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Method for constructing gene 2a type hepatitis c virus clinical isolates cell culture model

A hepatitis C virus, hepatitis C virus technology, applied in the fields of biotechnology and virology, can solve the problem of limited virus strains

Inactive Publication Date: 2015-04-29
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, currently available virus strains with high-efficiency replication and infection capabilities in in vitro culture systems are still very limited, so it is urgent to find more virus strains suitable for in vitro drug screening systems to help find a wider range of anti-HCV viruses. spectrum of potential drugs, or the development of more effective vaccines

Method used

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  • Method for constructing gene 2a type hepatitis c virus clinical isolates cell culture model
  • Method for constructing gene 2a type hepatitis c virus clinical isolates cell culture model
  • Method for constructing gene 2a type hepatitis c virus clinical isolates cell culture model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0072] Example 2. Core protein-NS3 protease region, NS3 helicase-NS5A region and NS5B region of PR63 are active in in vitro culture

[0073]Except for JFH-1, other HCV clones were inactive in cell culture: the RNA of wild-type MA, J6CF, and DH8CF could not replicate and produce infectious particles in cultured cells (Li, Y. et al., 2012, Proceedings of the National Academy of Sciences of the United States of America 109:E1101-1110; Murayama, A., T. et al., 2012, Journal of virology 86:2143-2152; Ramirez, S. et al., 2013, Hepatology (Baltimore, Md)). Therefore, the present inventors did not start from PR63 to construct its consensus sequence cDNA clone. On the contrary, they first used JFH-1 as the backbone to target the core protein-NS3 protease region containing the source of the PR63 sequence (the upstream of the SpeI enzyme cleavage point in the NS3 coding sequence, NS3p), NS3 helicase (downstream of the SpeI cut point in the NS3 coding sequence, NS3h)-NS5A region, NS5B reg...

Embodiment 3

[0078] Example 3. Combining functional regions of PR63 and confirming mutations that can promote virus production

[0079] The present inventors tried to combine PR63-derived FspA I-Spe I segment, Spe I-Rsr II segment and Rsr II-SgrA I segment to construct a nearly full-length infectious clone of PR63. However, these three segments were spliced ​​together to replace the corresponding region in the JFH-1 vector, resulting in clone PR63 C-NS5B / JFH-1 (selected from: FspA I-Spe I segment from S10 + Spe I-Rsr II segment from A6 + Rsr II-SgrA I segment from 5B13 (S10-A6-5B13) (from FspA I-SgrA The sequence between I is the clone derived from PR63, and the rest is still the sequence derived from JFH-1); FspA I-Spe I segment from S10 + Spe I-Rsr II segment from A6 + Rsr II-SgrA from 5B21 I segment (S10-A6-5B21) or FspA I-Spe I segment from S10 + Spe I-Rsr II segment from A6 + Rsr II-SgrA I segment from 5B23 (S10-A6-5B23)) after electroporation No HCV-positive cells were found in th...

Embodiment 4

[0082] Example 4, the untranslated regions at both ends of PR63 and the C-terminus of NS5B were replaced to obtain the full-length infectious genome of PR63

[0083] Thanks to clone PR63 C-5B_A1676S_W849G_Q862P_C2432R_A2941T_S2955F / JFH-1 can effectively amplify the virus in Huh7.5.1 cells without additional mutations, based on which the inventors further narrowed down the sequence of JFH-1. First, replace the 5' untranslated region of JFH-1 origin and the N-terminal sequence of the core protein with the sequence of PR63 (replace PR63 with the 5' UTR-FspA I segment derived from PR63 C-5B_A1676S_W849G_Q862P_C2432R_A2941T_S2955F / JFH-1 genome 5'UTR-FspA I segment). The 5'untranslated region of PR63 is the same as J6CF, and there are 3 nucleotide differences compared with JFH-1. The 5'untranslated region derived from PR63 and the N-terminus of the core protein can be easily replaced without affecting the infectivity. The obtained replacement product is abbreviated as PR63 5UTR...

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Abstract

The invention relates to a method for constructing a gene 2a type hepatitis c virus clinical isolates cell culture model. The invention further provides a recombined hepatitis c virus capable of efficient infection and reproduction in virus packaging cells. The technical scheme provided by the invention can be used for providing individual-based treatment for HCV patients, and developing vaccines with a wider spectrum of activity, filtrating specific anti-HCV drugs and the like.

Description

technical field [0001] The invention belongs to the fields of biotechnology and virology; more specifically, the invention relates to a method for constructing a cell culture model of a clinical isolate of hepatitis C virus genotype 2a. Background technique [0002] HCV belongs to the Flaviviridae family and is an enveloped, single-stranded, positive-sense RNA virus. Its genome is 9.6kb long, including 5'untranslated region, open reading frame and 3'untranslated region. Its open reading frame encodes a polyprotein, which can be cut to obtain structural proteins (core, E1, E2), p7 and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). HCV can be divided into at least 6 genotypes and many subtypes and isolates. The genotypes differ not only in sequence but also in response to antiviral therapy. 1b and 2a are the most common genotypes in Chinese HCV patients. Due to the low fidelity of HCV's RNA-dependent RNA polymerase, HCV exists as a quasi-species in the host, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C12N15/51C12N7/01A61K39/29C12Q1/70C12N15/63
CPCC07K14/005A61K39/00C12N2770/24222C12N2770/24234C12N2770/24252C12Q1/025
Inventor 钟劲卢捷向禹陶万银王娜
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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