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Full-length infectious clones of hepatitis C virus gene type 6a Chinese toxic strain and application thereof

A virus, full-length technology, applied in the direction of application, virus, genetic engineering, etc.

Pending Publication Date: 2019-08-16
SUN YAT SEN UNIV
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AI Technical Summary

Problems solved by technology

Genotype 6 is more prevalent in southern China, and there is no full-length cell culture system for genotype 6 Chinese strains, that is, full-length infectious clones of genotype 6

Method used

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  • Full-length infectious clones of hepatitis C virus gene type 6a Chinese toxic strain and application thereof
  • Full-length infectious clones of hepatitis C virus gene type 6a Chinese toxic strain and application thereof
  • Full-length infectious clones of hepatitis C virus gene type 6a Chinese toxic strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0023] 1. Extract the RNA of the virus CH6a from the serum, and reverse to obtain the cDNA of the virus.

[0024] The entire sequence of the virus was amplified by PCR and constructed into a plasmid vector. The full-length sequence was 9641bp (with sequence CH6a FL -WT).

[0025] 2. Construct the chimeric virus plasmid and add mutations (see the mutations shown in the attached tables of each accompanying drawing).

[0026] 3. The method of in vitro transcription Transcribe the viral RNA from the plasmid, and then transfect Huh7.5 and series cells.

[0027] 4. The virus titer and the adaptive mutation acquired by infection were detected.

[0028] 5. The sensitivity of the virus to current clinical drugs was tested.

[0029] Table 1. Sequence Analysis CH6a C-NS2 Recombinant Viruses Mutations Identified. The culture supernatant (the 11th day and the 13th day, figure 1 B) Inoculate into cells, and collect the supernatant when the virus spreads to infect most of the cells (def...

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Abstract

A hepatitis C virus (HCV) genome is highly heterogeneous, and can be divided into eight genotypes and multiple gene subtypes according to the difference of genomic bases. HCV from patients cannot be cultured directly in cells in vitro. Infectious clones of HCV for constructing epidemic strains by a reverse genetic method is an important technical operation platform for promoting HCV basic researchand drug and vaccine research and development. The gene type 6 HCV is mainly prevalent in China and Southeast Asia. At present, no full-length infectious clones of the gene type 6 Chinese strain exist. The main content comprises establishment of in-vitro cell infectious clones CH6acc of the HCV gene type 6a in China and demonstration of application of the CH6acc in drug research. A CH6acc cell culture model can be used in HCV basic research, and drug and vaccine research and development.

Description

technical field [0001] This patent relates to the construction of the hepatitis C virus cell culture system, and the main content is the establishment and application of the full-length infectious clone of the hepatitis C virus type 6a Chinese strain. Background technique [0002] Approximately 71 million people worldwide are infected with hepatitis C virus (HCV), and chronic HCV infection can lead to liver cirrhosis and even liver cancer. Currently, no vaccine against HCV has been developed. HCV is a single-stranded positive-sense RNA virus with a full length of about 9.6kb. The HCV genome consists of a large open reading frame (ORF) and non-coding sequences (UTR) at both ends. ORF is cleaved into 10 viral proteins by virus and host proteases, which are structural proteins: core protein, envelope protein 1, envelope protein 2 and non-structural proteins (NS): P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. [0003] The HCV genome is diverse, and can be divided into 4 different...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/51C12N7/00
CPCC12N7/00C12N2770/24221
Inventor 李义平陈明晓
Owner SUN YAT SEN UNIV
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