CN101889085A - Hcv ns3 protease replicon shuttle vectors - Google Patents

Hcv ns3 protease replicon shuttle vectors Download PDF

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CN101889085A
CN101889085A CN2008801195086A CN200880119508A CN101889085A CN 101889085 A CN101889085 A CN 101889085A CN 2008801195086 A CN2008801195086 A CN 2008801195086A CN 200880119508 A CN200880119508 A CN 200880119508A CN 101889085 A CN101889085 A CN 101889085A
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hcv replicon
shuttle vectors
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S·阿力
W-R·蒋
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Abstract

The present invention provides for novel HCV NS3 protease replicon shuttle vectors useful for cloning in HCV polynucleotide sequences from samples of HCV-infected patients and testing the resulting replicons for drug susceptibility.

Description

HCV NS3 protease replicon shuttle vectors
The present invention relates to new HCV NS3 protease replicon shuttle vectors, it can be used for screening, test and estimates HCV and other flavivirus proteinase inhibitor.
Hepatitis C virus is a whole world major health, and be chronic hepatopathy main diseases because of (Boyer, people such as N., J.Hepatol.200032:98-112).The patient that HCV infects has the danger that develops into liver cirrhosis and develop into hepatocellular carcinoma subsequently, so HCV is the main indications of liver transplantation.
According to the World Health Organization, world wide exist to surpass 200,000,000 infected individuals, and infect to 4 million peoples every year at least 3 1,000,000.In case infected, about 20% people removes this virus, but all the other people may carry HCV in the remaining years.10% to 20% chronic infection individuality finally develops into liver cirrhosis disease or the cancer of destroying liver.This virus disease is propagated, is propagated or propagated or vertically be transmitted to the offspring from infecting mother or carrying mother in the property mode by the syringe needle that pollutes by blood that pollutes and blood products in the parenteral mode.The existing therapy that infects at HCV has limited clinical benefit, especially for genotype 1, wherein said existing therapy be limited to recombinant interferon-α separately or with the immunotherapy of nucleoside analogues 'Libaweilin ' associating, press for the improved therapeutical agent of effectively defeating chronic HCV infection.
HCV has incorporated the member as flaviviridae (Flaviviridae) into, flaviviridae comprises Flavivirus (flaviviruses), pestivirus (pestiviruses) and hepatitis virus belong to (hepaciviruses), wherein said hepatitis virus belongs to and comprises hepatitis C virus (Rice, C.M., Flaviviridae:The viruses and their replication, in " Fields Virology ", Fields, B.N., Knipe, D.M. and Howley, P.M. writes, Lippincott-RavenPublishers, Philadelphia, Pa., the 30th chapter, 931-959,1996).HCV contains the genomic envelope virus of 9.4kb sense single stranded rna of having an appointment.Viral genome is by one 5 ' non-translational region (UTR), a long open reading-frame (ORF) (ORF) of about 3011 the amino acid whose polyprotein precursors of coding and 3 ' UTR of a weak point.5 ' UTR is the conservative part of topnotch in the HCV genome, and for the startup of polyprotein translation with regulate and control important.
The genetic analysis of HCV determined to present at dna sequence dna＞6 kinds of oligogene types of 30% divergence.Every kind of genotype contains a series of more closely-related hypotypes, wherein said hypotype nucleotides sequence list show 20%～25% divergence (Simmonds, P., 2004, J.Gen.Virol.85:3173-88).Distinguished more than 30 kinds of hypotypes.In the U.S., about 70% infected individuals suffers from the 1a type and the 1b type infects.The 1b type is the most popular hypotype (X.Forns and J.Bukh, Clinics in Liver Disease 1999, the 3:693-716 in the Asia; People such as J.Bukh, Semin.Liv.Dis., 1995,15:41-63).Unfortunately, 1 type infect to existing therapy reply than 2 types or 3 type genotype littler (N.N.Zein, Clin.Microbiol.Rev., 2000,13:223-235).
Processing is very similar with polyprotein in the gene organization of the Nonstructural Protein part of the ORF of pestivirus and hepatitis virus.These positive chain RNA virus have single big open reading-frame (ORF) (ORF), the essential whole viral proteins of coding virus replication.These protein are expressed as polyprotein, and wherein said polyprotein is processed to produce sophisticated viral protein after the proteolytic enzyme of leukoprotease and encoding viral is translated altogether and translated.The viral protein that responsible virus genome RNA duplicates is positioned carboxyl terminal.2/3rds of ORF is called non-structure (NS) albumen.For pestivirus and hepatitis virus, sophisticated non-structure (NS) albumen is made up of p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B from aminoterminal to the order successively of this ORF carboxyl terminal of Nonstructural Protein coding region.
The NS albumen of pestivirus and hepatitis virus is total to be the sequence domains of feature with the specified protein function.For example, the virus NS albumen in this two papova has the aminoacid sequence motif that belongs to the serine protease feature and belongs to the aminoacid sequence motif of helicase feature (people such as Gorbalenya, Nature 1988333:22; Bazan and Fletterick, Virology 1989,171:637-639; People such as Gorbalenya, Nucleic Acid Res.1989,17,3889-3897).Similarly, the NS5B albumen of pestivirus and hepatitis virus have the motif that belongs to RNA guide RNA polysaccharase feature (Koonin, E.V. and Dolja, V.V., Crit.Rev.Biochem.Molec.Biol.1993,28:375-430).
The NS albumen of pestivirus and hepatitis virus is directly similarly in the practical function and the function of virus in life cycle.In both cases, the NS3 serine protease is responsible for all proteolysis processing (Wiskerchen and Collett, Virology, 1991,184:341-350 of the polyprotein precursor in its downstream, position among the ORF; People such as Bartenschlager, J.Virol.1993,67:3835-3844; People such as Eckart, Biochem.Biophys.Res.Comm.1993,192:399-406; People such as Grakoui, J.Virol.1993,67:2832-2843; People such as Grakoui, Proc.Natl.Acad.Sci.USA 1993,90:10583-10587; People such as Ilijikata, J.Virol.1993,67:4665-4675; People such as Tome, J.Virol.1993,67:4017-4026).In both cases, NS4A albumen is all as cofactor and the effect of NS3 serine protease (people such as Bartenschlager, J.Virol.1994,68:5045-5055; People such as Failla, J.Virol.1994,68:3753-3760; People such as Xu, J Virol.1997,71:5312-5322).The NS3 albumen of this two papova is all also brought into play effect (people such as Kim, Biochem.Biophys.Res.Comm.1995, the 215:160-166 of helicase; Jin and Peterson, Arch.Biochem.Biophys.1995,323:47-53; Warrener and Collett, J.Virol.199569:1720-1726).At last, the NS5B albumen of pestivirus and hepatitis virus all have the rna polymerase activity that the RNA of prediction relies on (people such as Behrens, EMBO 1996,15:12-22; People such as Lechmann, J.Virol.1997,71:8416-8428; People such as Yuan, Biochem.Biophys.Res.Comm.1997,232:231-235; Hagedorn, PCT WO 97/12033; People such as Zhong, J.Virol.1998,72:9365-9369).
HCV NS albumen 3 (NS3) contains the serine protease of the most of viral enzyme of helpful processing and therefore is considered as virus replication and infection is essential.Known sudden change in yellow fever virus NS3 proteolytic enzyme reduce viral infection (people such as Chambers, Proc.Natl.Acad.Sci.USA, 1990,87:8898-8902).Shown that 181 amino acid (the 1027th～1207 residue of viral polyprotein) of NS3 contain the serine protease structural domain of NS3, the site, whole 4 downstreams of this structural domain processing HCV polyprotein (people such as Lin, J.Virol.1994,68:8147-8157).HCV NS3 serine protease and relevant cofactor NS4A thereof help the whole viral enzyme of processing, and therefore to be considered as virus replication be essential.As if this course of processing similar with the course of processing that human immunodeficiency virus's aspartyl protease is implemented, wherein said aspartyl protease also participates in viral enzyme processing hiv protease inhibitor, wherein said hiv protease inhibitor is the potent antiviral drug among the mankind, thereby this stage that shows break virus life cycle has caused the therapeutic activity agent.Therefore, it is an attractive target spot of finding medicine.
The current approved therapy that can be used for treating the HCV infection at present that has limited quantity.The new treatment and existing methods of treatment: the R.G.Gish of treatment HCV and inhibition HCV NS5B polysaccharase have been summarized, Sem.Liver.Dis., 1999,19:5; Di Besceglie, A.M. and Bacon, B.R, Scientific American, in October, 1999,80-85; G.Lake-Bakaar, " at chronic hcv virus hepatopathy now with therapy (Current and FutureTherapy for Chronic Hepatitis C Virus Liver Disease) in the future ", Curr.DrugTarg.Infect Dis.2003,3 (3): 247-253; People such as P.Hoffmann, " (1999-2002) " about the nearest patent (1999-2002) (Recent patents onexperimental therapy for hepatitis C virus infection) of the experimental therapy of hepatitis C virus, Exp.Opin.Ther.Patents 2003,13 (11): 1707-1723; People such as F.F.Poordad, " hepatitis C therapy progress (Developments in Hepatitis Ctherapy during 2000-2002) during the 2000-2002 ", Exp.Opin.Emerging Drugs 2003,8 (1): 9-25; People such as M.P.Walker, " be used for chronic hepatitis C treatment have prospect material standed for (Promising Candidates for the treatment of chronic hepatitis C) ", Exp.Opin.Investig.Drugs 2003,12 (8): 1269-1280; People such as S.-L.Tan, " Remedies for hepatitis C: situation and emerging strategy (Hepatitis C Therapeutics:Current Status and Emerging Strategies) " now, Nature Rev.DrugDiscov.2002,1:867-881; People such as R.De Francesco, " move towards new era of hepatitis C virus therapy: the RNA polymerase inhibitor (Approaching a new era for hepatitis C virus therapy:inhibitors of the NS3-4A serine protease and the NS5B RNA-dependentRNA polymerase) that NS3-4A serpin and NS5B RNA rely on; Antiviral Res.2003,58:1-16; People such as Q.M.Wang, " hepatitis C virus encoded protein matter: the target of antiviral therapy (Hepatitis C virus encodedproteins:targets for antiviral therapy) ", Drugs of the Future 2000,25 (9): 933-8-944; J.A.Wu and Z.Hong, " RNA polymerase that target NS5B relies on is used for anti-HCV chemotherapy (Targeting NS5B-Dependent RNA Polymerase forAnti-HCV Chemotherapy) ", Cur.Drug Targ.-Inf.Dis.20033:207-219.
Although make progress, be still unknown yet HCV duplicates the basic aspect that takes place with pathology in the function aspects of understanding this viral gene structure and viral protein.Obtaining the main challenge that the experimental HCV of understanding duplicates is to lack the effective cell culture systems that allows to produce infectious viral particle.Though reported primary cell culture and some human cell line's infection, yet it is too low so that do not allow detailed analysis to produce level that viral amount and HCV duplicate in those systems.
Reported the structure of the alternative subgene group HCV RNA that in Bel7402 Huh-7, duplicates with minimum efficiency.People such as Lohman report C-p7 or the C-NS2 district by the disappearance protein coding region makes up from clone's total length HCV and has genome (genotype 1b) deutero-replicon (I377/NS3-3 ') people such as (, Science 1999285:110-113) Lohman.This replicon contains following element: (i) the HCV 5 '-UTR that merges with 12 amino acid of capsid coding region; (ii) neomycin phosphotransferase gene (NPTID; (iii) insert NPTII gene downstream and guide HCV protein N S2 or NS3 to NS5B translation from the IRES of encephalomyocarditis virus (EMCV) and (iv) 3 '-UTR.Behind transfection Huh-7 cell, only those are supported the cell expressing NPTII albumen of HCV rna replicon and manifest resistance at medicine G418.Although contain the replicon rna and the viral protein of conspicuous level from the clone of this type of G418 resistance colony-derived, however in the Huh-7 cell of 106 transfections only 1 cell support that HCV duplicates.
Similar alternative HCV replicon makes up based on HCV-H genotype 1a infections clone people such as (, Science 2000290:1972-74) Blight.HCV-H deutero-replicon can not be set up effective HCV and duplicate, and shows that the replicon that Lohmann (1999) above early makes up depends on the specific gene type 1b community cDNA clone who uses in those experiments.People such as Blight above (2000) have repeated the structure of the replicon that undertaken by people (1999) such as Lohmann above and have obtained the Huh-7 cell colony of tolerance G418 by the gene assembly method of implementing PCR-based.Independently whether the order-checking of G418 tolerance cell clone needs replicon to adapt to host cell to determine that high-level HCV duplicates.A plurality of independently adaptive mutations of bunch collection in HCV Nonstructural Protein NS5A have been identified.The replication in vitro ability of increase is given in described sudden change, compares with the replicon that this area early makes up, and has the transduction efficiency of from 0.2% to 10% transfectional cell, and for example I377/NS3-3 ' replicon has 0.0001% transduction efficiency.
The present invention is a feature to develop new HCV replicon shuttle vectors, it imports unique Restriction Enzyme site at 5 ' and 3 ' end place of the proteolytic enzyme structural domain of NS3 gene, thus from the NS3 proteolytic enzyme sequence of the analyte derivative of HCV infected patient can in this shuttle vectors, clone and can estimate the gained replicon duplicate adaptability and to the susceptibility of HCV NS3 proteinase inhibitor.Because individual HCV infected patient generally contains because of the hereditary various viral colony due to the high error rate of NS5B RNA polymerase, so use shuttle vectors of the present invention will allow to characterize particular patient deutero-NS3 ease variants and these variants susceptibility or resistance to pharmacological agent.
Therefore, the invention provides the HCV replicon shuttle vectors that comprises the HCV polynucleotide sequence, wherein said HCV polynucleotide sequence comprises the unique Restriction Enzyme sequence between 10 5 ' Nucleotide being positioned at the proteic polynucleotide sequence of own coding NS3 5 ' end and 10 the 3 ' Nucleotide successively; The polynucleotide sequence of the proteic proteolytic enzyme structural domain of coding NS3; Be positioned at 10 5 ' Nucleotide of polynucleotide sequence 3 ' end of the proteic proteolytic enzyme structural domain of own coding NS3 and the unique Restriction Enzyme sequence between 10 3 ' Nucleotide; The polynucleotide sequence of the proteic helicase structural domain of coding NS3; The proteic polynucleotide sequence of coding NS4A; The proteic polynucleotide sequence of coding NS4B; The proteic polynucleotide sequence of coding NS5A; With the proteic polynucleotide sequence of coding NS5B.In one embodiment of the invention, the polynucleotide sequence of the modified or proteic proteolytic enzyme structural domain of disappearance coding NS3, thus the proteic proteolytic enzyme structural domain of NS3 is non-functional.
In another embodiment of the invention, at unique Restriction Enzyme recognition sequence EcoRV at coding NS3 proteic polynucleotide sequence 5 ' end place and at unique Restriction Enzyme recognition sequence AsiSI at polynucleotide sequence 3 ' the end place of the proteic proteolytic enzyme structural domain of coding NS3.Still in one embodiment of the invention, the HCV replicon shuttle vectors comprises the HCV polynucleotide sequence that is selected from SEQ ID NO:3 or SEQ IDNO:6.
Another embodiment of the present invention provides and has been used for assessing the method for HCV NS3 proteinase inhibitor in the validity of experimenter's control HCV infection, comprise step: provide sample from the experimenter of HCV infection, the polynucleotide sequence of the proteic proteolytic enzyme structural domain of pcr amplification coding NS3 in the sense strand primer that comprises unique Restriction Enzyme sequence by use and a plurality of HCV quasispecies that the antisense strand primer that comprises different unique Restriction Enzyme sequences exists from sample, the polynucleotide sequence of cloning described pcr amplification to the HCV replicon shuttle vectors to produce chimeric HCV replicon plasmid, the described chimeric HCV replicon plasmid of linearizing also carries out in-vitro transcription producing chimeric HCV replicon rna to described linearization plasmid, and with described HCV replicon rna transfection Huh7 clone and HCV NS3 proteinase inhibitor exist or not in the presence of measure the levels of replication of described HCV replicon rna.
An embodiment more of the present invention provides and has been used for assessing the method for HCV NS3 proteinase inhibitor in the validity of experimenter's control HCV infection, comprise step: provide sample from the experimenter of HCV infection, the polynucleotide sequence of the proteic proteolytic enzyme structural domain of pcr amplification coding NS3 in the sense strand primer that comprises unique Restriction Enzyme sequence by use and a plurality of HCV quasispecies that the antisense strand primer that comprises different unique Restriction Enzyme sequences exists from sample, the polynucleotide sequence of cloning described pcr amplification to the HCV replicon shuttle vectors to produce chimeric HCV replicon plasmid, transform described plasmid to the cell to produce a plurality of colonies of transformant, compile described colony and separate chimeric HCV replicon plasmid from the colony that compiles, the described chimeric HCV replicon plasmid of linearizing, described linearization plasmid is carried out in-vitro transcription producing chimeric HCV replicon rna, and with described HCV replicon rna transfection Huh7 clone and HCV NS3 proteinase inhibitor exist or not in the presence of measure the levels of replication of described HCV replicon rna.
Aforementioned and other advantages and feature of the present invention and the mode that obtains them will consider to become obvious easily after following the present invention's detailed description is together with the embodiment that follows, and wherein said embodiment has set forth exemplary.
The accompanying drawing summary
Fig. 1 is the synoptic diagram of the component of HCV replicon shuttle vectors.
Fig. 2 shows NS3 protease replicon shuttle vectors (A) pSC_1b_NS3/ proteolytic enzyme _ EcoRV_AsiSI (SEQ ID NO:3); (B) plasmid map of pSC_1b_NS3/ proteolytic enzyme/LacZ_EcoRV_AsiSI (SEQ ID NO:6).
Fig. 3 shows pSC_1b_NS3/ proteolytic enzyme _ EcoRV_AsiSI and contains replication from the replicon of patient's deutero-NS3 proteolytic enzyme of HCV genotype-1a or genotype 1b.RLU representative is at transfection observed Lampyridea luciferase signal level after 96 hours.
The term that uses among the present invention defines hereinafter.
Term " HCV replicon " refers to self copy the nucleic acid that produces from can instructing of hepatitis C virus.As used herein, term " replicon " comprises RNA and DNA and heterozygote thereof.For example, the genomic double-stranded DNA form of HCV can be used for producing the single stranded RNA transcript that constitutes the HCV replicon.The HCV replicon can comprise total length HCV genome or be also referred to as the HCV subgene group construct of " sub-genome duplication ".For example, described herein HCV sub-genome duplication contains most of genes of this virus Nonstructural Protein, but does not have the proteic most of genes of coding structure.Sub-genome duplication can instruct for viral subgenomic because of group is duplicated, sub-genome duplication duplicates necessary whole viral gene expression, and do not produce virion.
Basic HCV replicon is the subgene group construct that contains HCV 5 '-untranslated (UTR) district, HCV NS3-NS5B polyprotein coding region and HCV 3 '-UTR.Can there be those nucleic acid region and the non-HCV sequence of other nucleic acid region as HCV NS2, HCV structural protein are provided.
HCV 5 '-UTR district is provided for the internal ribosome entry site (IRES) of protein translation and duplicates required element.HCV 5 '-UTR district comprises the natural HCV of existence 5 '-UTR and its functional deriv that extends into about 36 Nucleotide in HCV core encoder district.5 '-UTR district can exist at different positions, as select the site in the sequence downstream of albumen, reporter protein or HCV polyprotein at coding.
Except HCV 5 '-UTR-PC district, non-HCV IRES element also may reside in the replicon.Non-HCV IRES element can exist at different positions, comprises the regional upstream of next-door neighbour's coding HCV polyprotein.The example of operable non-HCV IRES element is EMCV IRES, poliovirus IRES and bovine viral diarrhea virus IRES.
HCV 3 '-UTR helps HCV to duplicate.HCV 3 ' UTR comprises naturally occurring HCV3 '-UTR and its functional deriv.Naturally occurring 3 '-UTR comprises the additional areas of poly-U zone and about 100 Nucleotide.
NS3-NS5B polyprotein coding region provides the polyprotein that can be processed into different proteins in cell.The suitable NS3-NS5B polyprotein sequence that can be the part of replicon is included in those NS3-NS5B polyprotein sequences that exist in the different HCV strains and causes NS3-NS5B processing to produce its function equivalent of functional reproducing unit.Appropriate processing can be measured by test NS5B RNA RNA-dependent polysaccharase.
" carrier " is section of DNA, and as plasmid, phage or clay, wherein another segment DNA section can be attached thereto, thus cause connect duplicating, express or integrating of DNA section." shuttle vectors " refers to carrier, wherein can insert or excision DNA section from carrier in specific limited enzyme site.The DNA section that inserts shuttle vectors encode usually desired polypeptides or RNA, and design limit enzyme site is used to transcribe and translate to guarantee that the DNA section inserts in the appropriate open reading-frame (ORF).
Variety carrier can be used for the express nucleic acid molecule.Examples of such carriers comprises chromosomal pattern, additive type and viral deutero-carrier, for example from bacterial plasmid, from phage, from yeast episome, from yeast chromosomal element (comprising the yeast artificial chromosome) deutero-carrier, from virus as baculovirus, papovavirus such as SV40, vaccinia virus, adenovirus, poxvirus, Pseudorabies virus, simplexvirus and retrovirus deutero-carrier.Carrier also can be derived from the combination in these sources, as from plasmid and those carriers of phage genetic elements deutero-, for example clay and phagemid.Be used for the suitable clone of protokaryon and eucaryon host and expression vector people such as Sambrook, (1989) the 2nd edition .Cold Spring Harbor of Molecular Cloning:ALaboratory Manual. Laboratory Press, ColdSpring Harbor, NY describes among the USA.
The carrier that contains suitable nucleic acid molecule can use known technology to import suitable host cell with propagation or expression.Host cell can comprise bacterial cell, includes but not limited to intestinal bacteria (E.coli), streptomyces (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium); Eukaryotic cell includes but not limited to yeast, insect cell such as fruit bat (Drosophila), zooblast such as Huh-7, HeLa, COS, HEK 293, MT-2T, CEM-SS and Chinese hamster ovary celI, and vegetable cell.
Carrier generally includes the selective marker of the cell subsets that can select to contain the construction of recombinant vector body.This mark can comprise in containing the same vehicle of described nucleic acid molecule herein or may reside on the discrete carrier.Mark comprises tetracycline resistance gene or ampicillin resistance gene that is used for prokaryotic host cell and Tetrahydrofolate dehydrogenase or the neomycin resistance that is used for eukaryotic host cell.Yet any mark that provides phenotypic character to select will be effective.
" polynucleotide " or " nucleic acid molecule " are often referred to any polyribonucleotide or polydeoxyribonucleotide, and it can be the RNA of unmodified or the RNA or the DNA of DNA or modification." polynucleotide " include but not limited to strand and double-stranded DNA, as DNA, strand and the double-stranded RNA of the mixture of strand district and double stranded region with as the RNA of the mixture in strand district and double-stranded territory district, comprise the hybrid molecule of DNA and RNA, it can be strand or more generally be the two strands or the mixture of strand district and double stranded region.In addition, " polynucleotide " refer to comprise RNA or DNA or comprise RNA and three sequences of DNA." polynucleotide " also comprise the short relatively polynucleotide that often are called oligonucleotide.
In addition, term " dna molecular " only refers to the primary structure and the secondary structure of molecule, and dna molecular is not limited to any concrete tertiary structure form.Therefore, this term is included in the double-stranded DNA of finding in linear DNA molecule (for example restriction fragment), virus, plasmid and the karyomit(e) especially.When the structure of concrete double chain DNA molecule is discussed, can be in this article together with the routine custom of non transcribed DNA chain (that is, have with mRNA homologous sequence chain) sequence be described according to the sequence that only provides 5 ' to 3 ' direction.
" RNA molecule " refers to be in the poly form of the ribonucleotide of its single stranded form or double-stranded spiral form.When the structure of concrete RNA molecule is discussed, can according to the routine custom that provides 5 ' to 3 ' direction sequence sequence be described in this article.
Term " Restriction Enzyme sequence " refers to by near equal specific double chain DNA sequences of identification of the bacterial enzyme of cutting double-stranded DNA and cutting at the specific nucleotide sequence place or it.Restriction Enzyme " EcoRV " recognition sequence 5 ' GAT
Figure GPA00001151487500101
ATC 3 ' and shown in (with ▲Show) cutting of nucleotide position place
3’CTA ▲TAG?5’
Double-stranded DNA.Restriction Enzyme " AsiSI " recognition sequence 5 ' GCGAT
Figure GPA00001151487500103
CGC 3 ' and at recognition sequence
3’CGC ▲TAGCG?5’
The T residue after the cutting.
As used in this article term " primer " refer to RNA or DNA, strand or double-stranded, generate and from biosystem deutero-or the synthetic oligonucleotide that produces by Restriction Enzyme digestion, wherein said oligonucleotide is placing appropriate environment following time can functionally serve as template dependency nucleic acid synthetic releaser.When existing together with suitable nucleoside triphosphate precursors, polysaccharase, suitable cofactor and the condition of suitable nucleic acid-templated, nucleic acid such as suitable temperature and pH, primer can be by the polysaccharase effect or is produced that Nucleotide due to the similar activity of primer extension product adds and 3 ' the terminal extension of primer.Cause that thing can and require according to concrete application conditions changing aspect the length.For example in the PCR reaction, primer length generally is a 15-25 Nucleotide or longer.Primer must be fully complementary synthetic to cause the purpose extension products with the purpose template, promptly can with purpose template strand renaturation by this way, described mode be enough to provide be in suitable juxtaposed primer 3 '-hydroxylic moiety be used in startup by polysaccharase or similar enzyme cause synthetic in.Do not require that primer sequence represents the complete complement of purpose template.For example, non-complementary nucleotide sequence (for example Restriction Enzyme recognition sequence) can be connected to otherwise be 5 ' of complementary primer-end.Alternatively, non-complementary base can be dispersed in Oligonucleolide primers sequence inside, and condition is that this primer sequence and purpose template strand have enough complementarity and be used for synthetic extension products so that the templa-primer complex body functionally to be provided.
Term " chimeric " means from merging or montage dna molecular together that the DNA from two or more different sourcess produces as used in this article.
As used herein, term " quasispecies " means little variant set of advantage HCV genome sequence (being genotype), and described little variant forms because of the high mutation rate between the HCV replicative phase in the experimenter of single infection even in the individual cells clone.
Term " experimenter " refers to vertebrates as used in this article, the member of mammalian species especially, and include but not limited to rodents, rabbit, Shrew Murinus and primates, the latter comprises the people.
Term " sample " refers to from the sample of isolating tissue of experimenter or body fluid, includes but not limited to for example blood plasma, serum, spinal fluid, lymph liquid, skin cut into slices (the external sections of theskin), respiratory tract, enteron aisle and urogenital tract, tear, saliva, breast, hemocyte, tumour, organ and also have the sample that vitro cell culture forms (including but not limited to from the cell of institute's culturing cell, the virus infection of inferring, conditioned medium and the cellular component that the reconstitution cell growth produces) outward.
When with external source or allogeneic dna sequence DNA or RNA transfered cell inside, then cell is by this type of DNA or RNA " conversion " or " transfection ".Convertibility or transfection DNA or RNA can or can unconformability, and (covalently connecting) goes into to constitute the chromosomal DNA of cellular genome.For example in prokaryotic organism, yeast and mammalian cell, convertibility DNA can maintain on additive type element such as the plasmid.With regard to eukaryotic cell, the cell of stable conversion is such cell, and wherein convertibility DNA is integrated in the karyomit(e), thereby this DNA is inherited by the filial generation cell by chromosome duplication.The ability that this stability is set up clone or clone by eukaryotic cell shows that wherein said clone or clone are made up of the daughter cell colony of containing convertibility DNA.Under the situation that transforms the HCV replicon of mammalian cell described in the present invention, RNA molecule for example HCV RNA molecule has the ability of half self-replicating.The Huh-7 cell that carries the HCV replicon detects by selective marker or the reporter gene that exists on this replicon.
" clone " refers to usually by the mitotic division process from individual cells or common ancestor's deutero-cell colony.
Embodiment
Those skilled in the art provide following preparation and embodiment so that can more be expressly understood and implement the present invention.They should not be considered as limiting the scope of the invention, and only are considered as explanation and describe the present invention.
Embodiment 1
The structure of plasmid
The NS3 protease replicon shuttle vectors is derived from HCV replicon shuttle vectors pSC_1b_NS3_EcoRV, wherein said pSC_1b_NS3_EcoRV is used for producing the intermediate carrier of HCV NS3 replicon shuttle vectors pSC_1b_NS3_EcoRV_XbaI and the U.S. Patent application USSN 60/995 that is called " HCV NS3 replicon shuttle vectors " that submits on September 27th, 2007 people such as Chua, open in 558, complete being incorporated herein as a reference of described document mode by reference.The assembly of described replicon shuttle vectors shows and contains the HCV polynucleotide sequence from 5 '-UTR, NS3 to NS5B albumen and 3 '-UTR in Fig. 1.Described carrier also comprises the poliovirus internal ribosome entry site (IRES) of control Lampyridea luciferase genes translation.In the downstream of Lampyridea luciferase genes, control the translation of HCV nonstructural gene (NS3, NS4A, NS4B, NS5A and NS5B) from the IRES of encephalomyocarditis virus (EMCV).
Use the site-directed mutagenesis kit of QuickChange, according to manufacturer specification (Stratagene, La Jolla, CA, USA), importing pSC_1b_NS3_EcoRV_XbaI will suddenly change.For the proteolytic enzyme structural domain 3 ' end (corresponding to amino acid position Ser181) at the NS3 gene imports AsiSI Restriction Enzyme sequence, use following primer:
Adopted primer is arranged
5’-GAGTCTATGGGAACCACTATGCGATCGCCGGTCTTCACGGACAACTCGTC-3’(SEQ?ID?NO：1)
Antisense primer
5’-GACGAGTTGTCCGTGAAGACCGGCGATCGCATAGTGGTTCCCATAGACTC-3’(SEQ?ID?NO：2)
These sudden changes do not change the proteic amino acid coding of NS3 and cause generating NS3 proteolytic enzyme shuttle vectors pSC_1b_NS3/ proteolytic enzyme _ EcoRV_AsiSI (SEQ ID NO:3, Fig. 2 A).Generated another kind of NS3 protease replicon shuttle vectors, the sequence of the proteolytic enzyme structural domain of the NS3 gene of wherein encoding is substituted by beta-galactosidase enzymes (lacZ) encoding sequence from pUC19 (GenBank accession number M77789).Use following primer, import at the EcoRV restriction site of lacZ gene 5 ' end with at its 3 ' AsiSI restriction site of holding by pcr amplification
5’-ATCATCATCGATATCACCGCGTTGGCCGATTCATTAATG-3’(SEQ?ID?NO：4)(EcoRV)??????????????(LacZ)
5’-GATGATGATGCGATCGCCAGCTCCCGGAGACGGTCAC-3’(SEQ?ID?NO：5)(AsiSI)???????????????(LacZ)
To generate carrier pSC_1b_NS3/ proteolytic enzyme/LacZ_EcoRV_AsiSI (SEQ ID NO:6, Fig. 2 B).
Use the replication of the phenotype test method assessment NS3 protease replicon shuttle vectors of describing (with in Fig. 3, showing) among the embodiment 3.
Embodiment 2
The clone from the NS3 proteolytic enzyme structural domain PCR sample of infected patient amplification to the NS3 protease replicon shuttle vectors
Use the scheme of SuperScript III system (Invitrogen) according to manufacturers, produce the dna sequence dna of the proteolytic enzyme structural domain of coding NS3 gene by the reverse transcription polymerase chain reaction (RT-PCR) from the RNA of blood plasma, wherein said blood plasma obtains from the patient of HCV infection genotype-1a and genotype 1-b.The primer that is used for this RT-PCR step is as follows.
Genotype 1a:
Adopted primer (NS2) 5 '-CGTGCGGTGACATCATCAACGG-3 ' (SEQ ID NO:7) antisense primer (NS3/ helicase) 5 '-CTCGCCCCCGCAGTCTCTGC-3 ' (SEQ IDNO:8) is arranged
Genotype 1b:
Adopted primer (NS2) 5 '-GAGACCAAGATCATCACCTGG-3 ' (SEQ ID NO:9) is arranged
Antisense primer (NS3/ helicase) 5 '-GTCCAGGACTGTGCCGATGCC-3 ' (SEQ IDNO:10)
At first carry out renaturation and be the PCR circulation of 94 ℃ of sex change, 53 ℃ of renaturation and 68 ℃ of extensions subsequently at 50 ℃.Use PhusionTM high-fidelity DNA polymerase system (New EnglandBiolabs) subsequently, use the primer that imports unique Restriction Enzyme sequence at 5 ' and 3 ' end of the proteic proteolytic enzyme structural domain of NS3, amplified production is carried out second take turns PCR.What be used near patient NS3 gene order 5 ' end importing EcoRV Restriction Enzyme sequence has adopted primer as follows:
Genotype 1a
5’-CTGTCTGTCTGATATCACCATGGCGCCCATCACGGCGTACGC
C-3’(SEQ?ID?NO：11)
Genotype 1b
5’-CTGTCTGTCTGATATCACCATGGCGCCTATTACGGCCTACTC-3’(SEQ?ID?NO：12)
It is as follows to be used near the proteolytic enzyme structural domain 3 ' end (corresponding to amino acid position Ser181) of NS3 gene the primer of importing AsiSI Restriction Enzyme sequence:
Genotype 1a
5’-TAGTAGTAGGCGATCGCATGGTTGTCCCTAGGTTCTC-3’(SEOID?NO：13)
Genotype 1b
5’-TAGTAGTAGGCGATCGCATAGTGGTTCCCATAGACTCG-3’
(SEQ?ID?NO：14)
Amplification is implemented in PCR circulation with 98 ℃ of sex change, 53 ℃ of renaturation and 72 ℃ of extensions.The patient NS3 albumen enzyme dna of amplification digests with Qiagen PCR purification column purifying and with EcoRV and SgfI (isoschizomer of AsiSI) subsequently.
NS3 proteolytic enzyme shuttles back and forth replicon carrier pSC_1b_NS3/ proteolytic enzyme _ EcoRV_AsiSI or pSC_1b_NS3/ proteolytic enzyme/LacZ_EcoRV_AsiSI by preparing with restriction endonuclease EcoRV and SgfI double digested.Use MightMix to connect test kit (Takara BioInc.), the 25ng shuttle vectors is connected 1.5 hours with patient's amplicon of digestion at 16 ℃.The carrier that uses 1: 2 to 1: 4 routinely is to the ratio of inset.5 μ l reactants are converted in the Top10 cell (Invitrogen) of 50 μ l and at 37 ℃ of phenotypic expressions coated plate after 1 hour.
Choose 96 independent bacterium colonies are supplemented with 50 μ g/ml penbritins with inoculation 200 μ lTerrific meat soups (TB).This " original seed " 96 hole flat boards are 37 ℃ of overnight incubation.Next day, use this flat board to prepare one and repeat dull and stereotyped.Use 48 pin reproducers flat board is copied on two LB flat boards that are supplemented with 100 μ g/ml Pyocianils.With 96 parts independently 200 μ l cultures also be transferred to the 1.5ml TB substratum that is supplemented with 50 μ g/ml penbritins to be used for the DNA preparation.After 37 ℃ of night incubation, sprawl replica plate to obtain 96 clones' the heterogeneous thing that compiles with 6ml LB, the described thing that compiles is used for the minim DNA preparation subsequently.Patient's this DNA compiles thing and is used for follow-up replicon phenotype test method subsequently.After 37 ℃ of joltings of spending the night, with 96 parts independently 1.5ml TB culture centrifugally get off, decant and use the Qiaprep 96Turbo Mini-DNA test kit of Qiagen to extract plasmid DNA.These independently molecular cloning be used for sequencing reaction, the compound 69 6 that the representative of wherein said independently molecular cloning is used for replicon phenotype test method compiles thing.
To heterogeneous clone compile the thing plasmid DNA or independently molecular cloning check order with the identity that confirms patient's sample and in the sub-assay method of phenocopy check to the susceptibility of inhibitor.
Embodiment 3
The sub-assay method of phenocopy
A. the preparation of in-vitro transcription RNA
5 micrograms of DNA plasmids are by Sca I Restriction Enzyme (Roche) linearizing.After 37 ℃ of digested overnight, use this DNA of Qiagen PCR purification kit purifying.Use T7RiboMAX Express (Promega),, the linearizing DNA of 1 microgram is used for in-vitro transcription according to the scheme of manufacturers.37 ℃ hatch 2 hours after, carry out 30 minutes DNA enzymes at 37 ℃ and handle to remove dna profiling.Use the centrifugal post of RNeasy (Qiagen) then, according to the scheme of manufacturers, the RNA of purifying in-vitro transcription.
B. hepatoma cell line
With liver cancer Lunet Huh-7 clone at 37 ℃ in containing 5%CO 2Moistening atmosphere under cultivate and to be supplemented with Glutamax TMImprove in the Eagle substratum (DMEM) with the Dulbecco of 100mg/ml Sodium.alpha.-ketopropionate.This substratum further replenishes with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin.All reagent is all from Invitrogen/Gibco.
C. determining of instantaneous type replicon levels of replication
Use electroporation, with 400 ten thousand Lunet Huh7 of RNA transfection cell of 5 μ g in-vitro transcription.Cell be resuspended among the 7.2ml DMEM that contains 5%FBS subsequently and with 50000 cells/well (in 90 μ l final volume) cover plant in 96 hole flat boards.After adding inhibitor (when using inhibitor) with 3 times of extent of dilution with the 1%DMSO final concentration and adding inhibitor in 24 hours after the transfection, used luciferase assay method system (Promega) to read Lampyridea luciferase report signal in 72 hours.With IC 50Value is evaluated as such inhibitor concentration, and when described inhibitor concentration, the level of Lampyridea luciferase signal is compared when not adding compound, observes the Lampyridea luciferase and reports that proteic level reduces by 50%.
With luciferase signal verification replicon shuttle vectors pSC_1b_NS3/ proteolytic enzyme _ EcoRV_AsiSI and contain HCV genotype-1a and those replicatioies of the replicon of genotype-1b patient's deutero-NS3 proteolytic enzyme structural domain and display result in Fig. 3.Also on these replicons, check the restraining effect of HCV proteinase inhibitor BILN2061, VX-950 and NM107 and be displayed in Table 1 the result.
Table 1
Genotype Replicon Average IC 50
Contrast or patient's sample number ??BILN2061??nM ??VX-950??nM ??NM107??μM
PSC_1b_NS3/ proteolytic enzyme EcoRV AsiSI ??0.489 ??0.160 ??0.283
??GT-1a ??RO-191 ??0.798 ??0.057 ??0.539
??GT-1a ??RO-192 ??0.340 ??0.068 ??0.554
??GT-1a ??RO-193 ??0.298 ??0.132 ??0.640
??GT-1a ??RO-194 ??0.665 ??0.052 ??0.509
??GT-1a ??RO-195 ??0.629 ??0.028 ??0.312
??GT-1a ??RO-207 ??0.386 ??0.083 ??1.162
??GT-1b ??PC232 ??0.468 ??0.166 ??0.449
??GT-1b ??206 ??0.643 ??0.175 ??0.279
??GT-1b ??301 ??0.378 ??0.162 ??0.279
Sequence table
＜110〉Flax Huffmun-Laroqie Co., Ltd
 
＜120〉HCV NS3 protease replicon shuttle vectors
 
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＜170〉PatentIn version 3 .2
 
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＜223〉carrier A siSI has adopted primer
 
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gagtctatgg?gaaccactat?gcgatcgccg?gtcttcacgg?acaactcgtc????50
 
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＜213〉artificial sequence
 
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＜223〉carrier A siSI antisense primer
 
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gacgagttgt?ccgtgaagac?cggcgatcgc?atagtggttc?ccatagactc????50
 
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＜223〉pSC 1b NS3/ proteolytic enzyme EcoRV AsiSI
 
<400>3
cctgcaggta?atacgactca?ctatagccag?cccccgattg?ggggcgacac?tccaccatag????60
atcactcccc?tgtgaggaac?tactgtcttc?acgcagaaag?cgtctagcca?tggcgttagt????120
atgagtgtcg?tgcagcctcc?aggacccccc?ctcccgggag?agccatagtg?gtctgcggaa????180
ccggtgagta?caccggaatt?gccaggacga?ccgggtcctt?tcttggatca?acccgctcaa????240
tgcctggaga?tttgggcgtg?cccccgcgag?actgctagcc?gagtagtgtt?gggtcgcgaa????300
aggccttgtg?gtactgcctg?atagggtgct?tgcgagtgcc?ccgggaggtc?tcgtagaccg????360
tgcaccgttt?aaacccccgt?gctgctggaa?gtcgatttcg?cttagggtaa?ccgtggacct????420
cgaaaacaga?cgcacaaaac?caagttcaat?agaagggggt?acaaaccagt?accaccacga????480
acaagcactt?ctgtttcccc?ggtgatgtcg?tatagactgc?ttgcgtggtt?gaaagcgacg????540
gatccgttat?ccgcttatgt?acttcgagaa?gcccagtacc?acctcggaat?cttcgatgcg????600
ttgcgctcag?cactcaaccc?cagagtgtag?cttaggctga?tgagtctgga?catccctcac????660
cggtgacggt?ggtccaggct?gcgttggcgg?cctacctatg?gctaacgcca?tgggacgcta????720
gttgtgaaca?aggtgtgaag?agcctattga?gctacataag?aatcctccgg?cccctgaatg????780
cggctaatcc?caacctcgga?gcaggtggtc?acaaaccagt?gattggcctg?tcgtaacgcg????840
caagtccgtg?gcggaaccga?ctactttggg?tgtccgtgtt?tccttttatt?ttattgtggc????900
tgcttatggt?gacaatcaca?gattgttatc?ataaagcgaa?ttggattggc?catccggtga????960
aagtgagact?cattatctat?ctgtttgctg?gatccgctcc?attgagtgtg?tttactctaa????1020
gtacaatttc?aacagttatt?tcaatcagac?aattgtatca?taatggcggg?cccagaagac????1080
gccaaaaaca?taaaggaagg?cccggcgcca?ttctatcctc?ttgaggatgg?aaccgctgga????1140
gagcaactgc?ataaggctat?gaagagatac?gccctggttc?ctggaacaat?tgcttttaca????1200
gatgcacata?tcgaggtgaa?catcacgtac?gcggaatact?tcgaaatgtc?cgttcggttg????1260
gcagaagcta?tgaaacgata?tgggctgaat?acaaatcaca?gaatcgtcgt?atgcagtgaa????1320
aactctcttc?aattctttat?gccggtgttg?ggcgcgttat?ttatcggagt?tgcagttgcg????1380
cccgcgaacg?acatttataa?tgaacgtgaa?ttgctcaaca?gtatgaacat?ttcgcagcct????1440
accgtagtgt?ttgtttccaa?aaaggggttg?caaaaaattt?tgaacgtgca?aaaaaaatta????1500
ccaataatcc?agaaaattat?tatcatggat?tctaaaacgg?attaccaggg?atttcagtcg????1560
atgtacacgt?tcgtcacatc?tcatctacct?cccggtttta?atgaatacga?ttttgtacca????1620
gagtcctttg?atcgtgacaa?aacaattgca?ctgataatga?attcctctgg?atctactggg????1680
ttacctaagg?gtgtggccct?tccgcataga?actgcctgcg?tcagattctc?gcatgccaga????1740
gatcctattt?ttggcaatca?aatcattccg?gatactgcga?ttttaagtgt?tgttccattc????1800
catcacggtt?ttggaatgtt?tactacactc?ggatatttga?tatgtggatt?tcgagtcgtc????1860
ttaatgtata?gatttgaaga?agagctgttt?ttacgatccc?ttcaggatta?caaaattcaa????1920
agtgcgttgc?tagtaccaac?cctattttca?ttcttcgcca?aaagcactct?gattgacaaa????1980
tacgatttat?ctaatttaca?cgaaattgct?tctgggggcg?cacctctttc?gaaagaagtc????2040
ggggaagcgg?ttgcaaaacg?cttccatctt?ccagggatac?gacaaggata?tgggctcact????2100
gagactacat?cagctattct?gattacaccc?gagggggatg?ataaaccggg?cgcggtcggt????2160
aaagttgttc?cattttttga?agcgaaggtt?gtggatctgg?ataccgggaa?aacgctgggc????2220
gttaatcaga?gaggcgaatt?atgtgtcaga?ggacctatga?ttatgtccgg?ttatgtaaac????2280
aatccggaag?cgaccaacgc?cttgattgac?aaggatggat?ggctacattc?tggagacata????2340
gcttactggg?acgaagacga?acacttcttc?atagttgacc?gcttgaagtc?tttaattaaa????2400
tacaaaggtt?atcaggtggc?ccccgctgaa?ttggaatcga?tattgttaca?acaccccaac????2460
atcttcgacg?cgggcgtggc?aggtcttccc?gacgatgacg?ccggtgaact?tcccgccgcc????2520
gttgttgttt?tggagcacgg?aaagacgatg?acggaaaaag?agatcgtgga?ttacgtcgcc????2580
agtcaagtaa?caaccgcgaa?aaagttgcgc?ggaggagttg?tgtttgtgga?cgaagtaccg????2640
aaaggtctta?ccggaaaact?cgacgcaaga?aaaatcagag?agatcctcat?aaaggccaag????2700
aagggcggaa?agtccaaatt?gtaagcggcc?gcgttgttaa?acagaccaca?acggtttccc????2760
tctagcggga?tcaattccgc?cccccccccc?taacgttact?ggccgaagcc?gcttggaata????2820
aggccggtgt?gcgtttgtct?atatgttatt?ttccaccata?ttgccgtctt?ttggcaatgt????2880
gagggcccgg?aaacctggcc?ctgtcttctt?gacgagcatt?cctaggggtc?tttcccctct????2940
cgccaaagga?atgcaaggtc?tgttgaatgt?cgtgaaggaa?gcagttcctc?tggaagcttc????3000
ttgaagacaa?acaacgtctg?tagcgaccct?ttgcaggcag?cggaaccccc?cacctggcga????3060
caggtgcctc?tgcggccaaa?agccacgtgt?ataagataca?cctgcaaagg?cggcacaacc????3120
ccagtgccac?gttgtgagtt?ggatagttgt?ggaaagagtc?aaatggctct?cctcaagcgt????3180
attcaacaag?gggctgaagg?atgcccagaa?ggtaccccat?tgtatgggat?ctgatctggg????3240
gcctcggtgc?acatgcttta?catgtgttta?gtcgaggtta?aaaaaacgtc?taggcccccc????3300
gaaccacggg?gacgtggttt?tcctttgaaa?aacacgatat?caccatggcg?cctattacgg????3360
cctactccca?acagacgcga?ggcctacttg?gctgcatcat?cactagcctc?acaggccggg????3420
acaggaacca?ggtcgagggg?gaggtccaag?tggtctccac?cgcaacacaa?tctttcctgg????3480
cgacctgcgt?caatggcgtg?tgttggactg?tctatcatgg?tgccggctca?aagacccttg????3540
ccggcccaaa?gggcccaatc?acccaaatgt?acaccaatgt?ggaccaggac?ctcgtcggct????3600
ggcaagcgcc?ccccggggcg?cgttccttga?caccatgcac?ctgcggcagc?tcggaccttt????3660
acttggtcac?gaggcatgcc?gatgtcattc?cggtgcgccg?gcggggcgac?agcaggggga????3720
gcctactctc?ccccaggccc?gtctcctact?tgaagggctc?ttcgggcggt?ccactgctct????3780
gcccctcggg?gcacgctgtg?ggcatctttc?gggctgccgt?gtgcacccga?ggggttgcga????3840
aggcggtgga?ctttgtaccc?gtcgagtcta?tgggaaccac?tatgcgatcg?ccggtcttca????3900
cggacaactc?gtcccctccg?gccgtaccgc?agacattcca?ggtggcccat?ctacacgccc????3960
ctactggtag?cggcaagagc?actaaggtgc?cggctgcgta?tgcagcccaa?gggtataagg????4020
tgcttgtcct?gaacccgtcc?gtcgccgcca?ccctaggttt?cggggcgtat?atgtctaagg????4080
cacatggtat?cgaccctaac?atcagaatcg?gggtaaggac?catcaccacg?ggtgccccca????4140
tcacgtactc?cacctatggc?aagtttcttg?ccgacggtgg?ttgctctggg?ggcgcctatg????4200
acatcataat?atgtgatgag?tgccactcaa?ctgactcgac?cactatcctg?ggcatcggca????4260
cagtcctgga?ccaagcggag?acggctggag?cgcgactcgt?cgtgctcgcc?accgctacgc????4320
ctccgggatc?ggtcaccgtg?ccacatccaa?acatcgagga?ggtggctctg?tccagcactg????4380
gagaaatccc?cttttatggc?aaagccatcc?ccatcgagac?catcaagggg?gggaggcacc????4440
tcattttctg?ccattccaag?aagaaatgtg?atgagctcgc?cgcgaagctg?tccggcctcg????4500
gactcaatgc?tgtagcatat?taccggggcc?ttgatgtatc?cgtcatacca?actagcggag????4560
acgtcattgt?cgtagcaacg?gacgctctaa?tgacgggctt?taccggcgat?ttcgactcag????4620
tgatcgactg?caatacatgt?gtcacccaga?cagtcgactt?cagcctggac?ccgaccttca????4680
ccattgagac?gacgaccgtg?ccacaagacg?cggtgtcacg?ctcgcagcgg?cgaggcagga????4740
ctggtagggg?caggatgggc?atttacaggt?ttgtgactcc?aggagaacgg?ccctcgggca????4800
tgttcgattc?ctcggttctg?tgcgagtgct?atgacgcggg?ctgtgcttgg?tacgagctca????4860
cgcccgccga?gacctcagtt?aggttgcggg?cttacctaaa?cacaccaggg?ttgcccgtct????4920
gccaggacca?tctggagttc?tgggagagcg?tctttacagg?cctcacccac?atagacgccc????4980
atttcttgtc?ccagactaag?caggcaggag?acaacttccc?ctacctggta?gcataccagg????5040
ctacggtgtg?cgccagggct?caggctccac?ctccatcgtg?ggaccaaatg?tggaagtgtc????5100
tcatacggct?aaagcctacg?ctgcacgggc?caacgcccct?gctgtatagg?ctgggagccg????5160
ttcaaaacga?ggttactacc?acacacccca?taaccaaata?catcatggca?tgcatgtcgg????5220
ctgatctaga?ggtcgtcacg?agcacctggg?tgctggtagg?cggagtccta?gcagctctgg????5280
ccgcgtattg?cctgacaaca?ggcagcgtgg?tcattgtggg?caggatcatc?ttgtccggaa????5340
agccggccat?cattcccgac?agggaagtcc?tttaccggga?gttcgatgag?atggaagagt????5400
gcgcctcaca?cctcccttac?atcgaacagg?gaatgcagct?cgccgaacaa?ttcaaacaga????5460
aggcaatcgg?gttgctgcaa?acagccacca?agcaagcgga?ggctgctgct?cccgtggtgg????5520
aatccaagtg?gcggaccctc?gaagccttct?gggcgaagca?tatgtggaat?ttcatcagcg????5580
ggatacaata?tttagcaggc?ttgtccactc?tgcctggcaa?ccccgcgata?gcatcactga????5640
tggcattcac?agcctctatc?accagcccgc?tcaccaccca?acataccctc?ctgtttaaca????5700
tcctgggggg?atgggtggcc?gcccaacttg?ctcctcccag?cgctgcttct?gctttcgtag????5760
gcgccggcat?cgctggagcg?gctgttggca?gcataggcct?tgggacggtg?cttgtggata????5820
ttttggcagg?ttatggagca?ggggtggcag?gcgcgctcgt?ggcctttaag?gtcatgagcg????5880
gcgagatgcc?ctccaccgag?gacctggtta?acctactccc?tgctatcctc?tcccctggcg????5940
ccctagtcgt?cggggtcgtg?tgcgcagcga?tactgcgtcg?gcacgtgggc?ccaggggagg????6000
gggctgtgca?gtggatgaac?cggctgatag?cgttcgcttc?gcggggtaac?cacgtctccc????6060
ccacgcacta?tgtgcctgag?agcgacgctg?cagcacgtgt?cactcagatc?ctctctagtc????6120
ttaccatcac?tcagctgctg?aagaggcttc?accagtggat?caacgaggac?tgctccacgc????6180
catgctccgg?ctcgtggcta?agagatgttt?gggattggat?atgcacggtg?ttgactgatt????6240
tcaagacctg?gctccagtcc?aagctcctgc?cgcgattgcc?gggagtcccc?ttcttctcat????6300
gtcaacgtgg?gtacaaggga?gtctggcggg?gcgacggcat?catgcaaacc?acctgcccat????6360
gtggagcaca?gatcaccgga?catgtgaaaa?acggttccat?gaggatcgtg?gggcctagga????6420
cctgtagtaa?cacgtggcat?ggaacattcc?ccattaacgc?gtacaccacg?ggcccctgca????6480
cgccctcccc?ggcgccaaat?tattctaggg?cgctgtggcg?ggtggctgct?gaggagtacg????6540
tggaggttac?gcgggtgggg?gatttccact?acgtgacggg?catgaccact?gacaacgtaa????6600
agtgcccgtg?tcaggttccg?gcccccgaat?tcttcacaga?agtggatggg?gtgcggttgc????6660
acaggtacgc?tccagcgtgc?aaacccctcc?tacgggagga?ggtcacattc?ctggtcgggc????6720
tcaatcaata?cctggttggg?tcacagctcc?catgcgagcc?cgaaccggac?gtagcagtgc????6780
tcacttccat?gctcaccgac?ccctcccaca?ttacggcgga?gacggctaag?cgtaggctgg????6840
ccaggggatc?tcccccctcc?ttggccagct?catcagctag?ccagctgtct?gcgccttcct????6900
tgaaggcaac?atgcactacc?cgtcatgact?ccccggacgc?tgacctcatc?gaggccaacc????6960
tcctgtggcg?gcaggagatg?ggcgggaaca?tcacccgcgt?ggagtcagaa?aataaggtag????7020
taattttgga?ctctttcgag?ccgctccaag?cggaggagga?tgagagggaa?gtatccgttc????7080
cggcggagat?cctgcggagg?tccaggaaat?tccctcgagc?gatgcccata?tgggcacgcc????7140
cggattacaa?ccctccactg?ttagagtcct?ggaaggaccc?ggactacgtc?cctccagtgg????7200
tacacgggtg?tccattgccg?cctgccaagg?cccctccgat?accacctcca?cggaggaaga????7260
ggacggttgt?cctgtcagaa?tctaccgtgt?cttctgcctt?ggcggagctc?gccacaaaga????7320
ccttcggcag?ctccgaatcg?tcggccgtcg?acagcggcac?ggcaacggcc?tctcctgacc????7380
agccctccga?cgacggcgac?gcgggatccg?acgttgagtc?gtactcctcc?atgccccccc????7440
ttgaggggga?gccgggggat?cccgatctca?gcgacgggtc?ttggtctacc?gtaagcgagg????7500
aggctagtga?ggacgtcgtc?tgctgctcga?tgtcctacac?atggacaggc?gccctgatca????7560
cgccatgcgc?tgcggaggaa?accaagctgc?ccatcaatgc?actgagcaac?tctttgctcc????7620
gtcaccacaa?cttggtctat?gctacaacat?ctcgcagcgc?aagcctgcgg?cagaagaagg????7680
tcacctttga?cagactgcag?gtcctggacg?accactaccg?ggacgtgctc?aaggagatga????7740
aggcgaaggc?gtccacagtt?aaggctaaac?ttctatccgt?ggaggaagcc?tgtaagctga????7800
cgcccccaca?ttcggccaga?tctaaatttg?gctatggggc?aaaggacgtc?cggaacctat????7860
ccagcaaggc?cgttaaccac?atccgctccg?tgtggaagga?cttgctggaa?gacactgaga????7920
caccaattga?caccaccatc?atggcaaaaa?atgaggtttt?ctgcgtccaa?ccagagaagg????7980
ggggccgcaa?gccagctcgc?cttatcgtat?tcccagattt?gggggttcgt?gtgtgcgaga????8040
aaatggccct?ttacgatgtg?gtctccaccc?tccctcaggc?cgtgatgggc?tcttcatacg????8100
gattccaata?ctctcctgga?cagcgggtcg?agttcctggt?gaatgcctgg?aaagcgaaga????8160
aatgccctat?gggcttcgca?tatgacaccc?gctgttttga?ctcaacggtc?actgagaatg????8220
acatccgtgt?tgaggagtca?atctaccaat?gttgtgactt?ggcccccgaa?gccagacagg????8280
ccataaggtc?gctcacagag?cggctttaca?tcgggggccc?cctgactaat?tctaaagggc????8340
agaactgcgg?ctatcgccgg?tgccgcgcga?gcggtgtact?gacgaccagc?tgcggtaata????8400
ccctcacatg?ttacttgaag?gccgctgcgg?cctgtcgagc?tgcgaagctc?caggactgca????8460
cgatgctcgt?atgcggagac?gaccttgtcg?ttatctgtga?aagcgcgggg?acccaagagg????8520
acgaggcgag?cctacgggcc?ttcacggagg?ctatgactag?atactctgcc?ccccctgggg????8580
acccgcccaa?accagaatac?gacttggagt?tgataacatc?atgctcctcc?aatgtgtcag????8640
tcgcgcacga?tgcatctggc?aaaagggtgt?actatctcac?ccgtgacccc?accacccccc????8700
ttgcgcgggc?tgcgtgggag?acagctagac?acactccagt?caattcctgg?ctaggcaaca????8760
tcatcatgta?tgcgcccacc?ttgtgggcaa?ggatgatcct?gatgactcat?ttcttctcca????8820
tccttctagc?tcaggaacaa?cttgaaaaag?ccctagattg?tcagatctac?ggggcctgtt????8880
actccattga?gccacttgac?ctacctcaga?tcattcaacg?actccatggc?cttagcgcat????8940
tttcactcca?tagttactct?ccaggtgaga?tcaatagggt?ggcttcatgc?ctcaggaaac????9000
ttggggtacc?gcccttgcga?gtctggagac?atcgggccag?aagtgtccgc?gctaggctac????9060
tgtcccaggg?ggggagggct?gccacttgtg?gcaagtacct?cttcaactgg?gcagtaagga????9120
ccaagctcaa?actcactcca?atcccggctg?cgtcccagtt?ggatttatcc?agctggttcg????9180
ttgctggtta?cagcggggga?gacatatatc?acagcctgtc?tcgtgcccga?ccccgctggt????9240
tcatgtggtg?cctactccta?ctttctgtag?gggtaggcat?ctatctactc?cccaaccgat????9300
gaacggggag?ctaaacactc?caggccaata?ggccatcctg?tttttttccc?tttttttttt????9360
tctttttttt?tttttttttt?tttttttttt?ttttttctcc?tttttttttc?ctcttttttt????9420
ccttttcttt?cctttggtgg?ctccatctta?gccctagtca?cggctagctg?tgaaaggtcc????9480
gtgagccgct?tgactgcaga?gagtgctgat?actggcctct?ctgcagatca?agtactacta????9540
gtagaggcgg?tttgcgtatt?gggcgctctt?ccgcttcctc?gctcactgac?tcgctgcgct????9600
cggtcgttcg?gctgcggcga?gcggtatcag?ctcactcaaa?ggcggtaata?cggttatcca????9660
cagaatcagg?ggataacgca?ggaaagaaca?tgtgagcaaa?aggccagcaa?aaggccagga????9720
accgtaaaaa?ggccgcgttg?ctggcgtttt?tccataggct?ccgcccccct?gacgagcatc????9780
acaaaaatcg?acgctcaagt?cagaggtggc?gaaacccgac?aggactataa?agataccagg????9840
cgtttccccc?tggaagctcc?ctcgtgcgct?ctcctgttcc?gaccctgccg?cttaccggat????9900
acctgtccgc?ctttctccct?tcgggaagcg?tggcgctttc?tcatagctca?cgctgtaggt????9960
atctcagttc?ggtgtaggtc?gttcgctcca?agctgggctg?tgtgcacgaa?ccccccgttc????10020
agcccgaccg?ctgcgcctta?tccggtaact?atcgtcttga?gtccaacccg?gtaagacacg????10080
acttatcgcc?actggcagca?gccactggta?acaggattag?cagagcgagg?tatgtaggcg????10140
gtgctacaga?gttcttgaag?tggtggccta?actacggcta?cactagaagg?acagtatttg????10200
gtatctgcgc?tctgctgaag?ccagttacct?tcggaaaaag?agttggtagc?tcttgatccg????10260
gcaaacaaac?caccgctggt?agcggtggtt?tttttgtttg?caagcagcag?attacgcgca????10320
gaaaaaaagg?atctcaagaa?gatcctttga?tcttttctac?ggggtctgac?gctcagtgga????10380
acgaaaactc?acgttaaggg?attttggtca?tgagattatc?aaaaaggatc?ttcacctaga????10440
tccttttaaa?ttaaaaatga?agttttaaat?caatctaaag?tatatatgag?taaacttggt????10500
ctgacagtta?ccaatgctta?atcagtgagg?cacctatctc?agcgatctgt?ctatttcgtt????10560
catccatagt?tgcctgactc?cccgtcgtgt?agataactac?gatacgggag?ggcttaccat????10620
ctggccccag?tgctgcaatg?ataccgcgag?acccacgctc?accggctcca?gatttatcag????10680
caataaacca?gccagccgga?agggccgagc?gcagaagtgg?tcctgcaact?ttatccgcct????10740
ccatccagtc?tattaattgt?tgccgggaag?ctagagtaag?tagttcgcca?gttaatagtt????10800
tgcgcaacgt?tgttgccatt?gctacaggca?tcgtggtgtc?acgctcgtcg?tttggtatgg????10860
cttcattcag?ctccggttcc?caacgatcaa?ggcgagttac?atgatccccc?atgttgtgca????10920
aaaaagcggt?tagctccttc?ggtcctccga?tcgttgtcag?aagtaagttg?gccgcagtgt????10980
tatcactcat?ggttatggca?gcactgcata?attctcttac?tgtcatgcca?tccgtaagat????11040
gcttttctgt?gactggtgag?tactcaacca?agtcattctg?agaatagtgt?atgcggcgac????11100
cgagttgctc?ttgcccggcg?tcaatacggg?ataataccgc?gccacatagc?agaactttaa????11160
aagtgctcat?cattggaaaa?cgttcttcgg?ggcgaaaact?ctcaaggatc?ttaccgctgt????11220
tgagatccag?ttcgatgtaa?cccactcgtg?cacccaactg?atcttcagca?tcttttactt????11280
tcaccagcgt?ttctgggtga?gcaaaaacag?gaaggcaaaa?tgccgcaaaa?aagggaataa????11340
gggcgacacg?gaaatgttga?atactcatac?tcttcctttt?tcaatattat?tgaagcattt????11400
atcagggtta?ttgtctcatg?agcggataca?tatttgaatg?tatttagaaa?aataaacaaa????11460
taggggttcc?gcgcacattt??ccccgaaaag?tgccacctga?cgtctaagaa??accatt????11516
 
<210>4
<211>39
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉LacZ EcoRV has adopted primer
 
<400>4
atcatcatcg??atatcaccgc??gttggccgat??tcattaatg????39
 
<210>5
<211>37
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉LacZ AsiSI antisense primer
 
<400>5
gatgatgatg??cgatcgccag??ctcccggaga??cggtcac????37
 
<210>6
<211>11590
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉pSC_1b_NS3/ proteolytic enzyme/LacZ_EcoRV_AsiSI
 
<400>6
cctgcaggta?atacgactca?ctatagccag?cccccgattg?ggggcgacac?tccaccatag????60
atcactcccc?tgtgaggaac?tactgtcttc?acgcagaaag?cgtctagcca?tggcgttagt????120
atgagtgtcg?tgcagcctcc?aggacccccc?ctcccgggag?agccatagtg?gtctgcggaa????180
ccggtgagta?caccggaatt?gccaggacga?ccgggtcctt?tcttggatca?acccgctcaa????240
tgcctggaga?tttgggcgtg?cccccgcgag?actgctagcc?gagtagtgtt?gggtcgcgaa????300
aggccttgtg?gtactgcctg?atagggtgct?tgcgagtgcc?ccgggaggtc?tcgtagaccg????360
tgcaccgttt?aaacccccgt?gctgctggaa?gtcgatttcg?cttagggtaa?ccgtggacct????420
cgaaaacaga?cgcacaaaac?caagttcaat?agaagggggt?acaaaccagt?accaccacga????480
acaagcactt?ctgtttcccc?ggtgatgtcg?tatagactgc?ttgcgtggtt?gaaagcgacg????540
gatccgttat?ccgcttatgt?acttcgagaa?gcccagtacc?acctcggaat?cttcgatgcg????600
ttgcgctcag?cactcaaccc?cagagtgtag?cttaggctga?tgagtctgga?catccctcac????660
cggtgacggt?ggtccaggct?gcgttggcgg?cctacctatg?gctaacgcca?tgggacgcta????720
gttgtgaaca?aggtgtgaag?agcctattga?gctacataag?aatcctccgg?cccctgaatg????780
cggctaatcc?caacctcgga?gcaggtggtc?acaaaccagt?gattggcctg?tcgtaacgcg????840
caagtccgtg?gcggaaccga?ctactttggg?tgtccgtgtt?tccttttatt?ttattgtggc????900
tgcttatggt?gacaatcaca?gattgttatc?ataaagcgaa?ttggattggc?catccggtga????960
aagtgagact?cattatctat?ctgtttgctg?gatccgctcc?attgagtgtg?tttactctaa????1020
gtacaatttc?aacagttatt?tcaatcagac?aattgtatca?taatggcggg?cccagaagac????1080
gccaaaaaca?taaaggaagg?cccggcgcca?ttctatcctc?ttgaggatgg?aaccgctgga????1140
gagcaactgc?ataaggctat?gaagagatac?gccctggttc?ctggaacaat?tgcttttaca????1200
gatgcacata?tcgaggtgaa?catcacgtac?gcggaatact?tcgaaatgtc?cgttcggttg????1260
gcagaagcta?tgaaacgata?tgggctgaat?acaaatcaca?gaatcgtcgt?atgcagtgaa????1320
aactctcttc?aattctttat?gccggtgttg?ggcgcgttat?ttatcggagt?tgcagttgcg????1380
cccgcgaacg?acatttataa?tgaacgtgaa?ttgctcaaca?gtatgaacat?ttcgcagcct????1440
accgtagtgt?ttgtttccaa?aaaggggttg?caaaaaattt?tgaacgtgca?aaaaaaatta????1500
ccaataatcc?agaaaattat?tatcatggat?tctaaaacgg?attaccaggg?atttcagtcg????1560
atgtacacgt?tcgtcacatc?tcatctacct?cccggtttta?atgaatacga?ttttgtacca????1620
gagtcctttg?atcgtgacaa?aacaattgca?ctgataatga?attcctctgg?atctactggg????1680
ttacctaagg?gtgtggccct?tccgcataga?actgcctgcg?tcagattctc?gcatgccaga????1740
gatcctattt?ttggcaatca?aatcattccg?gatactgcga?ttttaagtgt?tgttccattc????1800
catcacggtt?ttggaatgtt?tactacactc?ggatatttga?tatgtggatt?tcgagtcgtc????1860
ttaatgtata?gatttgaaga?agagctgttt?ttacgatccc?ttcaggatta?caaaattcaa????1920
agtgcgttgc?tagtaccaac?cctattttca?ttcttcgcca?aaagcactct?gattgacaaa????1980
tacgatttat?ctaatttaca?cgaaattgct?tctgggggcg?cacctctttc?gaaagaagtc????2040
ggggaagcgg?ttgcaaaacg?cttccatctt?ccagggatac?gacaaggata?tgggctcact????2100
gagactacat?cagctattct?gattacaccc?gagggggatg?ataaaccggg?cgcggtcggt????2160
aaagttgttc?cattttttga?agcgaaggtt?gtggatctgg?ataccgggaa?aacgctgggc????2220
gttaatcaga?gaggcgaatt?atgtgtcaga?ggacctatga?ttatgtccgg?ttatgtaaac????2280
aatccggaag?cgaccaacgc?cttgattgac?aaggatggat?ggctacattc?tggagacata????2340
gcttactggg?acgaagacga?acacttcttc?atagttgacc?gcttgaagtc?tttaattaaa????2400
tacaaaggtt?atcaggtggc?ccccgctgaa?ttggaatcga?tattgttaca?acaccccaac????2460
atcttcgacg?cgggcgtggc?aggtcttccc?gacgatgacg?ccggtgaact?tcccgccgcc????2520
gttgttgttt?tggagcacgg?aaagacgatg?acggaaaaag?agatcgtgga?ttacgtcgcc????2580
agtcaagtaa?caaccgcgaa?aaagttgcgc?ggaggagttg?tgtttgtgga?cgaagtaccg????2640
aaaggtctta?ccggaaaact?cgacgcaaga?aaaatcagag?agatcctcat?aaaggccaag????2700
aagggcggaa?agtccaaatt?gtaagcggcc?gcgttgttaa?acagaccaca?acggtttccc????2760
tctagcggga?tcaattccgc?cccccccccc?taacgttact?ggccgaagcc?gcttggaata????2820
aggccggtgt?gcgtttgtct?atatgttatt?ttccaccata?ttgccgtctt?ttggcaatgt????2880
gagggcccgg?aaacctggcc?ctgtcttctt?gacgagcatt?cctaggggtc?tttcccctct????2940
cgccaaagga?atgcaaggtc?tgttgaatgt?cgtgaaggaa?gcagttcctc?tggaagcttc????3000
ttgaagacaa?acaacgtctg?tagcgaccct?ttgcaggcag?cggaaccccc?cacctggcga????3060
caggtgcctc?tgcggccaaa?agccacgtgt?ataagataca?cctgcaaagg?cggcacaacc????3120
ccagtgccac?gttgtgagtt?ggatagttgt?ggaaagagtc?aaatggctct?cctcaagcgt????3180
attcaacaag?gggctgaagg?atgcccagaa?ggtaccccat?tgtatgggat?ctgatctggg????3240
gcctcggtgc?acatgcttta?catgtgttta?gtcgaggtta?aaaaaacgtc?taggcccccc????3300
gaaccacggg?gacgtggttt?tcctttgaaa?aacacgatat?caccgcgttg?gccgattcat????3360
taatgcagct?ggcacgacag?gtttcccgac?tggaaagcgg?gcagtgagcg?caacgcaatt????3420
aatgtgagtt?agctcactca?ttaggcaccc?caggctttac?actttatgct?tccggctcgt????3480
atgttgtgtg?gaattgtgag?cggataacaa?tttcacacag?gaaacagcta?tgaccatgat????3540
tacgccaagc?ttgcatgcct?gcaggtcgac?tctagaggat?ccccgggtac?cgagctcgaa????3600
ttcactggcc?gtcgttttac?aacgtcgtga?ctgggaaaac?cctggcgtta?cccaacttaa????3660
tcgccttgca?gcacatcccc?ctttcgccag?ctggcgtaat?agcgaagagg?cccgcaccga????3720
tcgcccttcc?caacagttgc?gcagcctgaa?tggcgaatgg?cgcctgatgc?ggtattttct????3780
ccttacgcat?ctgtgcggta?tttcacaccg?catatggtgc?actctcagta?caatctgctc????3840
tgatgccgca?tagttaagcc?agccccgaca?cccgccaaca?cccgctgacg?cgccctgacg????3900
ggcttgtctg?ctcccggcat?ccgcttacag?acaagctgtg?accgtctccg?ggagctggcg????3960
atcgccggtc?ttcacggaca?actcgtcccc?tccggccgta?ccgcagacat?tccaggtggc????4020
ccatctacac?gcccctactg?gtagcggcaa?gagcactaag?gtgccggctg?cgtatgcagc????4080
ccaagggtat?aaggtgcttg?tcctgaaccc?gtccgtcgcc?gccaccctag?gtttcggggc????4140
gtatatgtct?aaggcacatg?gtatcgaccc?taacatcaga?atcggggtaa?ggaccatcac????4200
cacgggtgcc?cccatcacgt?actccaccta?tggcaagttt?cttgccgacg?gtggttgctc????4260
tgggggcgcc?tatgacatca?taatatgtga?tgagtgccac?tcaactgact?cgaccactat????4320
cctgggcatc?ggcacagtcc?tggaccaagc?ggagacggct?ggagcgcgac?tcgtcgtgct????4380
cgccaccgct?acgcctccgg?gatcggtcac?cgtgccacat?ccaaacatcg?aggaggtggc????4440
tctgtccagc?actggagaaa?tcccctttta?tggcaaagcc?atccccatcg?agaccatcaa????4500
gggggggagg?cacctcattt?tctgccattc?caagaagaaa?tgtgatgagc?tcgccgcgaa????4560
gctgtccggc?ctcggactca?atgctgtagc?atattaccgg?ggccttgatg?tatccgtcat????4620
accaactagc?ggagacgtca?ttgtcgtagc?aacggacgct?ctaatgacgg?gctttaccgg????4680
cgatttcgac?tcagtgatcg?actgcaatac?atgtgtcacc?cagacagtcg?acttcagcct????4740
ggacccgacc?ttcaccattg?agacgacgac?cgtgccacaa?gacgcggtgt?cacgctcgca????4800
gcggcgaggc?aggactggta?ggggcaggat?gggcatttac?aggtttgtga?ctccaggaga????4860
acggccctcg?ggcatgttcg?attcctcggt?tctgtgcgag?tgctatgacg?cgggctgtgc????4920
ttggtacgag?ctcacgcccg?ccgagacctc?agttaggttg?cgggcttacc?taaacacacc????4980
agggttgccc?gtctgccagg?accatctgga?gttctgggag?agcgtcttta?caggcctcac????5040
ccacatagac?gcccatttct?tgtcccagac?taagcaggca?ggagacaact?tcccctacct????5100
ggtagcatac?caggctacgg?tgtgcgccag?ggctcaggct?ccacctccat?cgtgggacca????5160
aatgtggaag?tgtctcatac?ggctaaagcc?tacgctgcac?gggccaacgc?ccctgctgta????5220
taggctggga?gccgttcaaa?acgaggttac?taccacacac?cccataacca?aatacatcat????5280
ggcatgcatg?tcggctgatc?tagaggtcgt?cacgagcacc?tgggtgctgg?taggcggagt????5340
cctagcagct?ctggccgcgt?attgcctgac?aacaggcagc?gtggtcattg?tgggcaggat????5400
catcttgtcc?ggaaagccgg?ccatcattcc?cgacagggaa?gtcctttacc?gggagttcga????5460
tgagatggaa?gagtgcgcct?cacacctccc?ttacatcgaa?cagggaatgc?agctcgccga????5520
acaattcaaa?cagaaggcaa?tcgggttgct?gcaaacagcc?accaagcaag?cggaggctgc????5580
tgctcccgtg?gtggaatcca?agtggcggac?cctcgaagcc?ttctgggcga?agcatatgtg????5640
gaatttcatc?agcgggatac?aatatttagc?aggcttgtcc?actctgcctg?gcaaccccgc????5700
gatagcatca?ctgatggcat?tcacagcctc?tatcaccagc?ccgctcacca?cccaacatac????5760
cctcctgttt?aacatcctgg?ggggatgggt?ggccgcccaa?cttgctcctc?ccagcgctgc????5820
ttctgctttc?gtaggcgccg?gcatcgctgg?agcggctgtt?ggcagcatag?gccttgggac????5880
ggtgcttgtg?gatattttgg?caggttatgg?agcaggggtg?gcaggcgcgc?tcgtggcctt????5940
taaggtcatg?agcggcgaga?tgccctccac?cgaggacctg?gttaacctac?tccctgctat????6000
cctctcccct?ggcgccctag?tcgtcggggt?cgtgtgcgca?gcgatactgc?gtcggcacgt????6060
gggcccaggg?gagggggctg?tgcagtggat?gaaccggctg?atagcgttcg?cttcgcgggg????6120
taaccacgtc?tcccccacgc?actatgtgcc?tgagagcgac?gctgcagcac?gtgtcactca????6180
gatcctctct?agtcttacca?tcactcagct?gctgaagagg?cttcaccagt?ggatcaacga????6240
ggactgctcc?acgccatgct?ccggctcgtg?gctaagagat?gtttgggatt?ggatatgcac????6300
ggtgttgact?gatttcaaga?cctggctcca?gtccaagctc?ctgccgcgat?tgccgggagt????6360
ccccttcttc?tcatgtcaac?gtgggtacaa?gggagtctgg?cggggcgacg?gcatcatgca????6420
aaccacctgc?ccatgtggag?cacagatcac?cggacatgtg?aaaaacggtt?ccatgaggat????6480
cgtggggcct?aggacctgta?gtaacacgtg?gcatggaaca?ttccccatta?acgcgtacac????6540
cacgggcccc?tgcacgccct?ccccggcgcc?aaattattct?agggcgctgt?ggcgggtggc????6600
tgctgaggag?tacgtggagg?ttacgcgggt?gggggatttc?cactacgtga?cgggcatgac????6660
cactgacaac?gtaaagtgcc?cgtgtcaggt?tccggccccc?gaattcttca?cagaagtgga????6720
tggggtgcgg?ttgcacaggt?acgctccagc?gtgcaaaccc?ctcctacggg?aggaggtcac????6780
attcctggtc?gggctcaatc?aatacctggt?tgggtcacag?ctcccatgcg?agcccgaacc????6840
ggacgtagca?gtgctcactt?ccatgctcac?cgacccctcc?cacattacgg?cggagacggc????6900
taagcgtagg?ctggccaggg?gatctccccc?ctccttggcc?agctcatcag?ctagccagct????6960
gtctgcgcct?tccttgaagg?caacatgcac?tacccgtcat?gactccccgg?acgctgacct????7020
catcgaggcc?aacctcctgt?ggcggcagga?gatgggcggg?aacatcaccc?gcgtggagtc????7080
agaaaataag?gtagtaattt?tggactcttt?cgagccgctc?caagcggagg?aggatgagag????7140
ggaagtatcc?gttccggcgg?agatcctgcg?gaggtccagg?aaattccctc?gagcgatgcc????7200
catatgggca?cgcccggatt?acaaccctcc?actgttagag?tcctggaagg?acccggacta????7260
cgtccctcca?gtggtacacg?ggtgtccatt?gccgcctgcc?aaggcccctc?cgataccacc????7320
tccacggagg?aagaggacgg?ttgtcctgtc?agaatctacc?gtgtcttctg?ccttggcgga????7380
gctcgccaca?aagaccttcg?gcagctccga?atcgtcggcc?gtcgacagcg?gcacggcaac????7440
ggcctctcct?gaccagccct?ccgacgacgg?cgacgcggga?tccgacgttg?agtcgtactc????7500
ctccatgccc?ccccttgagg?gggagccggg?ggatcccgat?ctcagcgacg?ggtcttggtc????7560
taccgtaagc?gaggaggcta?gtgaggacgt?cgtctgctgc?tcgatgtcct?acacatggac????7620
aggcgccctg?atcacgccat?gcgctgcgga?ggaaaccaag?ctgcccatca?atgcactgag????7680
caactctttg?ctccgtcacc?acaacttggt?ctatgctaca?acatctcgca?gcgcaagcct????7740
gcggcagaag?aaggtcacct?ttgacagact?gcaggtcctg?gacgaccact?accgggacgt????7800
gctcaaggag?atgaaggcga?aggcgtccac?agttaaggct?aaacttctat?ccgtggagga????7860
agcctgtaag?ctgacgcccc?cacattcggc?cagatctaaa?tttggctatg?gggcaaagga????7920
cgtccggaac?ctatccagca?aggccgttaa?ccacatccgc?tccgtgtgga?aggacttgct????7980
ggaagacact?gagacaccaa?ttgacaccac?catcatggca?aaaaatgagg?ttttctgcgt????8040
ccaaccagag?aaggggggcc?gcaagccagc?tcgccttatc?gtattcccag?atttgggggt????8100
tcgtgtgtgc?gagaaaatgg?ccctttacga?tgtggtctcc?accctccctc?aggccgtgat????8160
gggctcttca?tacggattcc?aatactctcc?tggacagcgg?gtcgagttcc?tggtgaatgc????8220
ctggaaagcg?aagaaatgcc?ctatgggctt?cgcatatgac?acccgctgtt?ttgactcaac????8280
ggtcactgag?aatgacatcc?gtgttgagga?gtcaatctac?caatgttgtg?acttggcccc????8340
cgaagccaga?caggccataa?ggtcgctcac?agagcggctt?tacatcgggg?gccccctgac????8400
taattctaaa?gggcagaact?gcggctatcg?ccggtgccgc?gcgagcggtg?tactgacgac????8460
cagctgcggt?aataccctca?catgttactt?gaaggccgct?gcggcctgtc?gagctgcgaa????8520
gctccaggac?tgcacgatgc?tcgtatgcgg?agacgacctt?gtcgttatct?gtgaaagcgc????8580
ggggacccaa?gaggacgagg?cgagcctacg?ggccttcacg?gaggctatga?ctagatactc????8640
tgccccccct?ggggacccgc?ccaaaccaga?atacgacttg?gagttgataa?catcatgctc????8700
ctccaatgtg?tcagtcgcgc?acgatgcatc?tggcaaaagg?gtgtactatc?tcacccgtga????8760
ccccaccacc?ccccttgcgc?gggctgcgtg?ggagacagct?agacacactc?cagtcaattc????8820
ctggctaggc?aacatcatca?tgtatgcgcc?caccttgtgg?gcaaggatga?tcctgatgac????8880
tcatttcttc?tccatccttc?tagctcagga?acaacttgaa?aaagccctag?attgtcagat????8940
ctacggggcc?tgttactcca?ttgagccact?tgacctacct?cagatcattc?aacgactcca????9000
tggccttagc?gcattttcac?tccatagtta?ctctccaggt?gagatcaata?gggtggcttc????9060
atgcctcagg?aaacttgggg?taccgccctt?gcgagtctgg?agacatcggg?ccagaagtgt????9120
ccgcgctagg?ctactgtccc?agggggggag?ggctgccact?tgtggcaagt?acctcttcaa????9180
ctgggcagta?aggaccaagc?tcaaactcac?tccaatcccg?gctgcgtccc?agttggattt????9240
atccagctgg?ttcgttgctg?gttacagcgg?gggagacata?tatcacagcc?tgtctcgtgc????9300
ccgaccccgc?tggttcatgt?ggtgcctact?cctactttct?gtaggggtag?gcatctatct????9360
actccccaac?cgatgaacgg?ggagctaaac?actccaggcc?aataggccat?cctgtttttt????9420
tccctttttt?tttttctttt?tttttttttt?tttttttttt?tttttttttt?ctcctttttt????9480
tttcctcttt?ttttcctttt?ctttcctttg?gtggctccat?cttagcccta?gtcacggcta????9540
gctgtgaaag?gtccgtgagc?cgcttgactg?cagagagtgc?tgatactggc?ctctctgcag????9600
atcaagtact?actagtagag?gcggtttgcg?tattgggcgc?tcttccgctt?cctcgctcac????9660
tgactcgctg?cgctcggtcg?ttcggctgcg?gcgagcggta?tcagctcact?caaaggcggt????9720
aatacggtta?tccacagaat?caggggataa?cgcaggaaag?aacatgtgag?caaaaggcca????9780
gcaaaaggcc?aggaaccgta?aaaaggccgc?gttgctggcg?tttttccata?ggctccgccc????9840
ccctgacgag?catcacaaaa?atcgacgctc?aagtcagagg?tggcgaaacc?cgacaggact????9900
ataaagatac?caggcgtttc?cccctggaag?ctccctcgtg?cgctctcctg?ttccgaccct????9960
gccgcttacc?ggatacctgt?ccgcctttct?cccttcggga?agcgtggcgc?tttctcatag????10020
ctcacgctgt?aggtatctca?gttcggtgta?ggtcgttcgc?tccaagctgg?gctgtgtgca????10080
cgaacccccc?gttcagcccg?accgctgcgc?cttatccggt?aactatcgtc?ttgagtccaa????10140
cccggtaaga?cacgacttat?cgccactggc?agcagccact?ggtaacagga?ttagcagagc????10200
gaggtatgta?ggcggtgcta?cagagttctt?gaagtggtgg?cctaactacg?gctacactag????10260
aaggacagta?tttggtatct?gcgctctgct?gaagccagtt?accttcggaa?aaagagttgg????10320
tagctcttga?tccggcaaac?aaaccaccgc?tggtagcggt?ggtttttttg?tttgcaagca????10380
gcagattacg?cgcagaaaaa?aaggatctca?agaagatcct?ttgatctttt?ctacggggtc????10440
tgacgctcag?tggaacgaaa?actcacgtta?agggattttg?gtcatgagat?tatcaaaaag????10500
gatcttcacc?tagatccttt?taaattaaaa?atgaagtttt?aaatcaatct?aaagtatata????10560
tgagtaaact?tggtctgaca?gttaccaatg?cttaatcagt?gaggcaccta?tctcagcgat????10620
ctgtctattt?cgttcatcca?tagttgcctg?actccccgtc?gtgtagataa?ctacgatacg????10680
ggagggctta?ccatctggcc?ccagtgctgc?aatgataccg?cgagacccac?gctcaccggc????10740
tccagattta?tcagcaataa?accagccagc?cggaagggcc?gagcgcagaa?gtggtcctgc????10800
aactttatcc?gcctccatcc?agtctattaa?ttgttgccgg?gaagctagag?taagtagttc????10860
gccagttaat?agtttgcgca?acgttgttgc?cattgctaca?ggcatcgtgg?tgtcacgctc????10920
gtcgtttggt?atggcttcat?tcagctccgg?ttcccaacga?tcaaggcgag?ttacatgatc????10980
ccccatgttg?tgcaaaaaag?cggttagctc?cttcggtcct?ccgatcgttg?tcagaagtaa????11040
gttggccgca?gtgttatcac?tcatggttat?ggcagcactg?cataattctc?ttactgtcat????11100
gccatccgta?agatgctttt?ctgtgactgg?tgagtactca?accaagtcat?tctgagaata????11160
gtgtatgcgg?cgaccgagtt?gctcttgccc?ggcgtcaata?cgggataata?ccgcgccaca????11220
tagcagaact?ttaaaagtgc?tcatcattgg?aaaacgttct?tcggggcgaa?aactctcaag????11280
gatcttaccg?ctgttgagat?ccagttcgat?gtaacccact?cgtgcaccca?actgatcttc????11340
agcatctttt?actttcacca?gcgtttctgg?gtgagcaaaa?acaggaaggc?aaaatgccgc????11400
aaaaaaggga?ataagggcga?cacggaaatg?ttgaatactc?atactcttcc?tttttcaata????11460
ttattgaagc?atttatcagg?gttattgtct?catgagcgga?tacatatttg?aatgtattta????11520
gaaaaataaa?caaatagggg?ttccgcgcac?atttccccga?aaagtgccac?ctgacgtcta????11580
agaaaccatt???????????????????????????????????????????????????????????11590
<210>7
<211>22
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉RT-PCR genotype 1a has adopted primer (NS2)
 
<400>7
cgtgcggtga?catcatcaac?gg????22
 
<210>8
<211>20
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉RT-PCR genotype 1a antisense primer (NS3 helicase)
 
<400>8
ctcgcccccg?cagtctctgc????20
 
<210>9
<211>21
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉RT-PCR genotype 1b has adopted primer (NS2)
 
<400>9
gagaccaaga?tcatcacctg?g????21
 
<210>10
<211>21
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉RT-PCR genotype 1b antisense primer (NS3 helicase)
 
<400>10
gtccaggact?gtgccgatgc?c????21
 
<210>11
<211>43
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉adopted primer genotype 1a EcoRV is arranged
 
<400>11
ctgtctgtct??gatatcacca?tggcgcccat?cacggcgtac?gcc????43
<210>12
<211>42
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉adopted primer genotype 1b EcoRV is arranged
 
<400>12
ctgtctgtct?gatatcacca?tggcgcctat?tacggcctac??tc????42
 
<210>13
<211>37
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉antisense primer genotype 1a AsiSI
 
<400>13
tagtagtagg??cgatcgcatg??gttgtcccta??ggttctc????37
 
<210>14
<211>38
<212>DNA
＜213〉artificial sequence
 
<220>
＜223〉antisense primer genotype 1b AsiSI
 
<400>14
tagtagtagg?cgatcgcata?gtggttccca?tagactcg????38

Claims (34)

1. the HCV replicon shuttle vectors that comprises the HCV polynucleotide sequence, described HCV polynucleotide sequence comprises successively:
(a) be arranged in 10 5 ' Nucleotide of the proteic polynucleotide sequence of own coding NS3 5 ' end and the unique Restriction Enzyme sequence between 10 3 ' Nucleotide;
(b) polynucleotide sequence of the proteic proteolytic enzyme structural domain of coding NS3;
(c) be arranged in 10 5 ' Nucleotide of polynucleotide sequence 3 ' end of the proteic proteolytic enzyme structural domain of own coding NS3 and the unique Restriction Enzyme sequence between 10 3 ' Nucleotide;
(d) polynucleotide sequence of the proteic helicase structural domain of coding NS3;
(e) the proteic polynucleotide sequence of coding NS4A;
(f) the proteic polynucleotide sequence of coding NS4B;
(g) the proteic polynucleotide sequence of coding NS5A; With
(h) the proteic polynucleotide sequence of coding NS5B.
2. the HCV replicon shuttle vectors of claim 1, the polynucleotide sequence of the wherein modified or proteic proteolytic enzyme structural domain of disappearance coding NS3, thus the proteic proteolytic enzyme structural domain of NS3 is non-functional.
3. the HCV replicon shuttle vectors of claim 1 or claim 2 is wherein at unique Restriction Enzyme recognition sequence EcoRV at the proteic polynucleotide sequence of coding NS3 5 ' end place and at unique Restriction Enzyme recognition sequence AsiSI at polynucleotide sequence 3 ' the end place of the proteic proteolytic enzyme structural domain of coding NS3.
4.HCV replicon shuttle vectors, it comprises the HCV polynucleotide sequence that is selected from SEQ ID NO:3 or SEQ ID NO:6.
5. be used for assessing the method for HCV NS3 proteinase inhibitor, comprise step in the validity of experimenter's control HCV infection:
(a) provide sample from the experimenter of HCV infection;
(b) comprise the sense strand primer and the antisense strand primer that comprises different unique Restriction Enzyme sequences of unique Restriction Enzyme sequence by use, the polynucleotide sequence of the proteic proteolytic enzyme structural domain of pcr amplification coding NS3 in a plurality of HCV quasispecies that from sample, exist;
(c) polynucleotide sequence of the described pcr amplification of clone to the HCV replicon shuttle vectors to produce chimeric HCV replicon plasmid;
(d) the described chimeric HCV replicon plasmid of linearizing and described linearization plasmid carried out in-vitro transcription to produce chimeric HCV replicon rna; With
(e) with described HCV replicon rna transfection Huh7 clone and HCV NS3 proteinase inhibitor exist or not in the presence of measure the levels of replication of described HCV replicon rna.
6. the method for claim 5, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 1.
7. the method for claim 5, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 2.
8. the method for claim 5, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 3.
9. the method for claim 5, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 4.
10. be used for assessing the method for HCV NS3 proteinase inhibitor, comprise step in the validity of experimenter's control HCV infection:
(a) provide sample from the experimenter of HCV infection;
(b) comprise the sense strand primer of the Restriction Enzyme sequence of discerning EcoRV and comprise the antisense strand primer of the Restriction Enzyme sequence of discern AsiSI by use, the pcr amplification polynucleotide sequence of the proteic proteolytic enzyme structural domain of NS3 of encoding in a plurality of HCV quasispecies that from sample, exist;
(c) polynucleotide sequence of the described pcr amplification of clone to the HCV replicon shuttle vectors to produce chimeric HCV replicon plasmid;
(d) the described chimeric HCV replicon plasmid of linearizing and described linearization plasmid carried out in-vitro transcription to produce chimeric HCV replicon rna; With
(e) with described HCV replicon rna transfection Huh7 clone and HCV NS3 proteinase inhibitor exist or not in the presence of measure the levels of replication of described HCV replicon rna.
11. the method for claim 10, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 1.
12. the method for claim 10, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 2.
13. the method for claim 5, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 3.
14. the method for claim 5, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 4.
15. be used for assessing the method for HCV NS3 proteinase inhibitor, comprise step in the validity of experimenter's control HCV infection:
(a) provide sample from the experimenter of HCV infection;
(b) comprise the sense strand primer of the nucleotide sequence that is selected from SEQ ID NO:11 or SEQ ID NO:12 and comprise the antisense strand primer of the nucleotide sequence that is selected from SEQ ID NO:13 or SEQ ID NO:14 by use, the polynucleotide sequence of the proteic proteolytic enzyme structural domain of pcr amplification coding NS3 in a plurality of HCV quasispecies that from sample, exist;
(c) polynucleotide sequence of the described pcr amplification of clone to the HCV replicon shuttle vectors to produce chimeric HCV replicon plasmid;
(d) the described chimeric HCV replicon plasmid of linearizing and described linearization plasmid carried out in-vitro transcription to produce chimeric HCV replicon rna; With
(e) with described HCV replicon rna transfection Huh7 clone and HCV NS3 proteinase inhibitor exist or not in the presence of measure the levels of replication of described HCV replicon rna.
16. the method for claim 15, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 1.
17. the method for claim 15, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 2.
18. the method for claim 15, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 3.
19. the method for claim 15, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 4.
20. be used for assessing the method for HCV NS3 proteinase inhibitor, comprise step in the validity of experimenter's control HCV infection:
(a) provide sample from the experimenter of HCV infection;
(b) comprise the sense strand primer and the antisense strand primer that comprises different unique Restriction Enzyme sequences of unique Restriction Enzyme sequence by use, the polynucleotide sequence of the proteic proteolytic enzyme structural domain of pcr amplification coding NS3 in a plurality of HCV quasispecies that from sample, exist;
(c) polynucleotide sequence of the described pcr amplification of clone to the HCV replicon shuttle vectors to produce chimeric HCV replicon plasmid;
(d) transform described plasmid to the cell to produce a plurality of colonies of transformant;
(e) compile described colony and the chimeric HCV replicon plasmid of colony separation from compiling;
(f) linearizing is carried out in-vitro transcription to produce chimeric HCV replicon rna from the described chimeric HCV replicon plasmid of step (e) and to described linearization plasmid; With
(g) with described HCV replicon rna transfection Huh7 clone and HCV NS3 proteinase inhibitor exist or not in the presence of measure the levels of replication of described HCV replicon rna.
21. the method for claim 20, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 1.
22. the method for claim 20, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 2.
23. the method for claim 20, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 3.
24. the method for claim 20, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 4.
25. be used for assessing the method for HCV NS3 proteinase inhibitor, comprise step in the validity of experimenter's control HCV infection:
(a) provide sample from the experimenter of HCV infection;
(b) comprise the sense strand primer of the Restriction Enzyme sequence of discerning EcoRV and comprise the antisense strand primer of the Restriction Enzyme sequence of discern AsiSI by use, the pcr amplification polynucleotide sequence of the proteic proteolytic enzyme structural domain of NS3 of encoding in a plurality of HCV quasispecies that from sample, exist;
(c) polynucleotide sequence of the described pcr amplification of clone to the HCV replicon shuttle vectors to produce chimeric HCV replicon plasmid;
(d) transform described plasmid to the cell to produce a plurality of colonies of transformant;
(e) compile described colony and the chimeric HCV replicon plasmid of colony separation from compiling;
(f) linearizing is carried out in-vitro transcription to produce chimeric HCV replicon rna from the described chimeric HCV replicon plasmid of step (e) and to described linearization plasmid; With
(g) with described HCV replicon rna transfection Huh7 clone and HCV NS3 proteinase inhibitor exist or not in the presence of measure the levels of replication of described HCV replicon rna.
26. the method for claim 25, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 1.
27. the method for claim 25, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 2.
28. the method for claim 25, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 3.
29. the method for claim 25, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 4.
30. be used for assessing the method for HCV NS3 proteinase inhibitor, comprise step in the validity of experimenter's control HCV infection:
(a) provide sample from the experimenter of HCV infection;
(b) comprise the sense strand primer of the nucleotide sequence that is selected from SEQ ID NO:11 or SEQ ID NO:12 and comprise the antisense strand primer of the nucleotide sequence that is selected from SEQ ID NO:13 or SEQ ID NO:14 by use, the polynucleotide sequence of the proteic proteolytic enzyme structural domain of pcr amplification coding NS3 in a plurality of HCV quasispecies that from sample, exist;
(c) polynucleotide sequence of the described pcr amplification of clone to the HCV replicon shuttle vectors to produce chimeric HCV replicon plasmid;
(d) transform described plasmid to the cell to produce a plurality of colonies of transformant;
(e) compile described colony and the chimeric HCV replicon plasmid of colony separation from compiling;
(f) linearizing is carried out in-vitro transcription to produce chimeric HCV replicon rna from the described chimeric HCV replicon plasmid of step (e) and to described linearization plasmid; With
(g) with described HCV replicon rna transfection Huh7 clone and HCV NS3 proteinase inhibitor exist or not in the presence of measure the levels of replication of described HCV replicon rna.
31. the method for claim 30, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 1.
32. the method for claim 30, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 2.
33. the method for claim 30, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 3.
34. the method for claim 30, wherein the HCV replicon shuttle vectors of step (c) comprises the HCV replicon shuttle vectors of claim 4.
CN2008801195086A 2007-12-07 2008-11-28 Hcv ns3 protease replicon shuttle vectors Pending CN101889085A (en)

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