Nucleic acid construct including nucleic acid derived from genotype 3[alpha] HCV genome

A hepatitis C virus and genome technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve the problems of different effects and low inhibitory effects

Inactive Publication Date: 2014-10-29
JAPAN AS REPRESENTED BY DIRECTOR GENERAL OF NAT INST OF INFECT IOUS DISEASES +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been reported that TMC435, an NS3 / 4 protease inhibitor of HCV, has a high inhibitory effect on genotypes 1-6 except for genotype 3a, but its inhibitory effect on genotype 3a is low, and its effect on HCV is based on genotype (Non-Patent Document 15)

Method used

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  • Nucleic acid construct including nucleic acid derived from genotype 3[alpha] HCV genome
  • Nucleic acid construct including nucleic acid derived from genotype 3[alpha] HCV genome
  • Nucleic acid construct including nucleic acid derived from genotype 3[alpha] HCV genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0239] (Example 1) Construction of the HCV subgenomic replicon RNA expression vector of the wild-type S310A strain

[0240] The HCV strain was isolated from a 71-year-old acute hepatitis C patient infected with HCV of genotype 3a, and named as S310A strain. The patient was diagnosed with HCV genotype 3a at the age of 59 and underwent liver transplantation 4 years later due to cirrhosis. Specifically, RNA was extracted and purified from patient serum using Isogen-LS (Nippon Gene), and cDNA was synthesized using random hexamer primers. PCR primers were designed according to the known conserved sequences of 4 genotype 3a HCV genomes (GenBank accession numbers AF046866, D28917, X76918 and D17763), and the synthesized cDNA was divided into 9 fragments to amplify the cDNA. For the sequence at the 5' end where amplification products are not easily obtained, amplification products were obtained by the 5' RACE method. Each fragment was cloned into a cloning vector for sequencing, pGE...

Embodiment 2

[0248] (Example 2) Preparation of HCV subgenomic replicon RNA of wild-type S310A strain

[0249] The expression vector pS310ASGR-Neo constructed in Example 1 was cleaved with restriction enzyme XbaI. Then, between 10 and 20 mu Add 20U mung bean nuclease to the XbaI cut-off fragment of g (the total reaction liquid volume is 50 mu L), incubated at 30°C for 30 minutes. Mung bean nuclease is an enzyme that catalyzes a reaction in which a single-stranded portion of double-stranded DNA is selectively decomposed and smoothed. Usually, when RNA transcription is performed with RNA polymerase using the above-mentioned XbaI cleaved fragment as it is as template DNA, a replicon RNA in which 4 nucleotides of CUAG, which is a part of the XbaI recognition sequence, are excessively added to the 3' end is synthesized. Therefore, in this example, four nucleotides of CTAG were removed from the XbaI cleavage fragment by treating the XbaI cleavage fragment with mung bean nuclease.

[0250] A...

Embodiment 3

[0252] (Example 3) Establishment of HCV subgenome replicon replication cell clone of S310A strain

[0253] The HCV subgenome replicon RNA (1 mu g, 3 mu g, 10 mu g or 30 mu g) Introduced into Huh7 cells. Electroporated Huh7 cells (3×10 6 1) were inoculated in a petri dish, and after culturing for 16 to 24 hours, G418 (neomycin) was added to the petri dish. Thereafter, the culture solution was exchanged twice a week while continuing the cultivation.

[0254] Viable cells were stained with crystal violet after 21 days of culture from the time of inoculation. As a result, about importing 10 mu g and 30 mu The cells of the HCV subgenome replicon RNA of the S310A strain of g can confirm colony formation ( figure 2 ). Colony formation indicates that the HCV subgenomic replicon RNA is replicated in the cell. The results showed that the HCV subgenomic replicon RNA produced using the genomic nonstructural region of the wild-type S310A strain has the ability to autonomo...

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Abstract

This invention provides a nucleic acid comprising, in the following order, a 5' untranslated region comprising a particular nucleotide sequence of the genome of hepatitis C virus genotype 3a; a nucleotide sequence encoding a particular amino acid sequence of an NS3 protein, a nucleotide sequence encoding a particular amino acid sequence of an NS4A protein, a nucleotide sequence encoding a particular amino acid sequence of an NS4B protein, a nucleotide sequence encoding a particular amino acid sequence of an NS5A protein, a nucleotide sequence encoding a particular amino acid sequence of an NS5B protein of the hepatitis C virus genotype 3a; and a 3' untranslated region comprising a particular nucleotide sequence of a genome of hepatitis C virus genotype 3a.

Description

technical field [0001] The present invention relates to a nucleic acid from the genome of hepatitis C virus of genotype 3a, a nucleic acid construct comprising the nucleic acid and a method for screening anti-hepatitis C virus substances. Background technique [0002] An experimental system capable of efficiently amplifying HCV is essential for basic research on hepatitis C virus (Hepatitis C virus; hereinafter referred to as HCV) and development of anti-HCV drugs. Specifically, a system for amplifying HCV in cultured cells or a system for evaluating the proliferation of HCV in cultured cells is required, and if these systems can be established, it is considered that the above research will advance dramatically. [0003] HCV is known to be a virus belonging to the Flaviviridae family and having a single-stranded (+) sense RNA as a genome, and is known to cause hepatitis C. HCV is divided into several types according to its genotype or serotype. According to the systematic a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/29C07K16/10C12N5/10C12N7/00C12Q1/02C12Q1/68
CPCC12N2770/24221G01N33/5008C12N7/00C07K16/109C12N2770/24234G01N33/5767C12N2770/24222G01N2333/186C07K14/005C12Q1/18A61P31/14
Inventor 胁田隆字M.萨伊德P.莫雷C.冈迪奧横川宽
Owner JAPAN AS REPRESENTED BY DIRECTOR GENERAL OF NAT INST OF INFECT IOUS DISEASES
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