A Study On Anti-wt-HBV Dominant Negative Mutants Constructed By Interfering Mutation In Replication

Objectives Chronic infection with the hepatitis B virus (HBV) is a major problem of public health and a common disease in our country and currently available therapies have limited efficacy. Gene therapy is a hot point in antiviral therapy. It is a key to get a vector targeted the infected tissues or cell in gene therapy. HBV is of hepatotropic specificity. It can specifically infect the liver and continuously replicate in the hepatocytes without obvious cytotoxicity by itself. Hepatitis B virus (HBV; is naturally provided with the prerequisite for being transformed as the liver-targeting gene therapeutic vector. HBV has more distinct advantages than other viral vectors on theory. If it can express anti-HBV products and meanwhile be packaged, the antiviral function will be amplified in vivo. The fact that a large number of HBV-infected humans appear continuously tc harbor the HBV genome and express HBsAg without pathogenic consequences offers a great advantage in terms of long-term maintenance and expression of a recombinant HBV vector bearing a desired gene. The recombinant HBV genome might be attenuated to the extent that the HBV replication level is reduced. So the limitation of other vectors for HBV gene therapy might be overcome. In this study, the HBV genome was modified as follow: (1) The C gene-truncated HBV vectors were experimentally constructed. (2) The HBV yectors with mutated envelope protein were constructed. (3) The HBV vector with core protein and envelope protein combinablenably mutated was corstructed. These vectors were transfected solely or co-transfected with adwR9 to test its had anti-HBV effect in molecular level. At the same time we had investigated the package of adw subtype C gene-truncated HBV mutant with aid of the ayw subtype helper construct pHBV3142 devoid of the encapsidation signal e.Methods 1. PcDNA3-AC, pcDNA3-C and pHBV-AC vectors were constructed by molecular cloning and PCR-based deletion from the wild HBV plasmid of adwR9. After transfection with pcDNA3-C and pcDNA3-C by using theSupported by the National Nature Science Foundation of China, NO: 30371287liposome method the protein was extracted from the cytoplasm of HepG2 cell line. The expression of mutant protein in the cytoplasmic lysates was detected by Western blot assay. The S protein of HBV was assay by ELISA in the supernatant and viral DNA was detected by Real-time fluorescence quantitative PCR from the supernatant and cytoplasm after co-transfection of pcDNA3-AC and pcDNA3-C with adwR9 respectively into HepG2 cell. The core particles in the transfected cytoplasm were assay by Native Western blot after co-transfection of pcDNA3-AC and pcDNA3 with pcDNA3-C respectively into HepG2 cell. The S protein expression and secret on were assay by ELISA in the supernatant and cytoplasm after transfection w th pHBV-AC into HepG2 cell. The S protein expression was detected by ELISA from the supernatant and the DNA was detected by Real time fluorescence quantitative PCR in the supernatant and cytoplasm after co-transfection with pHBV-AC and adwR9 into HepG2 cell.2. These envelope protein mutants, pHBV-mSl, pHBV-mS, pHBV-mSIS and pcDNA3-mS, were constructed by molecular cloning and PCR-based mutation from the wild HBV plasmid of adwR9. To observe protein expression and secretion of mutant after transfection with pcDNA3-mS and pcDNA3-S respectively into HepG2 cell the S protein in the supernatant and cytoplasm was assay by ELISA and Western blot was used to detect the protein expression in the cytoplasm. After co-transfection of pcDNA3-mS and pcDNA3 with adwR9 respectively into HepG2 cell the DNA was detected by Real-time fluorescence quantitative PCR in the cytoplasm and supernatant. The S protein was assay by ELISA in the supernatant after co-transfectic n of above both vectors with adwR9. The S protein in the supernatant and cytoplasm was assay by ELISA and the DNA was detected by Real-time fluorescence quantitative PCR in the cytoplasm and supernatant after transfectiou with pHBV-mSl, pHBV-mS, pHBV-mSIS and adwR9 into HepG...