そこで本実施例では、XbaI切断断片をMung Bean Nucleaseで処理することにより、XbaI切断断片からCTAGの4塩基を除去した。
実施例2で作製した野生型S310A株のHCVサブゲノムレプリコンRNA(1μg、3μg、10μg又は30μg)をエレクトロポレーション法によりHuh7細胞に導入した。エレクトロポレーション処理を行ったHuh7細胞(3×106個)を培養ディッシュに播種し、16時間~24時間培養した後に、培養ディッシュにG418(ネオマイシン)を添加した。その後、週に2回培養液を交換しながら培養を継続した。
樹立した10クローン(クローン番号:1~10)のS310Aサブゲノムレプリコン複製細胞を用い、細胞内のHCVサブゲノムレプリコンRNAのコピー数を定量した。HCVサブゲノムレプリコンRNAのコピー数の定量はTakeuchiらの文献(Gastroenterology、1999年、第116巻、p.636-642)及びKatoらの文献(Gastroenterology、2003年、第125巻、p.1808-1817)に示された手法に基づいて行った。
樹立したS310Aサブゲノムレプリコン複製細胞(クローン)におけるレプリコンRNA複製に対する抗ウイルス剤の効果を調べた。
実施例3で樹立したS310Aサブゲノムレプリコン複製細胞(クローン)内に存在するHCVサブゲノムレプリコンRNAの配列解析を行った。
実施例6で見出されたアミノ酸置換を引き起こす塩基置換、すなわち塩基変異が野生型S310A株のHCVサブゲノムレプリコンRNAの細胞内での複製に影響を与えるかどうかを以下のようにして調べた。
実施例7、図11に示したレプリコン複製細胞の検出では、細胞が生存できる最低限のネオマイシン耐性を付与できるだけのレプリコン複製能があれば検出される。したがって、実施例7で用いた方法はレプリコン複製量の解析には不向きである。そこで、変異体S310A株のHCVサブゲノムレプリコンRNAの複製効率をより定量的に評価するために、pS310ASGR-Neo中のネオマイシン耐性遺伝子をルシフェラーゼ遺伝子に組み換えた、HCVサブゲノムレプリコンRNA発現ベクターpS310ASGR-Lucを作製した。HCVサブゲノムレプリコンRNA発現ベクターpS310ASGR-Luc及び、pS310ASGR-Lucから合成されるHCVサブゲノムレプリコンRNAの構造を図12に示す。
実施例6で見出された各アミノ酸置換を有するS310A株のHCVフルゲノムレプリコンRNAの、培養細胞におけるHCV粒子産生能を評価するため、全長HCVゲノム配列を有するHCVフルゲノムレプリコンRNA発現ベクターの構築を行った。
実施例1で作製したpS310A及び実施例9で作製した各発現ベクターを、制限酵素XbaIで切断し、フェノール/クロロホルム抽出、エタノール沈殿を行った。次いで、XbaI切断断片を、Mung Bean Nuclease処理し、XbaI切断断片からXbaI認識配列由来の余分な3’末端のCTAGの4塩基を除去した。次いで、XbaI切断断片を含むMung Bean Nuclease処理後溶液について、proteinaseK処理、フェノール/クロロホルム抽出、エタノール沈殿を行ってDNA断片を精製した。これを鋳型DNAとして、MEGAscript(登録商標) T7 kit(Ambion社)を用いてRNAの合成を行った。
配列番号3:野生型S310A株ゲノムRNAのCoreタンパク質コード配列のcDNA配列
配列番号4:野生型S310A株ゲノムRNAのE1タンパク質コード配列のcDNA配列
配列番号5:野生型S310A株ゲノムRNAのE2タンパク質コード配列のcDNA配列
配列番号6:野生型S310A株ゲノムRNAのp7タンパク質コード配列のcDNA配列
配列番号7:野生型S310A株ゲノムRNAのNS2タンパク質コード配列のcDNA配列
配列番号8:野生型S310A株ゲノムRNAのNS3タンパク質コード配列のcDNA配列
配列番号9:野生型S310A株ゲノムRNAのNS4Aタンパク質コード配列のcDNA配列
配列番号10:野生型S310A株ゲノムRNAのNS4Bタンパク質コード配列のcDNA配列
配列番号11:野生型S310A株ゲノムRNAのNS5Aタンパク質コード配列のcDNA配列
配列番号12:野生型S310A株ゲノムRNAのNS5Bタンパク質コード配列のcDNA配列
配列番号13:野生型S310A株ゲノムRNAの3’非翻訳領域(3’UTR)のcDNA配列
配列番号14:野生型S310A株の前駆体タンパク質のアミノ酸配列。1~191位はCoreタンパク質、192~383位はE1タンパク質、384~752位はE2タンパク質、753~815位はp7タンパク質、816~1032位はNS2タンパク質、1033~1663位はNS3タンパク質、1664~1717位はNS4Aタンパク質、1718~1978位はNS4Bタンパク質、1979~2430位はNS5Aタンパク質、2431~3021位はNS5Bタンパク質のアミノ酸配列である。
配列番号16:野生型S310A株のHCVサブゲノムレプリコンRNAのcDNA配列
配列番号17:pS310ASGR-Neo T1286Iから合成される、変異体S310A T1286I HCVサブゲノムレプリコンRNAのcDNA配列
配列番号18:pS310ASGR-Neo R2198Hから合成される、変異体S310A R2198H HCVサブゲノムレプリコンRNAのcDNA配列
配列番号19:pS310ASGR-Neo R2895Kから合成される、変異体S310A R2895K HCVサブゲノムレプリコンRNAのcDNA配列
配列番号20:pS310ASGR-Neo T2496I/R2895Kから合成される、変異体S310A T2496I/R2895K HCVサブゲノムレプリコンRNAのcDNA配列
配列番号21:pS310ASGR-Neo T2188Aから合成される、変異体S310A T2188A HCVサブゲノムレプリコンRNAのcDNA配列
配列番号22:pS310ASGR-Neo T2496Iから合成される、変異体S310A T2496I HCVサブゲノムレプリコンRNAのcDNA配列
配列番号23:pS310ASGR-Neo R2895Gから合成される、変異体S310A R2895G HCVサブゲノムレプリコンRNAのcDNA配列
配列番号24:TaqManプローブ法プライマー
配列番号25:TaqManプローブ法プライマー
配列番号26:TaqManプローブ法プローブ
配列番号27:プライマー
配列番号28:プライマー
配列番号29:プライマー Neo-S4
配列番号30:プライマー 1286R
配列番号31:プライマー 1286F
配列番号32:プライマー 5546R
配列番号33:プライマー 5240F
配列番号34:プライマー 2188R
配列番号35:プライマー 2188F
配列番号36:プライマー 7601R
配列番号37:プライマー 2198R
配列番号38:プライマー 2198F
配列番号39:プライマー 7276F
配列番号40:プライマー 2496R
配列番号41:プライマー 2496F
配列番号42:プライマー 8579R
配列番号43:プライマー 7988F
配列番号44:プライマー R2895G-R
配列番号45:プライマー R2895G-F
配列番号46:プライマー 3X-54R-2a
配列番号47:プライマー R2895K-R
配列番号48:プライマー R2895K-F
配列番号49:変異体S310A S2210I HCVフルゲノムレプリコンRNAのcDNA配列
配列番号50:変異体S310A R2198H HCVフルゲノムレプリコンRNAのcDNA配列
配列番号51:変異体S310A R2895K HCVフルゲノムレプリコンRNAのcDNA配列
配列番号52:プライマー 2210R
配列番号53:プライマー 2210F
配列番号54:pS310ASGR-Neo S2210Iから合成される、変異体S310A S2210I HCVサブゲノムレプリコンRNAのcDNA配列
Claims (19)
- 遺伝子型3aのC型肝炎ウイルスゲノムの、配列番号1の塩基番号1~340の塩基配列を含む5’非翻訳領域と、配列番号14のアミノ酸番号1033~1663のNS3タンパク質のアミノ酸配列、アミノ酸番号1664~1717のNS4Aタンパク質のアミノ酸配列、アミノ酸番号1718~1978のNS4Bタンパク質のアミノ酸配列、アミノ酸番号1979~2430のNS5Aタンパク質のアミノ酸配列、及びアミノ酸番号2431~3021のNS5Bタンパク質のアミノ酸配列をコードする塩基配列と、配列番号1の塩基番号9407~9655の塩基配列を含む3’非翻訳領域とをこの順番で含む、核酸(ただし、該核酸がRNAの場合は、塩基配列中のチミン(t)をウラシル(u)に読み替える)。
- 前記NS3タンパク質のアミノ酸配列をコードする塩基配列が配列番号1の塩基番号3437~5329の塩基配列を含み、
前記NS4Aタンパク質のアミノ酸配列をコードする塩基配列が配列番号1の塩基番号5330~5491の塩基配列を含み、
前記NS4Bタンパク質のアミノ酸配列をコードする塩基配列が配列番号1の塩基番号5492~6274の塩基配列を含み、
前記NS5Aタンパク質のアミノ酸配列をコードする塩基配列が配列番号1の塩基番号6275~7630の塩基配列を含み、
前記NS5Bタンパク質のアミノ酸配列をコードする塩基配列が配列番号1の塩基番号7631~9406の塩基配列を含む、請求項1記載の核酸。 - 前記5’非翻訳領域が、配列番号1の塩基番号1~340の塩基配列中に、1又は複数の塩基の欠失、置換又は付加を含む、請求項1又は2記載の核酸。
- 請求項1~3のいずれか一項記載の核酸の塩基配列に塩基変異を有する核酸であり、該塩基変異が配列番号14に示されるアミノ酸配列を基準とした場合に以下の(a)~(g)の少なくとも1つのアミノ酸置換を引き起こす塩基変異を含む、核酸。
(a) 第1286番目のスレオニンのイソロイシンへの置換
(b) 第2188番目のスレオニンのアラニンへの置換
(c) 第2198番目のアルギニンのヒスチジンへの置換
(d) 第2210番目のセリンのイソロイシンへの置換
(e) 第2496番目のスレオニンのイソロイシンへの置換
(f) 第2895番目のアルギニンのグリシンへの置換
(g) 第2895番目のアルギニンのリジンへの置換 - C型肝炎ウイルスゲノムの、Coreタンパク質をコードする塩基配列、E1タンパク質をコードする塩基配列、E2タンパク質をコードする塩基配列、p7タンパク質をコードする塩基配列、及びNS2タンパク質をコードする塩基配列をさらに含む、請求項1~4のいずれか一項記載の核酸。
- Coreタンパク質をコードする塩基配列が、配列番号14のアミノ酸番号1~191のCoreタンパク質のアミノ酸配列をコードする塩基配列であり、
E1タンパク質をコードする塩基配列が、配列番号14のアミノ酸番号192~383のE1タンパク質のアミノ酸配列をコードする塩基配列であり、
E2タンパク質をコードする塩基配列が、配列番号14のアミノ酸番号384~752のE2タンパク質のアミノ酸配列をコードする塩基配列であり、
p7タンパク質をコードする塩基配列が、配列番号14のアミノ酸番号753~815のp7タンパク質のアミノ酸配列をコードする塩基配列であり、
NS2タンパク質をコードする塩基配列が、配列番号14のアミノ酸番号816~1032のNS2タンパク質のアミノ酸配列をコードする塩基配列である、
請求項5記載の核酸。 - 配列番号1及び49~51のいずれかに示される塩基配列を含む、請求項5記載の核酸。
- 配列番号17~23及び54のいずれかに示される塩基配列を含む、請求項4記載の核酸。
- 請求項1~4及び8のいずれか一項記載の核酸を含む、C型肝炎ウイルスのサブゲノムレプリコンRNA。
- 請求項5~7のいずれか一項記載の核酸を含む、C型肝炎ウイルスのフルゲノムレプリコンRNA。
- 請求項5~7のいずれか一項記載の核酸をウイルスゲノムとして含有する、C型肝炎ウイルス粒子。
- 請求項1~8のいずれか一項記載の核酸が導入された、細胞。
- 請求項11記載のC型肝炎ウイルス粒子又はその一部分を含有する、C型肝炎ウイルスワクチン。
- 請求項11記載のC型肝炎ウイルス粒子を抗原として認識する、C型肝炎ウイルスの抗体。
- 被験物質の存在下及び非存在下の双方で、請求項12記載の細胞、又は、請求項11記載のC型肝炎ウイルス粒子とC型肝炎ウイルス感受性細胞との混合物、を培養する培養ステップと、
前記培養ステップで培養物中に得られたサブゲノムレプリコンRNA、フルゲノムレプリコンRNA又はC型肝炎ウイルス粒子の量を定量する定量ステップと、
前記定量ステップの結果、被験物質の存在下で培養した培養物中で定量されたサブゲノムレプリコンRNA、フルゲノムレプリコンRNA又はC型肝炎ウイルス粒子の量が、それぞれ前記被験物質の非存在下で培養した培養物中で定量されたサブゲノムレプリコンRNA、フルゲノムレプリコンRNA又はC型肝炎ウイルス粒子の量より少ない場合に、前記被験物質は抗C型肝炎ウイルス活性を有すると評価する評価ステップと、
を含む、抗C型肝炎ウイルス物質をスクリーニングする方法。 - 前記核酸が2以上のC型肝炎ウイルス株のゲノムに由来するキメラ核酸であり、
配列番号1のC型肝炎ウイルスゲノム以外のC型肝炎ウイルスゲノムのCoreタンパク質をコードする塩基配列、E1タンパク質をコードする塩基配列、E2タンパク質をコードする塩基配列及びp7タンパク質をコードする塩基配列と、
配列番号1の塩基番号2786~3436の配列からなるNS2タンパク質をコードする塩基配列、配列番号1のC型肝炎ウイルスゲノム以外のC型肝炎ウイルスゲノムのNS2タンパク質をコードする塩基配列、又は、配列番号1の塩基番号2786~3436の塩基配列からなるNS2タンパク質をコードする塩基配列の一部と配列番号1のC型肝炎ウイルスゲノム以外のC型肝炎ウイルスゲノムのNS2タンパク質をコードする塩基配列の一部とが連結されたキメラ型のNS2タンパク質をコードする塩基配列と、
配列番号1における、塩基番号3437~5329の配列からなるNS3タンパク質をコードする塩基配列、塩基番号5330~5491の配列からなるNS4Aタンパク質をコードする塩基配列、塩基番号5492~6274の配列からなるNS4Bタンパク質をコードする塩基配列、塩基番号6275~7630の配列からなるNS5Aタンパク質をコードする塩基配列及び塩基番号7631~9406の配列からなるNS5Bタンパク質をコードする塩基配列とを、
5’側から3’側に向かって、この順で含む、請求項5記載の核酸。 - 請求項16記載の核酸の塩基配列に塩基変異を有する核酸であり、配列番号14に示されるアミノ酸配列を基準とした場合に以下の(a)~(g)の少なくとも1つのアミノ酸置換を引き起こす塩基変異を有する、核酸。
(a) 第1286番目のスレオニンのイソロイシンへの置換
(b) 第2188番目のスレオニンのアラニンへの置換
(c) 第2198番目のアルギニンのヒスチジンへの置換
(d) 第2210番目のセリンのイソロイシンへの置換
(e) 第2496番目のスレオニンのイソロイシンへの置換
(f) 第2895番目のアルギニンのグリシンへの置換
(g) 第2895番目のアルギニンのリジンへの置換 - 前記配列番号1の塩基番号1~340の塩基配列を含む5’非翻訳領域に代えて、配列番号1のC型肝炎ウイルスゲノム以外のC型肝炎ウイルスゲノムの5’非翻訳領域を含む、請求項16又は17記載の核酸。
- 請求項16~18のいずれか一項記載の核酸を含む、C型肝炎ウイルスのキメラフルゲノムレプリコンRNA。
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP12827627.6A EP2752485A4 (en) | 2011-08-31 | 2012-08-31 | NUCLEIC ACID RECOMBINANT PRODUCT COMPRISING NUCLEIC ACID DERIVED FROM GENOTYPE 3A GENE VHC GENOME |
US14/342,129 US9453056B2 (en) | 2011-08-31 | 2012-08-31 | Nucleic acid construct comprising nucleic acid derived from genome of hepatitis C virus of genotype 3a |
AU2012302666A AU2012302666B2 (en) | 2011-08-31 | 2012-08-31 | Nucleic acid construct including nucleic acid derived from genotype 3a HCV genome |
CA2847115A CA2847115A1 (en) | 2011-08-31 | 2012-08-31 | Nucleic acid construct comprising nucleic acid derived from genome of hepatitis c virus of genotype 3a |
JP2013531425A JP6026419B2 (ja) | 2011-08-31 | 2012-08-31 | 遺伝子型3aのC型肝炎ウイルスゲノム由来の核酸を含む核酸構築物 |
CN201280042305.8A CN104126008B (zh) | 2011-08-31 | 2012-08-31 | 包含来自基因型3a的丙型肝炎病毒基因组的核酸的核酸构建物 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2011189695 | 2011-08-31 | ||
JP2011-189695 | 2011-08-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013031956A1 true WO2013031956A1 (ja) | 2013-03-07 |
Family
ID=47756430
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2012/072179 WO2013031956A1 (ja) | 2011-08-31 | 2012-08-31 | 遺伝子型3aのC型肝炎ウイルスゲノム由来の核酸を含む核酸構築物 |
Country Status (7)
Country | Link |
---|---|
US (1) | US9453056B2 (ja) |
EP (1) | EP2752485A4 (ja) |
JP (1) | JP6026419B2 (ja) |
CN (1) | CN104126008B (ja) |
AU (1) | AU2012302666B2 (ja) |
CA (1) | CA2847115A1 (ja) |
WO (1) | WO2013031956A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018172759A1 (en) | 2017-03-20 | 2018-09-27 | Queen Mary University Of London | Method for identification of sofosbuvir resistant patients |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113862231B (zh) * | 2021-10-08 | 2024-02-13 | 中山大学 | 一种3a型丙肝病毒亚基因组复制子及应用 |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001017187A (ja) | 1999-04-03 | 2001-01-23 | Ralf Bartenschlager | C型肝炎ウイルス細胞培養系、c型肝炎ウイルス−rna−構築物、細胞培養系または構築物の使用、c型肝炎ウイルス−rna−構築物の細胞培養に適合した突然変異体を獲得する方法、c型肝炎ウイルス−全長ゲノム、c型肝炎ウイルス−部分ゲノム、または任意のc型肝炎ウイルス−構築物の突然変異体の製法、細胞培養に適合したc型肝炎ウイルス−構築物、その突然変異体、c型肝炎ウイルス−全長ゲノムの突然変異体、c型肝炎ウイルス粒子またはウイルス様粒子、およびこれで感染した細胞 |
JP2004000179A (ja) | 2002-03-29 | 2004-01-08 | Toray Ind Inc | C型慢性肝炎、肝硬変、肝癌病態を呈するモデル動物及びその作製方法 |
WO2004104198A1 (ja) | 2003-05-26 | 2004-12-02 | Toray Industries, Inc. | 遺伝子型2aのC型肝炎ウイルス(HCV)ゲノム由来の核酸を含む核酸構築物、及び該核酸構築物を導入した細胞 |
WO2005028652A1 (ja) * | 2003-09-19 | 2005-03-31 | Tokyo Metropolitan Organization For Medical Research | 新規hcv株由来の核酸、遺伝子、及び該遺伝子を利用したレプリコン複製細胞 |
WO2005080575A1 (ja) | 2004-02-20 | 2005-09-01 | Tokyo Metropolitan Organization For Medical Research | ヒトc型肝炎ウイルスの全長ゲノムを含む核酸構築物及び該核酸構築物を導入した組換え全長ウイルスゲノム複製細胞、並びにc型肝炎ウイルス粒子の作製方法 |
WO2006022422A1 (ja) | 2004-08-24 | 2006-03-02 | Tokyo Metropolitan Organization For Medical Research | 自律複製能を有する改変されたヒトc型肝炎ウイルスゲノムrna |
WO2010074249A1 (ja) * | 2008-12-26 | 2010-07-01 | 東レ株式会社 | C型肝炎ウイルス由来の核酸並びにそれを用いた発現ベクター、形質転換細胞及びc型肝炎ウイルス粒子 |
JP2011189695A (ja) | 2010-03-16 | 2011-09-29 | Ube Machinery Corporation Ltd | 型内コーティング成形装置及び型内コーティング成形方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014522648A (ja) * | 2011-07-06 | 2014-09-08 | ギリアード サイエンシーズ, インコーポレイテッド | Hcv遺伝子型3レプリコン |
-
2012
- 2012-08-31 AU AU2012302666A patent/AU2012302666B2/en not_active Ceased
- 2012-08-31 CN CN201280042305.8A patent/CN104126008B/zh not_active Expired - Fee Related
- 2012-08-31 EP EP12827627.6A patent/EP2752485A4/en not_active Withdrawn
- 2012-08-31 CA CA2847115A patent/CA2847115A1/en not_active Abandoned
- 2012-08-31 US US14/342,129 patent/US9453056B2/en not_active Expired - Fee Related
- 2012-08-31 JP JP2013531425A patent/JP6026419B2/ja not_active Expired - Fee Related
- 2012-08-31 WO PCT/JP2012/072179 patent/WO2013031956A1/ja active Application Filing
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001017187A (ja) | 1999-04-03 | 2001-01-23 | Ralf Bartenschlager | C型肝炎ウイルス細胞培養系、c型肝炎ウイルス−rna−構築物、細胞培養系または構築物の使用、c型肝炎ウイルス−rna−構築物の細胞培養に適合した突然変異体を獲得する方法、c型肝炎ウイルス−全長ゲノム、c型肝炎ウイルス−部分ゲノム、または任意のc型肝炎ウイルス−構築物の突然変異体の製法、細胞培養に適合したc型肝炎ウイルス−構築物、その突然変異体、c型肝炎ウイルス−全長ゲノムの突然変異体、c型肝炎ウイルス粒子またはウイルス様粒子、およびこれで感染した細胞 |
JP2004000179A (ja) | 2002-03-29 | 2004-01-08 | Toray Ind Inc | C型慢性肝炎、肝硬変、肝癌病態を呈するモデル動物及びその作製方法 |
WO2004104198A1 (ja) | 2003-05-26 | 2004-12-02 | Toray Industries, Inc. | 遺伝子型2aのC型肝炎ウイルス(HCV)ゲノム由来の核酸を含む核酸構築物、及び該核酸構築物を導入した細胞 |
WO2005028652A1 (ja) * | 2003-09-19 | 2005-03-31 | Tokyo Metropolitan Organization For Medical Research | 新規hcv株由来の核酸、遺伝子、及び該遺伝子を利用したレプリコン複製細胞 |
WO2005080575A1 (ja) | 2004-02-20 | 2005-09-01 | Tokyo Metropolitan Organization For Medical Research | ヒトc型肝炎ウイルスの全長ゲノムを含む核酸構築物及び該核酸構築物を導入した組換え全長ウイルスゲノム複製細胞、並びにc型肝炎ウイルス粒子の作製方法 |
WO2006022422A1 (ja) | 2004-08-24 | 2006-03-02 | Tokyo Metropolitan Organization For Medical Research | 自律複製能を有する改変されたヒトc型肝炎ウイルスゲノムrna |
WO2010074249A1 (ja) * | 2008-12-26 | 2010-07-01 | 東レ株式会社 | C型肝炎ウイルス由来の核酸並びにそれを用いた発現ベクター、形質転換細胞及びc型肝炎ウイルス粒子 |
JP2011189695A (ja) | 2010-03-16 | 2011-09-29 | Ube Machinery Corporation Ltd | 型内コーティング成形装置及び型内コーティング成形方法 |
Non-Patent Citations (46)
Title |
---|
"Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY |
AKAZAWA ET AL., J. VIROL., vol. 81, 2007, pages 5036 - 5045 |
APPAIAHGARI, M. B.; VRATI, S., VACCINE, vol. 22, 2004, pages 3669 - 3675 |
BLIGHT ET AL., SCIENCE, vol. 290, 2000, pages 1972 - 1974 |
CHOO ET AL., SCIENCE, vol. 244, 1989, pages 359 - 362 |
CLARK ET AL., JOURNAL OF MEDICINAL CHEMISTRY, vol. 48, 2005, pages 5504 - 5508 |
CRISTINA, J.; COLINA, R., VIROLOGY JOURNAL, vol. 3, 2006, pages 1 - 8 |
DANIEL ET AL., NATURE, vol. 426, 2003, pages 186 - 189 |
ED HARLOW ET AL.: "Antibodies: A Laboratory Manual", 1988 |
EVANS ET AL., NATURE, vol. 446, 2007, pages 801 - 805 |
FRIEBE ET AL., JOURNAL OF VIROLOGY, vol. 75, 2001, pages 12047 - 12057 |
GOTTWEIN, J. M. ET AL.: "Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies", J. VIROL., vol. 84, no. 10, 2010, pages 5277 - 5293, XP009140732 * |
GOTTWEIN, J. M. ET AL.: "Robust Hepatitis C Genotype 3a Cell Culture Releasing Adapted Intergenotypic 3a/2a (S52/JFH1) Viruses", GASTROENTEROLOGY, vol. 133, 2007, pages 1614 - 1626, XP022407067 * |
GRAVITZ, NATURE, vol. 474, 2011, pages S2 - S4 |
HIRASHIMA ET AL., JOURNAL OF MEDICINAL CHEMISTRY, vol. 49, 2006, pages 4721 - 4736 |
IKEDA ET AL., JOURNAL OF VIROLOGY, vol. 76, 2002, pages 2997 - 3006 |
KATO ET AL., GASTROENTEROLOGY, vol. 125, 2003, pages 1 808 - 1 81,7 |
KATO ET AL., GASTROENTEROLOGY, vol. 125, 2003, pages 1808 - 1817 |
KATO ET AL., J. CLIN. MICROBIOL., vol. 43, 2005, pages 5679 - 5684 |
KATO ET AL., JOURNAL OF CLINICAL MICROBIOLOGY, vol. 43, 2005, pages 5679 - 5684 |
KATO ET AL., JOURNAL OF MEDICAL VIROLOGY, vol. 64, 1992, pages 334 - 339 |
KIM ET AL., POLOS ONE., vol. 6, no. 4, 2011, pages EL 8556 |
LANFORD, R. E. ET AL.: "Hepatitis C virus genotype 1b chimeric replicon containing genotype 3 NS5A domain", VIROLOGY, vol. 355, 2006, pages 192 - 202, XP024896546 * |
LIN ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, 2004, pages 17508 - 17514 |
LINDENBACH ET AL., SCIENCE, 2005, pages 623 - 626 |
LINDENBACH ET AL., SCIENCE, vol. 309, 2005, pages 623 - 626 |
LOHMANN ET AL., JOURNAL OF VIROLOGY, vol. 75, 2001, pages 1437 - 1449 |
LOHMANN ET AL., SCIENCE, vol. 285, 1999, pages 110 - 113 |
LOMANN ET AL., SCIENCE, vol. 285, 1999, pages 110 - 113 |
MORI ET AL., BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 183, 1992, pages 334 - 342 |
OKAMOTO ET AL., JOURNAL OF GENERAL VIROLOGY, vol. 73, 1992, pages 673 - 679 |
PIETSCHMANN ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 103, 2007, pages 7408 - 7413 |
REESINK ET AL., GASTROENTEROLOGY, vol. 138, 2010, pages 913 - 921 |
REHMAN ET AL., GENETIC VACCINES AND THERAPY, vol. 9, 2011, pages 2 - 5 |
SAEED, M. ET AL.: "Efficient replication of genotype 3a and 4a HCV replicons in human hepatoma cells", ANTIMICROB. AGENTS. CHEMOTHER., vol. 56, no. 10, October 2012 (2012-10-01), pages 5365 - 5373, XP055147406 * |
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989 |
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001 |
See also references of EP2752485A4 * |
SIMMONDS ET AL., HEPATOLOGY, vol. 10, 1994, pages 1321 - 1324 |
TAKEUCHI ET AL., GASTROENTEROLOGY, vol. 116, 1999, pages 636 - 642 |
TOKITA ET AL., JOURNAL OF GENERAL VIROLOGY, vol. 79, 1998, pages 1847 - 1857 |
WAKITA ET AL., J. BIOL. CHEM., vol. 269, 1994, pages 14205 - 14210 |
WAKITA ET AL., NATURE MEDICINE, 2005, pages 791 - 796 |
WAKITA ET AL., NATURE MEDICINE, vol. 11, 2005, pages 791 - 796 |
XIANG, Z. ET AL.: "Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Spl.", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 402, 2010, pages 549 - 553, XP027503245 * |
YOSHIOKA ET AL., HEPATOLOGY, vol. 16, 1992, pages 293 - 299 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018172759A1 (en) | 2017-03-20 | 2018-09-27 | Queen Mary University Of London | Method for identification of sofosbuvir resistant patients |
Also Published As
Publication number | Publication date |
---|---|
CN104126008A (zh) | 2014-10-29 |
CA2847115A1 (en) | 2013-03-07 |
EP2752485A4 (en) | 2015-06-24 |
CN104126008B (zh) | 2017-03-22 |
AU2012302666A1 (en) | 2014-04-17 |
EP2752485A1 (en) | 2014-07-09 |
US20140286995A1 (en) | 2014-09-25 |
AU2012302666B2 (en) | 2017-05-25 |
JPWO2013031956A1 (ja) | 2015-03-23 |
US9453056B2 (en) | 2016-09-27 |
JP6026419B2 (ja) | 2016-11-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4921164B2 (ja) | ヒトc型肝炎ウイルスの全長ゲノムを含む核酸構築物及び該核酸構築物を導入した組換え全長ウイルスゲノム複製細胞、並びにc型肝炎ウイルス粒子の作製方法 | |
US9175269B2 (en) | Modified human hepatitis C virus genomic RNA that can be autonomously replicated | |
JP5593220B2 (ja) | C型肝炎ウイルス由来のキメラ遺伝子を含む核酸 | |
JP5816614B2 (ja) | 感染性c型肝炎ウイルス高生産hcv変異体及びその利用 | |
US8834893B2 (en) | Nucleic acid derived from hepatitis C virus and expression vector, transformed cell, and hepatitis C virus particles each prepared by using the same | |
JP6026419B2 (ja) | 遺伝子型3aのC型肝炎ウイルスゲノム由来の核酸を含む核酸構築物 | |
WO2012133735A1 (ja) | 遺伝子型1bのC型肝炎ウイルスゲノム由来の核酸を含む核酸構築物、及び該核酸構築物が導入されたC型肝炎ウイルスゲノム複製細胞、並びに感染性C型肝炎ウイルス粒子の製造方法 | |
JP5927185B2 (ja) | C型肝炎ウイルスj6cf株ゲノム由来の変異体レプリコン |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12827627 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2013531425 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2847115 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012827627 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2012302666 Country of ref document: AU Date of ref document: 20120831 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14342129 Country of ref document: US |