CN101889085A - HCV NS3 Protease Replicon Shuttle Vector - Google Patents

Abstract

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

Description

HCV NS3 Protease Replicon Shuttle Vector

The present invention relates to novel HCV NS3 protease replicon shuttle vectors that can be used for screening, testing and evaluating HCV and other flavivirus protease inhibitors.

Hepatitis C virus is a major health problem worldwide and is a major cause of chronic liver disease (Boyer, N. et al., J. Hepatol. 2000 32:98-112). HCV-infected patients are at risk of developing cirrhosis and subsequently hepatocellular carcinoma, so HCV is the main indication for liver transplantation.

According to the World Health Organization, there are more than 200 million infected individuals worldwide, and at least 3 to 4 million people are infected each year. Once infected, about 20% of people clear the virus, but the rest may carry HCV for the rest of their lives. Ten to 20 percent of chronically infected individuals eventually develop cirrhosis or cancer that destroys the liver. The viral disease is transmitted parenterally by contaminated blood and blood products, by contaminated needles, or sexually or vertically from an infected or carrier mother to her offspring. Existing therapies against HCV infection, which are limited to immunotherapy with recombinant interferon-α alone or in combination with the nucleoside analog ribavirin, have limited clinical benefit, particularly for genotype 1, and urgently Improved therapeutics that effectively combat chronic HCV infection are needed.

HCV has been classified as a member of the Flaviviridae family, which includes the genera flaviviruses, pestiviruses and hepaciviruses, which includes the hepatitis C virus (Rice, C.M., Flaviviridae: The viruses and their replication, in Fields Virology, eds. Fields, B.N., Knipe, D.M. and Howley, P.M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 30, 931-959, 1996). HCV is an enveloped virus with a positive-sense single-stranded RNA genome of approximately 9.4 kb. The viral genome consists of a 5' untranslated region (UTR), a long open reading frame (ORF) encoding a polyprotein precursor of approximately 3011 amino acids, and a short 3' UTR. The 5'UTR is the most highly conserved part of the HCV genome and is important for the initiation and regulation of polyprotein translation.

Genetic analysis of HCV has identified 6 major genotypes exhibiting >30% divergence in DNA sequence. Each genotype contains a series of more closely related subtypes, wherein the subtypes show 20%-25% divergence in nucleotide sequence (Simmonds, P., 2004, J.Gen.Virol.85:3173 -88). More than 30 subtypes have been distinguished. In the United States, approximately 70% of infected individuals have type 1a and 1b infections. Type 1b is the most prevalent subtype in Asia (X. Forns and J. Bukh, Clinics in Liver Disease 1999, 3: 693-716; J. Bukh et al., Semin. Liv. Dis., 1995, 15: 41- 63). Unfortunately, Type 1 infection is less responsive to existing therapies than Type 2 or Type 3 genotypes (N.N. Zein, Clin. Microbiol. Rev., 2000, 13:223-235).

The gene organization and polyprotein processing of the nonstructural protein portions of the ORFs of pestiviruses and hepaciviruses are very similar. These positive-strand RNA viruses have a single large open reading frame (ORF) encoding all viral proteins necessary for viral replication. These proteins are expressed as polyproteins that are co-translationally and post-translationally processed by cellular and virally encoded proteases to produce mature viral proteins. Viral proteins responsible for viral genomic RNA replication are located at the carboxyl terminus. Two-thirds of the ORFs are called nonstructural (NS) proteins. For pestiviruses and hepatitis viruses, the mature nonstructural (NS) protein consists of p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B in sequence from the amino terminus of the nonstructural protein coding region to the carboxyl terminus of the ORF.

The NS proteins of pestiviruses and hepaciviruses share sequence domains that characterize specific protein functions. For example, the viral NS3 protein in both groups of viruses has an amino acid sequence motif that is characteristic of a serine protease and an amino acid sequence motif that is characteristic of a helicase (Gorbalenya et al., Nature 1988 333:22; Bazan and Fletterick, Virology 1989, 171 : 637-639; Gorbalenya et al., Nucleic Acid Res. 1989, 17, 3889-3897). Similarly, the NS5B proteins of pestiviruses and hepaciviruses have motifs that are characteristic of RNA-guiding RNA polymerases (Koonin, E.V. and Dolja, V.V., Crit. Rev. Biochem. Molec. Biol. 1993, 28: 375-430).

The actual roles and functions of the NS proteins of pestiviruses and hepatitis viruses in the viral life cycle are directly similar. In both cases, the NS3 serine protease is responsible for all proteolytic processing of the polyprotein precursor downstream of its position in the ORF (Wiskerchen and Collett, Virology, 1991, 184:341-350; Bartenschlager et al., J. Virol. 1993, 67: 3835-3844; Eckart et al., Biochem. Biophys. Res. Comm. 1993, 192: 399-406; Grakoui et al., J. Virol. 1993, 67: 2832-2843; Grakoui et al., Proc. USA 1993, 90: 10583-10587; Ilijikata et al., J. Virol. 1993, 67: 4665-4675; Tome et al., J. Virol. 1993, 67: 4017-4026). In both cases, the NS4A protein acts as a cofactor with the NS3 serine protease (Bartenschlager et al., J. Virol. 1994, 68:5045-5055; Failla et al., J. Virol. 1994, 68: 3753-3760 ; Xu et al., J Virol. 1997, 71:5312-5322). The NS3 proteins of both groups of viruses also function as helicases (Kim et al., Biochem. Biophys. Res. Comm. 1995, 215:160-166; Jin and Peterson, Arch. Biochem. Biophys. 1995, 323: 47-53; Warrener and Collett, J. Virol. 199569:1720-1726). Finally, both the NS5B proteins of pestiviruses and hepaciviruses have predicted RNA-dependent RNA polymerase activity (Behrens et al., EMBO 1996, 15: 12-22; Lechmann et al., J. Virol. 1997, 71: 8416-8428 Yuan et al., Biochem. Biophys. Res. Comm. 1997, 232: 231-235; Hagedorn, PCT WO 97/12033; Zhong et al., J. Virol. 1998, 72: 9365-9369).

HCV NS protein 3 (NS3) contains serine protease activity that helps process most viral enzymes and is therefore considered essential for viral replication and infection. Mutations in the yellow fever virus NS3 protease are known to reduce viral infectivity (Chambers et al., Proc. Natl. Acad. Sci. USA, 1990, 87:8898-8902). The first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3, which processes all four downstream sites of the HCV polyprotein (Lin et al. J. Virol. 1994, 68: 8147-8157). The HCV NS3 serine protease and its associated cofactor NS4A help process a repertoire of viral enzymes and are therefore considered essential for viral replication. This processing appears to be similar to that performed by the human immunodeficiency virus aspartyl protease, which is also involved in the viral enzyme processing of HIV protease inhibitors, wherein the HIV protease inhibitors are human potent antiviral drug in , thus suggesting that disrupting this stage of the viral life cycle leads to therapeutically active agents. Therefore, it is an attractive target for drug discovery.

There are currently a limited number of approved therapies currently available for the treatment of HCV infection. New and existing treatments for HCV and inhibition of HCV NS5B polymerase have been reviewed: R.G. Gish, Sem. Liver. Dis., 1999, 19:5; Di Besceglie, A.M. and Bacon, B.R, Scientific American, 1999 October, 80-85; G. Lake-Bakaar, "Current and Future Therapy for Chronic Hepatitis C Virus Liver Disease", Curr.DrugTarg.Infect Dis.2003, 3 (3): 247-253; P. Hoffmann et al., "Recent patents on experimental therapy for hepatitis C virus infection (1999-2002)", Exp.Opin.Ther.Patents 2003, 13(11):1707-1723; F.F.Poordad et al., "Developments in Hepatitis Ctherapy during 2000-2002", Exp.Opin. Emerging Drugs 2003, 8(1):9-25; M.P. Walker et al., "Promising Candidates for the treatment of chronic hepatitis C", Exp.Opin.Investig. Drugs 2003, 12(8): 1269-1280; S.-L. Tan et al., "Hepatitis C Therapeutics: Current Status and Emerging Strategies", Nature Rev . DrugDiscov. 2002, 1: 867-881; R. De Francesco et al., "Towards a new era of hepatitis C virus therapy: NS3-4A serine protease inhibitors and NS5B RNA-dependent RNA polymerase inhibitors (Approaching a new era for hepatitis C virus therapy: inhibitors of t he NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase), Antiviral Res.2003, 58:1-16; Q.M.Wang et al., "Hepatitis C virus encoded proteins: targets for antiviral therapy : targets for antiviral therapy)", Drugs of the Future 2000, 25(9): 933-8-944; J.A.Wu and Z.Hong, "Targeting NS5B-dependent RNA polymerase for anti-HCV chemotherapy (Targeting NS5B -Dependent RNA Polymerase for Anti-HCV Chemotherapy), Cur.Drug Targ.-Inf.Dis.20033:207-219.

Despite advances in understanding the genetic architecture of the virus and the function of viral proteins, fundamental aspects of HCV replication and pathogenesis remain unknown. A major challenge in gaining an experimental understanding of HCV replication is the lack of efficient cell culture systems that allow the production of infectious virus particles. Although infection of primary cell cultures and certain human cell lines has been reported, the amount of virus produced and the level of HCV replication in those systems was too low to permit detailed analysis.

The construction of an alternative subgenomic HCV RNA that replicates with minimal efficiency in the human hepatoma cell line Huh-7 has been reported. Lohman et al. reported the construction of a replicon (I377/NS3-3') derived from the cloned full-length HCV consensus genome (genotype 1b) by deleting the C-p7 or C-NS2 region of the protein coding region (Lohman et al., Science 1999285:110-113). This replicon contains the following elements: (i) HCV 5'-UTR fused to 12 amino acids of the capsid coding region; (ii) neomycin phosphotransferase gene (NPTID); (iii) inserted downstream of the NPTII gene and directs HCV IRES and (iv) 3'-UTR from encephalomyocarditis virus (EMCV) translated by proteins NS2 or NS3 to NS5B. After transfection of Huh-7 cells, only those cells supporting HCV RNA replication expressed NPTII protein and appeared to be drug specific Resistance to G418. Although cell lines derived from such G418-resistant colonies contained significant levels of replicon RNA and viral proteins, only 1 of 106 transfected Huh-7 cells supported HCV replication.

A similar selectable HCV replicon was constructed based on the HCV-H genotype la infectious clone (Blight et al., Science 2000 290:1972-74). HCV-H derived replicons were not able to establish efficient HCV replication, suggesting that the replicon constructed earlier by Lohmann (1999), supra, relied on the specific genotype 1b consensus cDNA clone used in those experiments. Blight et al. (2000) supra repeated the replicon construction performed by Lohmann et al. (1999) supra by performing a PCR-based gene assembly approach and obtained G418-tolerant Huh-7 cell colonies. Independent G418-tolerant cell clones were sequenced to determine whether high-level HCV replication requires adaptation of the replicon to the host cell. Multiple independent adaptive mutations clustered in the HCV nonstructural protein NS5A were identified. The mutation confers increased replication capacity in vitro, with transduction efficiencies ranging from 0.2% to 10% of transfected cells compared to replicons constructed earlier in the art, e.g. the I377/NS3-3' replicon has 0.0001% transduction conduction efficiency.

The present invention is characterized by the development of a novel HCV replicon shuttle vector, which introduces unique restriction enzyme sites at the 5' and 3' ends of the protease domain of the NS3 gene, whereby NS3 protease derived from samples of HCV-infected patients Sequences can be cloned in this shuttle vector and the resulting replicon can be evaluated for replication fitness and susceptibility to HCV NS3 protease inhibitors. Because individual HCV-infected patients generally contain genetically diverse viral populations due to the high error rate of NS5B RNA polymerase, the use of the shuttle vectors of the present invention will allow the characterization of specific patient-derived NS3 protease variants and the effect of these variants on drug therapy. sensitivity or resistance.

Therefore, the present invention provides an HCV replicon shuttle vector comprising an HCV polynucleotide sequence, wherein said HCV polynucleotide sequence sequentially comprises 10 5' nucleotides located at the 5' end of a polynucleotide sequence encoding an NS3 protein A unique restriction enzyme sequence between the 10 3' nucleotides; the polynucleotide sequence encoding the protease domain of the NS3 protein; 10 5's located at the 3' end of the polynucleotide sequence encoding the protease domain of the NS3 protein Unique restriction enzyme sequence between ' nucleotides and 10 3' nucleotides; polynucleotide sequence encoding the helicase domain of NS3 protein; polynucleotide sequence encoding NS4A protein; polynucleotide encoding NS4B protein a nucleotide sequence; a polynucleotide sequence encoding an NS5A protein; and a polynucleotide sequence encoding an NS5B protein. In one embodiment of the invention, the polynucleotide sequence encoding the protease domain of the NS3 protein has been modified or deleted such that the protease domain of the NS3 protein is non-functional.

In another embodiment of the invention, the unique restriction enzyme sequence at the 5' end of the polynucleotide sequence encoding the NS3 protein recognizes EcoRV and at the 3' end of the polynucleotide sequence encoding the protease domain of the NS3 protein A unique restriction enzyme sequence recognizes AsiSI. Still in one embodiment of the present invention, the HCV replicon shuttle vector comprises an HCV polynucleotide sequence selected from SEQ ID NO: 3 or SEQ ID NO: 6.

Yet another embodiment of the present invention provides a method for assessing the effectiveness of an HCV NS3 protease inhibitor in controlling HCV infection in a subject comprising the steps of: providing a sample from a subject infected with HCV by using a A sense strand primer of a sex enzyme sequence and an antisense strand primer comprising a different unique restriction enzyme sequence PCR amplifies the polynucleotide sequence encoding the protease domain of the NS3 protein from multiple HCV quasispecies present in the sample, cloned The polynucleotide sequence amplified by PCR is inserted into the HCV replicon shuttle vector to generate a chimeric HCV replicon plasmid, and the linearized HCV replicon plasmid is linearized and the linearized plasmid is transcribed in vitro To generate chimeric HCV replicon RNA, and use the HCV replicon RNA transfection Huh7 cell line and measure the replication level of the HCV replicon RNA in the presence or absence of HCV NS3 protease inhibitor.

Yet another embodiment of the present invention provides a method for assessing the effectiveness of an HCV NS3 protease inhibitor in controlling HCV infection in a subject comprising the steps of: providing a sample from a subject infected with HCV by using a A sense strand primer of a sex enzyme sequence and an antisense strand primer comprising a different unique restriction enzyme sequence PCR amplifies the polynucleotide sequence encoding the protease domain of the NS3 protein from multiple HCV quasispecies present in the sample, cloned The polynucleotide sequence amplified by PCR is inserted into the HCV replicon shuttle vector to generate a chimeric HCV replicon plasmid, the plasmid is transformed into cells to generate multiple colonies of transformed cells, the colonies are pooled and A chimeric HCV replicon plasmid is isolated from pooled colonies, the chimeric HCV replicon plasmid is linearized, the linearized plasmid is transcribed in vitro to produce a chimeric HCV replicon RNA, and the HCV replicon RNA was transfected into Huh7 cell line and the replication level of said HCV replicon RNA was measured in the presence or absence of HCV NS3 protease inhibitor.

The foregoing and other advantages and features of the invention, and the manner in which they are obtained, will become readily apparent upon consideration of the following detailed description of the invention together with the accompanying examples, which illustrate exemplary embodiments.

A brief description of the drawings

Figure 1 is a schematic diagram of the components of the HCV replicon shuttle vector.

Figure 2 shows the plasmid map of the NS3 protease replicon shuttle vector (A) pSC_1b_NS3/Protease_EcoRV_AsiSI (SEQ ID NO: 3); (B) pSC_1b_NS3/Protease/LacZ_EcoRV_AsiSI (SEQ ID NO: 6).

Figure 3 shows the replication competence of pSC_1b_NS3/Protease_EcoRV_AsiSI and replicons containing patient-derived NS3 protease from HCV genotype-1a or genotype 1b. RLU represents the firefly luciferase signal level observed 96 hours after transfection.

Terms used in the present invention are defined below.

The term "HCV replicon" refers to a nucleic acid from the hepatitis C virus capable of directing the production of copies of itself. As used herein, the term "replicon" includes RNA as well as DNA and hybrids thereof. For example, the double-stranded DNA form of the HCV genome can be used to generate single-stranded RNA transcripts that make up the HCV replicon. The HCV replicon may comprise the full length HCV genome or a subgenomic construct of HCV also referred to as a "subgenomic replicon". For example, the HCV subgenomic replicons described herein contain most of the genes for the nonstructural proteins of the virus, but not most of the genes encoding structural proteins. The subgenomic replicon is capable of directing the expression of all viral genes necessary for viral subgenomic replication, subgenomic replicon replication, without the production of viral particles.

The basic HCV replicon is a subgenomic construct containing the HCV 5'-untranslated (UTR) region, the HCV NS3-NS5B polyprotein coding region, and the HCV 3'-UTR. Other nucleic acid regions such as those providing HCV NS2, HCV structural proteins, and non-HCV sequences may be present.

The HCV 5'-UTR region provides an internal ribosome entry site (IRES) for protein translation and elements required for replication. The HCV 5'-UTR region includes the naturally occurring HCV 5'-UTR and its functional derivatives extending approximately 36 nucleotides into the HCV core coding region. The 5'-UTR region may be present at various positions, such as a position downstream of a sequence encoding a selectin, reporter protein or HCV polyprotein.

In addition to the HCV 5'-UTR-PC region, non-HCV IRES elements can also be present in the replicon. Non-HCV IRES elements can be present at various locations, including immediately upstream of the region encoding the HCV polyprotein. Examples of non-HCV IRES elements that can be used are EMCV IRES, poliovirus IRES, and bovine viral diarrhea virus IRES.

HCV 3'-UTR helps HCV to replicate. The HCV 3'UTR includes the naturally occurring HCV 3'-UTR and its functional derivatives. The naturally occurring 3'-UTR includes a poly-U region and an additional region of about 100 nucleotides.

The NS3-NS5B polyprotein coding region provides polyproteins that can be processed into different proteins in the cell. Suitable NS3-NS5B polyprotein sequences that may be part of the replicon include those NS3-NS5B polyprotein sequences that are present in different HCV strains and their functional equivalents that result in the processing of NS3-NS5B to generate a functional replication apparatus. Proper processing can be measured by testing the NS5B RNA-dependent RNA polymerase.

A "vector" is a piece of DNA, such as a plasmid, phage or cosmid, to which another DNA segment can be ligated, thereby causing replication, expression or integration of the ligated DNA segment. "Shuttle vector" refers to a vector in which DNA segments can be inserted or excised from the vector at specific restriction enzyme sites. The DNA segment inserted into the shuttle vector usually encodes a polypeptide or RNA of interest, and restriction enzyme sites are designed to ensure insertion of the DNA segment in the proper open reading frame for transcription and translation.

A variety of vectors can be used to express nucleic acid molecules. Such vectors include chromosomal, episomal, and virally derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements (including yeast artificial chromosomes), from viruses such as baculoviruses, milk Polyvaviruses such as SV40, vaccinia virus, adenovirus, poxvirus, pseudorabies virus, herpesvirus, and retrovirus-derived vectors. Vectors can also be derived from combinations of these sources, such as those derived from plasmids and bacteriophage genetic elements, such as cosmids and phagemids. Suitable cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

Vectors containing suitable nucleic acid molecules can be introduced into suitable host cells for propagation or expression using known techniques. Host cells can include bacterial cells including but not limited to Escherichia coli (E.coli), Streptomyces (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium); eukaryotic cells including but not limited to yeast, insect cells Drosophila ), animal cells such as Huh-7, HeLa, COS, HEK 293, MT-2T, CEM-SS and CHO cells, and plant cells.

Vectors typically include a selectable marker that enables selection of a subpopulation of cells containing the recombinant vector construct. The marker may be contained on the same vector containing the nucleic acid molecule described herein or may be present on a separate vector. Markers include tetracycline resistance gene or ampicillin resistance gene for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

"Polynucleotide" or "nucleic acid molecule" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA as a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA as a mixture of single- and double-stranded regions , A hybrid molecule comprising DNA and RNA, which may be single-stranded or more generally double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. "Polynucleotide" also includes relatively short polynucleotides, often referred to as oligonucleotides.

Additionally, the term "DNA molecule" refers only to the primary and secondary structure of the molecule, and does not limit the DNA molecule to any particular form of tertiary structure. Thus, the term specifically includes double-stranded DNA found in linear DNA molecules (eg, restriction fragments), viruses, plasmids and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, it may be described herein according to the conventional convention of giving only the sequence in the 5' to 3' direction together with the non-transcribed DNA strand (i.e., the strand having sequence homology to the mRNA) sequence.

"RNA molecule" refers to a polymeric form of ribonucleotides in their single-stranded or double-stranded helical form. In discussing the structure of a particular RNA molecule, sequences may be described herein according to the conventional convention for giving sequences in the 5' to 3' direction.

ATC 3’并在所示(以 _▲显示的)核苷酸位置处切割The term "restriction enzyme sequence" refers to a specific double-stranded DNA sequence recognized and cut by bacterial enzymes that all cut double-stranded DNA at or near a specific nucleotide sequence. Restriction enzyme "EcoRV" recognition sequence 5'GAT

ATC 3' and as shown (with _▲ shown) cleavage at the nucleotide position

3'CTA _▲ TAG 5'

CGC 3’并在识别序列double-stranded DNA. Restriction enzyme "AsiSI" recognition sequence 5'GCGAT

CGC 3' and the recognition sequence

3'CGC _▲ TAGCG 5'

Cleavage after the T residue.

The term "primer" as used herein refers to an oligonucleotide of RNA or DNA, single- or double-stranded, derived by restriction enzyme digestion, derived from a biological system, or produced synthetically, wherein the oligonucleotide Capable of functioning as a primer for template-dependent nucleic acid synthesis when placed in the right environment. When present together with a suitable nucleic acid template, a suitable nucleoside triphosphate precursor of the nucleic acid, a polymerase, suitable cofactors and conditions such as suitable temperature and pH, the primer can be induced by the action of a polymerase or similar activity that produces primer extension products Nucleotides are added to extend the 3' end of the primer. The elicitors can vary in length according to specific application conditions and requirements. For example, in PCR reactions, primers are typically 15-25 nucleotides or longer in length. The primer must be sufficiently complementary to the template of interest to prime the synthesis of the extension product of interest, i.e. capable of annealing with the template strand of interest in a manner sufficient to provide the 3'-hydroxyl portion of the primer in an appropriate juxtaposition for use in priming by the polymerase. Or in the synthesis caused by similar enzymes. There is no requirement that the primer sequences represent the perfect complement of the template of interest. For example, a non-complementary nucleotide sequence (such as a restriction enzyme recognition sequence) can be ligated to the 5'-end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence is sufficiently complementary to the template strand of interest to functionally provide a template-primer complex for synthesis of extension products.

The term "chimeric" as used herein means a DNA molecule produced from DNA from two or more different sources that has been fused or spliced together.

As used herein, the term "quasispecies" means a collection of minor variants of the dominant HCV genomic sequence (i.e. genotype) that occur during HCV replication in a single infected subject or even in a single cell clone. high mutation rate formation.

The term "subject" as used herein refers to a vertebrate, particularly a member of the mammalian species, and includes, but is not limited to, rodents, rabbits, shrews and primates, the latter including humans.

The term "sample" refers to a sample of tissue or body fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary samples consisting of tracts, tears, saliva, milk, blood cells, tumors, organs, and also in vitro cell cultures (including but not limited to conditioned medium and cells from cultured cells, putative virus-infected cells, recombinant cell growth components).

A cell has been "transformed" or "transfected" with exogenous or heterologous DNA or RNA when such DNA or RNA is introduced into the interior of the cell. Transforming or transfecting DNA or RNA may or may not be integrated (covalently linked) into the chromosomal DNA that makes up the genome of the cell. For example, in prokaryotes, yeast and mammalian cells, transforming DNA can be maintained on episomal elements such as plasmids. With respect to eukaryotic cells, a stably transformed cell is one in which transforming DNA has become integrated into a chromosome so that the DNA is inherited by daughter cells through chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones consisting of a population of progeny cells containing the transforming DNA. In the case of an HCV replicon that transforms mammalian cells as described in the present invention, RNA molecules such as HCV RNA molecules have the ability to replicate semiautonomously. Huh-7 cells carrying the HCV replicon are detected by the presence of a selectable marker or reporter gene on the replicon.

"Clone"refers to a population of cells derived from a single cell or common ancestor, usually by the process of mitosis.

Example

The following preparations and examples are given to enable those skilled in the art to more clearly understand and practice the present invention. They should not be considered as limiting the scope of the invention, but only as illustrating and describing the invention.

Example 1

Plasmid construction

The NS3 protease replicon shuttle vector was derived from the HCV replicon shuttle vector pSC_1b_NS3_EcoRV, which was an intermediate vector used to generate the HCV NS3 replicon shuttle vector pSC_1b_NS3_EcoRV_XbaI and was named in Chua et al., September 27, 2007. "HCV NS3 Replicon Shuttle Vector" is disclosed in U.S. Patent Application USSN 60/995,558, which is hereby incorporated by reference in its entirety. The components of the replicon shuttle vector are shown in Figure 1 and contain the HCV polynucleotide sequence from the 5'-UTR, NS3 to NS5B proteins and the 3'-UTR. The vector also includes a poliovirus internal ribosome entry site (IRES) that controls translation of the firefly luciferase gene. Downstream of the firefly luciferase gene, an IRES from encephalomyocarditis virus (EMCV) controls the translation of HCV nonstructural genes (NS3, NS4A, NS4B, NS5A, and NS5B).

Mutations were introduced into pSC_1b_NS3_EcoRV_XbaI using the QuickChange Site-Directed Mutagenesis Kit following the manufacturer's instructions (Stratagene, La Jolla, CA, USA). To introduce the AsiSI restriction enzyme sequence at the 3' end of the protease domain of the NS3 gene (corresponding to amino acid position Ser181), the following primers were used:

sense primer

5'-GAGTCTATGGGAACCACTATGCGATCGCCGGTCTTCACGGACAACTCGTC-3' (SEQ ID NO: 1)

antisense primer

5'-GACGAGTTGTCCGTGAAGACCGGCGATCGCATAGTGGTTCCCATAGACTC-3' (SEQ ID NO: 2)

These mutations did not alter the amino acid coding sequence of the NS3 protein and resulted in the generation of the NS3 protease shuttle vector pSC_1b_NS3/Protease_EcoRV_AsiSI (SEQ ID NO: 3, Figure 2A). Another NS3 protease replicon shuttle vector was generated in which the sequence encoding the protease domain of the NS3 gene was replaced by the β-galactosidase (lacZ) coding sequence from pUC19 (GenBank accession number M77789). An EcoRV restriction site at the 5' end of the lacZ gene and an AsiSI restriction site at its 3' end were introduced by PCR amplification using the following primers

5'-ATCATCATCGATATCACCGCGTTGGCCGATTCATTAATG-3' (SEQ ID NO: 4) (EcoRV) (LacZ)

5'-GATGATGATGCGATCGCCAGCTCCCGGAGACGGTCAC-3' (SEQ ID NO: 5) (AsiSI) (LacZ)

To generate the vector pSC_1b_NS3/Protease/LacZ_EcoRV_AsiSI (SEQ ID NO: 6, Figure 2B).

The replicative capacity of the NS3 protease replicon shuttle vector was assessed using the phenotypic assay described in Example 3 (and shown in Figure 3).

Example 2

Cloning of NS3 protease domain PCR samples amplified from infected patients into NS3 protease replicon shuttle vectors

The DNA sequence encoding the protease domain of the NS3 gene was generated by reverse transcription-polymerase chain reaction (RT-PCR) of RNA from plasma obtained from infection using the SuperScript III system (Invitrogen) according to the manufacturer's protocol. Patients with HCV genotype-1a and genotype 1-b were acquired. Primers used in this RT-PCR step are as follows.

Genotype 1a:

Sense primer (NS2) 5'-CGTGCGGTGACATCATCAACGG-3' (SEQ ID NO: 7) antisense primer (NS3/helicase) 5'-CTCGCCCCCGCAGTCTCTGC-3' (SEQ ID NO: 8)

Genotype 1b:

Sense primer (NS2) 5'-GAGACCAAAGATCATCACCTGG-3' (SEQ ID NO: 9)

Antisense primer (NS3/helicase) 5'-GTCCAGGACTGTGCCGATGCC-3' (SEQ IDNO: 10)

Annealing was performed first at 50°C followed by PCR cycles of denaturation at 94°C, annealing at 53°C and extension at 68°C. The amplified product was then subjected to a second round of PCR using the PhusionTM high-fidelity DNA polymerase system (New England Biolabs) using primers introducing unique restriction enzyme sequences at the 5' and 3' ends of the protease domain of the NS3 protein. The sense primers used to import the EcoRV restriction enzyme sequence near the 5' end of the patient's NS3 gene sequence are as follows:

Genotype 1a

5'-CTGTCTGTCTGATATCACCATGGCGCCCATCACGGCGTACGC

C-3' (SEQ ID NO: 11)

Genotype 1b

5'-CTGTCTGTCTGATATCACCATGGCGCCTATTACGGCCTACTC-3' (SEQ ID NO: 12)

The primers used to introduce the AsiSI restriction enzyme sequence near the 3' end of the protease domain of the NS3 gene (corresponding to amino acid position Ser181) are as follows:

Genotype 1a

5'-TAGTAGTAGGCGATCGCATGGTTGTCCCTAGGTTCTC-3'(SEOID NO: 13)

Genotype 1b

5'-TAGTAGTAGGCGATCGCATAGTGGTTCCCATAGACTCG-3'

(SEQ ID NO: 14)

Amplification was performed with a PCR cycle of denaturation at 98°C, annealing at 53°C, and extension at 72°C. Amplified patient NS3 protease DNA was subsequently purified using Qiagen PCR purification columns and digested with EcoRV and SgfI (isoschimozyme of AsiSI).

The NS3 protease shuttle replicon vector pSC_1b_NS3/Protease_EcoRV_AsiSI or pSC_1b_NS3/Protease/LacZ_EcoRV_AsiSI was prepared by double digestion with restriction endonucleases EcoRV and SgfI. Using the MightMix ligation kit (Takara BioInc.), 25 ng of the shuttle vector was ligated to the digested patient amplicon for 1.5 hours at 16°C. Vector to insert ratios of 1:2 to 1:4 are routinely used. 5 μl of the reaction was transformed into 50 μl of Top10 cells (Invitrogen) and plated after 1 hour of phenotypic expression at 37°C.

96 independent colonies were picked to inoculate 200 μl Terrific broth (TB) supplemented with 50 μg/ml ampicillin. This "stock" 96-well plate was incubated overnight at 37°C. The next day, use this plate to prepare a duplicate plate. A 48-pin replicator was used to replicate the plate onto two LB plates supplemented with 100 μg/ml carbenicillin. Ninety-six separate 200 μl cultures were also transferred to 1.5 ml TB medium supplemented with 50 μg/ml ampicillin for DNA preparation. After overnight incubation at 37°C, replica plates were spread with 6 ml LB to obtain a heterogeneous pool of 96 clones, which was then used for mini DNA preps. This pool of DNA from the patient is then used in subsequent replicon phenotyping assays. After overnight shaking at 37°C, 96 separate 1.5 ml TB cultures were centrifuged down, decanted and plasmid DNA was extracted using Qiagen's Qiaprep 96 Turbo Mini-DNA kit. These independent molecular clones were used in sequencing reactions, wherein the independent molecular clones represented the pool of composite 96 for replicon phenotyping assays.

Heterogeneous clonal pooled plasmid DNA or independent molecular clones were sequenced to confirm the identity of patient samples and tested for inhibitor sensitivity in phenotypic replicon assays.

Example 3

Phenotypic Replicon Assays

A. Preparation of in vitro transcribed RNA

5 μg of DNA plasmid was linearized by Sca I restriction enzyme (Roche). After overnight digestion at 37°C, the DNA was purified using the Qiagen PCR purification kit. 1 µg of linearized DNA was used for in vitro transcription using T7RiboMAX Express (Promega) following the manufacturer's protocol. After 2 hours of incubation at 37°C, DNase treatment was performed at 37°C for 30 minutes to remove the DNA template. In vitro transcribed RNA was then purified using RNeasy spin columns (Qiagen) following the manufacturer's protocol.

B. Liver cancer cell lines

The liver cancer Lunet Huh-7 cell line was cultured at 37°C in a humidified atmosphere containing 5% CO ₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with Glutamax ^™ and 100 mg/ml sodium pyruvate. The medium was further supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. All reagents were from Invitrogen/Gibco.

C. Determination of Replication Levels of Transient Replicons

4 million Lunet Huh7 cells were transfected with 5 μg of in vitro transcribed RNA using electroporation. Cells were then resuspended in 7.2 ml DMEM containing 5% FBS and plated in 96-well plates at 50000 cells/well (in 90 μl final volume). Inhibitors (when inhibitors were used) were added at 3-fold dilutions at a final concentration of 1% DMSO 24 hours after transfection and firefly fireflies were read 72 hours after addition of inhibitors using a luciferase assay system (Promega). Luciferase reporter signal. The _IC50 value was estimated as the inhibitor concentration at which a 50% reduction in the level of the firefly luciferase reporter protein was observed compared to the level of the firefly luciferase signal when no compound was added .

Those replication abilities of the replicon shuttle vector pSC_1b_NS3/Protease_EcoRV_AsiSI and the replicon containing the HCV genotype-1a and genotype-1b patient-derived NS3 protease domains were examined with the luciferase signal and the results are shown in FIG. 3 . The inhibitory effect of HCV protease inhibitors BILN2061, VX-950 and NM107 was also tested on these replicons and the results are shown in Table 1.

Table 1

genotype Replicon Average _IC50 Number of Control or Patient Samples BILN2061nM VX-950nM NM107μM pSC_1b_NS3/Protease 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 listing

<110> F. Huffman-La Roche Co., Ltd.

the

<120>HCV NS3 protease replicon shuttle vector

the

<130>R0382A-PRO

the

<160>14

the

<170> PatentIn version 3.2

the

<210>1

<211>50

<212>DNA

<213> Artificial sequence

the

<220>

<223> vector AsiSI sense primer

the

<400>1

gagtctatgg gaaccactat gcgatcgccg gtcttcacgg acaactcgtc 50

the

<210>2

<211>50

<212>DNA

<213> Artificial sequence

the

<220>

<223> vector AsiSI antisense primer

the

<400>2

gacgagttgt ccgtgaagac cggcgatcgc atagtggttc ccatagactc 50

the

<210>3

<211>11516

<212>DNA

<213> Artificial sequence

the

<220>

<223>pSC 1b NS3/Protease EcoRV AsiSI

the

<400>3

cctgcaggta atacgactca ctatagccag cccccgattg ggggcgacac tccaccatag 60

atcactcccc tgtgaggaac tactgtcttc acgcagaaag cgtctagcca tggcgttagt 120

atgagtgtcg tgcagcctcc aggaccccccc 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 ttatgtggc 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 attaccagggg 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

tacgattat 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 ggtacccat 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 ctgcacggggc caacgcccct gctgtatagg ctgggagccg 5160

ttcaaaacga ggttactacc acaccacccca 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 ccagggggagg 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 cggagggagga 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 accaccccccc 8700

ttgcgcgggc tgcgtgggag acagctagac acactccagt caattcctgg ctaggcaaca 8760

tcatcatgta tgcgcccacc ttgtgggcaa ggatgatcct gatgactcat ttcttctcca 8820

tccttctagc tcaggaacaa cttgaaaaag ccctagattg tcagatctac ggggcctgtt 8880

actccatga gccacttgac ctacctcaga tcattcaacg actccatggc cttagcgcat 8940

tttcactcca tagttactct ccaggtgaga tcaatagggt ggcttcatgc ctcaggaaac 9000

ttggggtacc gcccttgcga gtctgggac atcgggccag aagtgtccgc gctaggctac 9060

tgtcccaggg ggggagggct gccacttgtg gcaagtacct cttcaactgg gcagtaagga 9120

ccaagctcaa actcactcca atcccggctg cgtcccagtt ggatttatcc agctggttcg 9180

ttgctggtta cagcggggga gacatatac acagcctgtc tcgtgcccga ccccgctggt 9240

tcatgtggtg cctactccta ctttctgtag gggtaggcat ctatctactc cccaaccgat 9300

gaacggggag ctaaacactc caggccaata ggccatcctg tttttttccc tttttttttt 9360

tctttttttt ttttttttttttttttttttttttttctcc 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 accacgctc 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 tattagaaa aataaacaaa 11460

taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accatt 11516

the

<210>4

<211>39

<212>DNA

<213> Artificial sequence

the

<220>

<223>LacZ EcoRV sense primer

the

<400>4

atcatcatcg atatcaccgc gttggccgat tcattaatg 39

the

<210>5

<211>37

<212>DNA

<213> Artificial sequence

the

<220>

<223>LacZ AsiSI Antisense Primer

the

<400>5

gatgatgatg cgatcgccag ctcccggaga cggtcac 37

the

<210>6

<211>11590

<212>DNA

<213> Artificial sequence

the

<220>

<223>pSC_1b_NS3/Protease/LacZ_EcoRV_AsiSI

the

<400>6

cctgcaggta atacgactca ctatagccag cccccgattg ggggcgacac tccaccatag 60

atcactcccc tgtgaggaac tactgtcttc acgcagaaag cgtctagcca tggcgttagt 120

atgagtgtcg tgcagcctcc aggaccccccc 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 ttatgtggc 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 attaccagggg 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

tacgattat 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 ggtacccat 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

cccttacgcat 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

tggggggcgcc 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 atttaccgg 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 taccaacacac cccataacca aatacatcat 5280

ggcatgcatg tcggctgatc tagagtcgt 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 agacaacactc 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 aggggggggag 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 tttttttttttttttttttttttttttttt 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 agggatttg 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

the

<220>

<223>RT-PCR genotype 1a sense primer (NS2)

the

<400>7

cgtgcggtga catcatcaac gg 22

the

<210>8

<211>20

<212>DNA

<213> Artificial sequence

the

<220>

<223> RT-PCR genotype 1a antisense primer (NS3 helicase)

the

<400>8

ctcgcccccg cagtctctgc 20

the

<210>9

<211>21

<212>DNA

<213> Artificial sequence

the

<220>

<223>RT-PCR genotype 1b sense primer (NS2)

the

<400>9

gagaccaaga tcatcacctg g 21

the

<210>10

<211>21

<212>DNA

<213> Artificial sequence

the

<220>

<223> RT-PCR genotype 1b antisense primer (NS3 helicase)

the

<400>10

gtccaggact gtgccgatgc c 21

the

<210>11

<211>43

<212>DNA

<213> Artificial sequence

the

<220>

<223> sense primer genotype 1a EcoRV

the

<400>11

ctgtctgtct gatatcacca tggcgcccat cacggcgtac gcc 43

<210>12

<211>42

<212>DNA

<213> Artificial sequence

the

<220>

<223> sense primer genotype 1b EcoRV

the

<400>12

ctgtctgtct gatatcacca tggcgcctat tacggcctac tc 42

the

<210>13

<211>37

<212>DNA

<213> Artificial sequence

the

<220>

<223> Antisense Primer Genotype 1a AsiSI

the

<400>13

tagtagtagg cgatcgcatg gttgtcccta ggttctc 37

the

<210>14

<211>38

<212>DNA

<213> Artificial sequence

the

<220>

<223> Antisense Primer Genotype 1b AsiSI

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Claims (34)

1. the HCV replicon shuttle vector that comprises HCV polynucleotide sequence, described HCV polynucleotide sequence comprises successively: (a) a unique restriction enzyme sequence located between 10 5' nucleotides and 10 3' nucleotides in the 5' end from the polynucleotide sequence encoding the NS3 protein; (b) a polynucleotide sequence encoding the protease domain of the NS3 protein; (c) a unique restriction enzyme sequence located between 10 5' nucleotides and 10 3' nucleotides in the 3' end of the polynucleotide sequence from the protease domain encoding the NS3 protein; (d) a polynucleotide sequence encoding the helicase domain of the NS3 protein; (e) a polynucleotide sequence encoding an NS4A protein; (f) a polynucleotide sequence encoding an NS4B protein; (g) a polynucleotide sequence encoding an NS5A protein; and (h) Polynucleotide sequence encoding NS5B protein. 2. The HCV replicon shuttle vector of claim 1, wherein the polynucleotide sequence encoding the protease domain of the NS3 protein has been modified or deleted such that the protease domain of the NS3 protein is non-functional. 3. The HCV replicon shuttle carrier of claim 1 or claim 2, wherein the unique restriction enzyme sequence recognition EcoRV at the polynucleotide sequence 5' end of the coding NS3 protein and at the polynucleoside of the protease domain of the coding NS3 protein A unique restriction enzyme sequence at the 3' end of the acid sequence recognizes AsiSI. 4. An HCV replicon shuttle vector comprising an HCV polynucleotide sequence selected from SEQ ID NO: 3 or SEQ ID NO: 6. 5. A method for evaluating the effectiveness of an HCV NS3 protease inhibitor in controlling HCV infection in a subject, comprising the steps of: (a) providing a sample from a subject infected with HCV; (b) PCR amplification of the protease encoding the NS3 protein from multiple HCV quasispecies present in the sample by using a sense strand primer comprising a unique restriction enzyme sequence and an antisense strand primer comprising a different unique restriction enzyme sequence the polynucleotide sequence of the domain; (c) cloning the polynucleotide sequence amplified by PCR into an HCV replicon shuttle vector to generate a chimeric HCV replicon plasmid; (d) linearizing said chimeric HCV replicon plasmid and performing in vitro transcription of said linearized plasmid to produce chimeric HCV replicon RNA; and (e) Huh7 cell line was transfected with the HCV replicon RNA and the replication level of the HCV replicon RNA was measured in the presence or absence of HCV NS3 protease inhibitor. 6. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1. 7. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2. 8. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3. 9. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. 10. A method for evaluating the effectiveness of an HCV NS3 protease inhibitor in controlling HCV infection in a subject, comprising the steps of: (a) providing a sample from a subject infected with HCV; (b) PCR amplification of proteins encoding NS3 from multiple HCV quasispecies present in the sample by using a sense strand primer comprising a restriction enzyme sequence recognizing EcoRV and an antisense strand primer comprising a restriction enzyme sequence recognizing AsiSI The polynucleotide sequence of the protease domain; (c) cloning the polynucleotide sequence amplified by PCR into an HCV replicon shuttle vector to generate a chimeric HCV replicon plasmid; (d) linearizing said chimeric HCV replicon plasmid and performing in vitro transcription of said linearized plasmid to produce chimeric HCV replicon RNA; and (e) Huh7 cell line was transfected with the HCV replicon RNA and the replication level of the HCV replicon RNA was measured in the presence or absence of HCV NS3 protease inhibitor. 11. The method of claim 10, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1. 12. The method of claim 10, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2. 13. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3. 14. The method of claim 5, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. 15. A method for evaluating the effectiveness of an HCV NS3 protease inhibitor in controlling HCV infection in a subject, comprising the steps of: (a) providing a sample from a subject infected with HCV; (b) by using a sense strand primer comprising a nucleotide sequence selected from SEQ ID NO: 11 or SEQ ID NO: 12 and a primer comprising a nucleotide sequence selected from SEQ ID NO: 13 or SEQ ID NO: 14 An antisense strand primer for PCR amplifying the polynucleotide sequence encoding the protease domain of the NS3 protein from multiple HCV quasispecies present in the sample; (c) cloning the polynucleotide sequence amplified by PCR into an HCV replicon shuttle vector to generate a chimeric HCV replicon plasmid; (d) linearizing said chimeric HCV replicon plasmid and performing in vitro transcription of said linearized plasmid to produce chimeric HCV replicon RNA; and (e) Huh7 cell line was transfected with the HCV replicon RNA and the replication level of the HCV replicon RNA was measured in the presence or absence of HCV NS3 protease inhibitor. 16. The method of claim 15, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1. 17. The method of claim 15, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2. 18. The method of claim 15, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3. 19. The method of claim 15, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. 20. A method for evaluating the effectiveness of an HCV NS3 protease inhibitor in controlling HCV infection in a subject, comprising the steps of: (a) providing a sample from a subject infected with HCV; (b) PCR amplification of the protease encoding the NS3 protein from multiple HCV quasispecies present in the sample by using a sense strand primer comprising a unique restriction enzyme sequence and an antisense strand primer comprising a different unique restriction enzyme sequence the polynucleotide sequence of the domain; (c) cloning the polynucleotide sequence amplified by PCR into an HCV replicon shuttle vector to generate a chimeric HCV replicon plasmid; (d) transforming said plasmid into cells to produce multiple colonies of transformed cells; (e) pooling said colonies and isolating a chimeric HCV replicon plasmid from the pooled colonies; (f) linearizing said chimeric HCV replicon plasmid from step (e) and performing in vitro transcription of said linearized plasmid to produce chimeric HCV replicon RNA; and (g) Transfect the Huh7 cell line with the HCV replicon RNA and measure the replication level of the HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor. 21. The method of claim 20, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1. 22. The method of claim 20, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2. 23. The method of claim 20, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3. 24. The method of claim 20, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. 25. A method for evaluating the effectiveness of an HCV NS3 protease inhibitor in controlling HCV infection in a subject, comprising the steps of: (a) providing a sample from a subject infected with HCV; (b) PCR amplification of proteins encoding NS3 from multiple HCV quasispecies present in the sample by using a sense strand primer comprising a restriction enzyme sequence recognizing EcoRV and an antisense strand primer comprising a restriction enzyme sequence recognizing AsiSI The polynucleotide sequence of the protease domain; (c) cloning the polynucleotide sequence amplified by PCR into an HCV replicon shuttle vector to generate a chimeric HCV replicon plasmid; (d) transforming said plasmid into cells to produce multiple colonies of transformed cells; (e) pooling said colonies and isolating a chimeric HCV replicon plasmid from the pooled colonies; (f) linearizing said chimeric HCV replicon plasmid from step (e) and performing in vitro transcription of said linearized plasmid to produce chimeric HCV replicon RNA; and (g) Transfect the Huh7 cell line with the HCV replicon RNA and measure the replication level of the HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor. 26. The method of claim 25, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1. 27. The method of claim 25, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2. 28. The method of claim 25, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3. 29. The method of claim 25, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4. 30. A method for evaluating the effectiveness of an HCV NS3 protease inhibitor in controlling HCV infection in a subject, comprising the steps of: (a) providing a sample from a subject infected with HCV; (b) by using a sense strand primer comprising a nucleotide sequence selected from SEQ ID NO: 11 or SEQ ID NO: 12 and a primer comprising a nucleotide sequence selected from SEQ ID NO: 13 or SEQ ID NO: 14 An antisense strand primer for PCR amplifying the polynucleotide sequence encoding the protease domain of the NS3 protein from multiple HCV quasispecies present in the sample; (c) cloning the polynucleotide sequence amplified by PCR into an HCV replicon shuttle vector to generate a chimeric HCV replicon plasmid; (d) transforming said plasmid into cells to produce multiple colonies of transformed cells; (e) pooling said colonies and isolating a chimeric HCV replicon plasmid from the pooled colonies; (f) linearizing said chimeric HCV replicon plasmid from step (e) and performing in vitro transcription of said linearized plasmid to produce chimeric HCV replicon RNA; and (g) Transfect the Huh7 cell line with the HCV replicon RNA and measure the replication level of the HCV replicon RNA in the presence or absence of an HCV NS3 protease inhibitor. 31. The method of claim 30, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 1. 32. The method of claim 30, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 2. 33. The method of claim 30, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 3. 34. The method of claim 30, wherein the HCV replicon shuttle vector of step (c) comprises the HCV replicon shuttle vector of claim 4.

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