Construction method of HBV (Hepatitis B Virus) isolating strain replicating-form plasmid

Abstract

The invention discloses a construction method of HBV (Hepatitis B Virus) isolating strain replicating-form plasmid, comprising the following steps of: 1, extraction of HBV full-length genome, PCR (Polymerase Chain Reaction) amplification and clone construction; 2, construction of a recombinant transition vector; 3, enzyme cutting and dephosphorylation of the recombinant transition vector; and 4, construction of replicating-form plasmid, wherein the recombinant transition vector is synchronously and directionally connected by three connection segments, two reverse BspQI enzyme cutting sites are formed between two inserted HBV segments, and the clone of the HBV full-length genome and the bacterial colony cultivation and amplification of the replicating-form plasmid are carried out by an SOC culture medium. The invention applies a novel full-length genome clone vector, improves the enzyme cutting solution and adopts a novel bacterial colony culture medium. The invention has the advantages of simple performance, high efficiency and stability, can be applied to the comprehensive phenotype research on a China HBV clinic isolating strain whole genome and has very important application value.

Description

technical field [0001] The invention relates to the technical field of in vitro phenotype research of hepatitis B virus, in particular to the construction of a replication-type plasmid of a hepatitis B virus isolate. Background technique [0002] In my country, hepatitis B virus (HBV) infection is the main cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HBV replication is achieved through the process of reverse transcription, during which mutations are most likely to occur. In recent years, with the application and popularization of antiviral drugs and vaccines, the problems of drug resistance and immune escape due to virus mutation have attracted more and more attention. Phenotypic research on clinical isolates of HBV variants is an important means of HBV variation research. [0003] Cell models are the most widely used in the in vitro phenotype study of HBV variants. Cell model research methods include vector-mediated and non-vector-mediated tr...

AI Technical Summary

Problems solved by technology

At present, most of the HBV replicating plasmids used at home and abroad are 1.1-HBV vectors from laboratories in France and the United States, but the elements that initiate transcription on the vectors are all exogenous promoters and POLYA signals, which have certain limitations. : (1) The effect of foreign elements on HBV replication and expression is not clear; (2) The promoter of HBV is not a strictly conserved region, and the common clinical mutations of 1762 and 1764 occur in the promoter region. 1.1-HBV vectors that cannot phenotype variants in the above regions

Moreover, most of the HBV strains of domestic replicating plasmids come from foreign samples, and their serotypes and genotypes are quite different from those of domestic strains.

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