Construction of hepatitis B virus siRNA expression vector and its application in antivirus treatment

Abstract

The present invention relates to construction of hepatitis B virus siRNA expression vector and its antiviral therapeutic application. According to the design principle said construction includes the following steps: respectively selecting nucleotide sequence of HBV envelope protein (MS) coding region 69nt-87nt, polymerase(p) coding region 708nt-726nt and core protein (c) coding region 2052 nt-2070nt, designing correspondent siRNA sequence, using BLAST software to analyze homogenously to confirm that it is HBV highly conservative sequence, then chemically synthesizing single-stranded oligonucleotide, external annearing to form double-stranded DNA, connecting it with eukaryotic expression vector pTZU6+1, enzyme excision identification and sequence determination and analysis show that siRNA extression vectors of three target hepatitis B virus (HBV) gene sequences are constructed. The tests show that HBV siRNA has the strong action of inhibiting virus replication and expression.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering. Background technique: [0002] At present, the antiviral treatment plan for hepatitis B (HBV) is still in the process of continuous exploration and practice. The existing nucleoside analogs and interferon cannot fundamentally solve the antiviral problem, so it is urgent to think about antiviral from a new perspective. treatment measures. In recent years, gene blocking technology has made great progress in gene function research, and clinical treatment research has also shown good prospects, providing us with a reference technology for designing HBV antiviral treatment programs. At present, the gene knockdown technology that inhibits virus replication by finding and knocking down key enzymes or RNA molecules in the replication of HBV virus is becoming the focus of many scholars. [0003] RNA interference (RNAi) is a new gene blocking technology discovered and developed in recent yea...

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Problems solved by technology

[0004] There are only 4-5 reports on RNAi antiviral treatment at home and abroad. The results of in vitro experiments have confirmed that the inhibitory effect is specific and efficient. However, RNAi blocking technology is used to design siRNA sequences targeting different coding regions of HBV genes. , to further construct the HBV siRNA expression vector, so far there is no relevant report at home and abroad

Simultaneously in vitro HBV transfected HepG 2 In the cell model, microparticle enzyme immunoassay (MEIA) was used to study the secretion of HBV siRNA specifically inhibiting the secretion of viral proteins; Cy-3 labeled antibody was used to perform immunofluorescent staining on the expression of HBcAg and HBsAg in transfected cells to achieve intuitive and localized observation. So far no reports

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