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Nucleic acid and gene originating in novel hcv strain and replicon-replicating cell using the gene

一种复制子、核酸的技术，应用在基因工程、植物基因改良、生物化学设备和方法等方向，能够解决不适于建立自主感染并复制的细胞培养体系等问题，达到高概率复制效率的效果

Inactive Publication Date: 2006-12-20
TOKYO METROPOLITAN INST OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, because the susceptibility to HCV infection has not been confirmed, it is also considered that Huh7 cells are not suitable for establishing a cell culture system that enables HCV to infect and replicate autonomously.

Method used

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  • Nucleic acid and gene originating in novel hcv strain and replicon-replicating cell using the gene
  • Nucleic acid and gene originating in novel hcv strain and replicon-replicating cell using the gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Replication of replicon RNA

[0130] 1. Construction of expression vector

[0131] The DNA corresponding to the complete region of the genome of the hepatitis C virus JFH-2.1 strain and JFH-2.2 strain isolated from patients with fulminant hepatitis was obtained from the JFH-2.1 and JFH-2.2 clones containing the full-length genome cDNA of the virus strain, and the T7 RNA A synthetic DNA of a promoter sequence was attached to the 5' end of each clone, and then inserted into a plasmid (pUC19 plasmid). The plasmid DNAs thus constructed are hereinafter referred to as pJFH-2.1 and pJFH-2.2, respectively. It should be noted that the above JFH-2.1 and JFH-2.2 clones were prepared according to published reports (Lehmann et al., Science, (1999)).

[0132] The structures of the thus constructed plasmid DNAs pJFH-2.1 and pJFH-2.2 are shown in figure 1 line above. "T7" indicates the T7 RNA promoter, and "G" indicates the dGTP inserted upstream of the 5' end of pJFH-2.1 and pJFH-...

Embodiment 2

[0139] Establishment of replicon-replicating cell clones

[0140] For each synthetic replicon RNA rSGREP-JFH2.1 and rSGREP-JFH2.2 prepared in Example 1, 0.01 ng to 10 μg of replicon RNA was mixed with total cellular RNA extracted from Huh7 cells to adjust the concentration of total RNA The amount is 10 μg. The mixed RNA was then introduced into Huh7 cells by electroporation. The electroporated Huh7 cells were seeded in culture dishes and cultured for 16 hours to 24 hours, and then various concentrations of G418 (neomycin) were added to the dishes. Thereafter, the culture was continued while changing the medium twice a week. After 21 days of culture from the time of inoculation, surviving cells were stained with crystal violet. The result is as figure 2 Colony formation is confirmed as shown.

[0141] For cells transfected with rSGREP-JFH2.1 and rSGREP-JFH2.2 and showing colony formation, surviving cell colonies were further cloned from the culture dish after 21 days of c...

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Abstract

The present invention relates to a gene derived from a novel fulminant hepatitis C virus strain, an HCV replicon RNA with a high replication efficiency obtained using the gene, and an HCV replicon-replicating cell transfected with the replicon RNA. When the HCV replicon RNA and the HCV replicon-replicating cell of the present invention are used, HCV proteins can be continuously produced in a large amount.

Description

technical field [0001] The present invention relates to nucleic acids and genes derived from novel fulminant strains of hepatitis C virus, HCV replicons and replicon-replicating cells using the genes. Background technique [0002] For virus research and the research and development of antiviral drugs, experimental systems that allow efficient amplification of viruses are absolutely necessary. In addition, when there is a system for amplifying viruses by using cultured cells or a system for evaluating virus proliferation by using cultured cells, virus research and research and development of antiviral drugs show remarkable progress. [0003] Hepatitis C virus (HCV) is a virus belonging to the Flaviviridae family having a single-stranded (+) sense-strand RNA as its genome, and is known to cause hepatitis C. Recent studies have revealed that the hepatitis C virus can be classified into many types according to genotype or serotype. According to Simmonds et al.'s systematic ana...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/51C12N15/10C12Q1/68C12N5/08G01N33/50G01N33/15C07K14/18C12N7/00C12N15/67C12N15/86
CPCC12N2840/203C12N2770/24221C12N7/00C12N2770/24222C12N15/67C12N15/86C07K14/005A61P31/14
Inventor 胁田隆字加藤孝宣伊达朋子宫本道子
Owner TOKYO METROPOLITAN INST OF MEDICAL SCI
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