| Hepatitis B virus (HBV) causes both acute transient and chronic hepatitis. Chronic HBV infection can lead to the development of liver cirrhosis and hepatocellular carcinoma. Among the 350 million people with chronic HBV infection, the risk of dying from HBV-related complications is between 15% to 25%. Despite availability of effective recombinant vaccines, current therapies for chronic HBV infection are limited in their effect on viral gene expression and replication. Effective therapies against viral infection are not available at present. There are some problems involved in the available techniques of HBV gene therapies, such as antioligonuclitide and ribozyme and deoxyribozyme. The pregenomic RNA is 3.5kb in length, which serves both as the template for reverse transcription and the generation of viral DNA. HBV genome overlap obviously, and synthesis of HBV proteins could be inhibited by cut its mRNA and pregenomic RNA on proper site. RNA interference (RNAi) is a process during which double stranded RNA (dsRNA) induces the sequence-specific degradation of homologous single-stranded RNA. As a result, the expression of the target gene is suppressed. RNAi can therefore be used to develop specific dsRNA based gene therapeutics by knocking down specific genes such as the gene related to viral infection, carcinoma, and the others diseases. In cultured mammalian cells, the sequence-specific gene silencing can be accomplished by introducing small interfering RNA (siRNA) that is cleaved from dsRNA. Only RNA molecules<30 bases in length can be used to exclusively induce RNAi in mammalian cells because longer molecules also activate the nonspecific double-stranded RNA-dependent IFN response. With the advance of producing siRNA, several laboratories made progress in using RNAi to control viral infection and showed that siRNA can inhibit infection by polio, HIV, HCV and influenza virus. Recently it was reported that RNAi could also reduced HBV replication and expression in the post-transcriptional level. HepG2.2.15 cell line, a derivative of the human HepG2 hepatoma cell line, contains integrated HBV ayw DNA. This line reappears a state that cell has been infected by HBV and HBV genome has integrated into the genome of host cell. This cell line supports viral gene expression and replication and serves as an in vitro model for treating of HBV infections. There is no report about anti-HBV by using PCR producing siRNA expression cassettes (SECs) inHepG2.2.15 cells. The aims of this study were to develop an RNAi approach that specifically targets the precore and core regions and S regions of hepatitis B virus (HBV) by synthesizing short interfering RNA (siRNA) in vivo, and to explore the inhibitory effect of siRNAs on HBV replication and expression, and to provide some evidences for anti-HBV therapy by RNAi. The sequences of HBV S region and preC/C region were amplified by PCR from DNA of HepG2.2.15cells with primers designed and synthesized. PCR products were purified and recombined with vectors of pMD18-T or pGEM T-easy. The recombinant plasmid was transformed into E. coli strain JM 109. DNA sequence analysis proved that the inserted fragment was HBV DNA sequence encoding ayw HBV phenotype. The downstream primers of SECs were designed and synthesized according to the target sequences selected in HBV S and preC/C sequences analyzed in HepG2.2.15 cells following the protocol provided by the manafacturer. 10 DNA fragments contained the sequences related with selected target sites was amplified by PCR with gene-specific downstream primers, the upstream primers and DNA templates provided by the manufacturers. The PCR products were combined and purified, and then transfected intoHepG2.2.15cells that plated one day before transfection. We researched the effect of HBV siRNA producing system specific for the S and preC/C gene of HBV genome in the different time points post-transfection at one-day intervals. According above results, the siRNA had the best effect of inhibition was selected to entra... |
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