Hepatitis C virus chimeric replicon and its construction method

A hepatitis C virus and a construction method technology, applied in the field of hepatitis C virus chimeric replicon and its construction, can solve the problems of low transient transfection efficiency of the chimeric replicon, and achieve improved replication efficiency, rapid technical route, Efficient effect

Inactive Publication Date: 2013-07-24
辉源生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the transient transfection efficiency of the chimeric replicon with GT1b as the backbone is low, and the replication efficiency of the replicon must be improved by constructing a stable cell line

Method used

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  • Hepatitis C virus chimeric replicon and its construction method
  • Hepatitis C virus chimeric replicon and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Comparison of replication kinetics of chimeric replicons

[0023] 1) Experimental method

[0024] Huh7 cells were transiently transfected with HCV GT1b / GT3a-NS5b and HCV GT2a / GT3a-NS5b, respectively, at 37℃, 5% CO 2 Culture in an incubator. The transfected cells were collected at 4 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours to test their replication efficiency. The Renilla luciferase activity at each time point was tested according to the kit instructions.

[0025] 2) Experimental results

[0026] The experimental results are shown in Table 1:

[0027] Table 1 The replication rate of each time point relative to the 4-hour time point

[0028] 4 hours 24 hours 48 hours 72 hours 96 hours HCV GT1b / GT3a-NS5b10.480.70.670.16 HCV GT2a / GT3a-NS5b 10.661.071.523.84

[0029] It can be seen from Table 1 that the replication rate of HCV GT2a / GT3a-NS5b is higher than that of HCV GT1b / GT3a-NS5b, which is more suitable for transient expression.

Embodiment 2

[0030] Example 2 Activity of HCV796 in different chimeric replicons

[0031] 1) Experimental method

[0032] Huh7 cells were transiently transfected with HCV GT1b / GT3a-NS5b and HCV GT2a / GT3a-NS5b, respectively, at 37℃, 5% CO 2 Incubate in an incubator for 4 hours, transfer 50μl of the compound to the transfected cell culture plate, and incubate at 37°C, 5% CO 2 After 72 hours of culture, the Renilla luciferase activity in each well was tested according to the kit instructions.

[0033] 2) Experimental results

[0034] The experimental results are shown in Table 2:

[0035] Table 2 The activity of compound HCV796 in different chimeric replicons

[0036]

[0037] It can be seen from Table 2 that the activity of compound HCV796 against HCV GT2a / GT3a-NS5b is better than that against HCV GT1b / GT3a-NS5b. It can be seen that the HCV GT1b / GT3a-NS5b replicon we constructed not only has a high replication rate, but also is sensitive to reference compounds.

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Abstract

The invention provides a hepatitis C virus chimeric replicon and its construction method. The hepatitis C virus (HCV) chimeric replicon is a chimeric replicon of HCV genotype 2a and genotype 3a, and non-structural protein 5b, and is named as HCV GT2a/GT3aNS5b. The construction method includes: taking pRep-JFH-1 as the basis, and replacing its NS5B gene with GT3a NS5B gene, thus obtaining an NS5B chimeric replicon. Compared with the existing chimeric replicons taking GT1b as the skeleton, the hepatitis C virus chimeric replicon provided in the invention has high transient transfection efficiency, and the replication efficiency of the replicon does not need to be improved by constructing a stale cell line. Thus, the invention provides a rapid technical route for construction of more HCV clinical sample chimeric replicons in the future.

Description

technical field [0001] The invention relates to biotechnology, in particular to a hepatitis C virus chimeric replicon and its construction method. Background technique [0002] Epidemiological reports show that about 170 million people worldwide are infected with hepatitis C (HCV). After HCV infects the human body, it can remain latent for a long time, leading to liver cirrhosis and liver cancer. According to different genotypes, HCV is divided into 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a, 6a and other subtypes, among which, genotype 1 (GT1a / 1b) accounts for 74% of clinical infection subtypes, and genotype Types 2 and 3 accounted for 22% of clinically infected subtypes, and genotypes 4 / 5 / 6 accounted for 4% of clinically infected subtypes. In China, clinically isolated hepatitis C is mainly genotypes 1, 2, and 3, among which genotypes GT1b and GT2a are the main ones. [0003] In the fight against hepatitis C, human beings are constantly exploring and researching new therapeutic dru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/51C12N15/10C12R1/93
Inventor 陈景才江海清李忠张晓霁张宏达谈璐瑶
Owner 辉源生物科技(上海)有限公司
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