AIM: To study the effect of a novel targeted ribonuclease(TN), the fusion protein of HBVc and human eosinophil-derived neurotoxin (hEDN), on the HBV replication in vitro.METHODS: The gene encoding the targeted ribonuc...AIM: To study the effect of a novel targeted ribonuclease(TN), the fusion protein of HBVc and human eosinophil-derived neurotoxin (hEDN), on the HBV replication in vitro.METHODS: The gene encoding the targeted ribonucleasewas cloned into pcDNA3.1 (-) to form recombinant eukaryoticexpression vector p/TN. Control plasmids, including p/hEDN,p/HBVc, and p/TNmut in which a Lys113→Arg mutation wasintroduced by sequential PCR to eliminate the ribonucleaseactivity of hEDN, were also constructed. Liposome-mediatedtransfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection wasperformed. After that, RT-PCR was used to verify thetransgene expression. Morphology of the transfected cellswas observed and MTT assay was performed to detect thecytotoxicity of transgene expression. Concentration of HBsAgin the supernatant of the transfected cells was measuredusing solid-phase radioimmunoassay.RESULTS: Transgenes were successfully expressed in 2.2.15cells. No obvious cytotoxic effect of transgene expressionon 2.2.15 cells was found. The HBsAg concentration in thep/TN transfected cells was reduced by 58 % compared withthat of mock transfected cells. No such an effect was foundin all other controls.CONCLUSION: The targeted ribonuclease can inhibit HBVreplication in vitro while it has no cytotoxicity on host cells.The targeted ribonuclease may be used as a novel antiviralagent for human HBV infection.展开更多
AIM: To study the effect of siRNA expressed from DNA vector on HBV replication.METHODS: Human U6 promoter was amplified from genomic DNA and cloned into plasmid pUC18 to construct a mammalian siRNA expression vector p...AIM: To study the effect of siRNA expressed from DNA vector on HBV replication.METHODS: Human U6 promoter was amplified from genomic DNA and cloned into plasmid pUC18 to construct a mammalian siRNA expression vector pUC18U6. Then oligonucleotides coding for a short hairpin RNA against HBV were cloned into pUC18U6 to form pUC18U6HBVsir which was introduced into 2.2.15 cells by using liposome-mediated transfection.2.2.15 cells transfected by pUC18U6 and pUC18U6GFPsir which expressed siRNA against green fluorescent protein and mock-transfected 2.2.15 cells were used as controls.Concentration of HBsAg in the supernatant of the transfected cells was measured by using solid-phase radioimmunoassay.RESULTS: A mammalian siRNA expression vector pUC18U6 was constructed successfully. Compared with controls,pUC18U6HBVsir which expressed siRNA against HBV decreased concentration of HBsAg significantly by 44%(P<0.05).CONCLUSION: HBV replication in 2.2.15 cells is inhibited by siRNA expressed from the DNA vector.展开更多
基金National Natural Science Foundation of China,No. 30100157Medical Research Fund of PLA,No.01MA184Innovation Project of FMMU,No.CX99005
文摘AIM: To study the effect of a novel targeted ribonuclease(TN), the fusion protein of HBVc and human eosinophil-derived neurotoxin (hEDN), on the HBV replication in vitro.METHODS: The gene encoding the targeted ribonucleasewas cloned into pcDNA3.1 (-) to form recombinant eukaryoticexpression vector p/TN. Control plasmids, including p/hEDN,p/HBVc, and p/TNmut in which a Lys113→Arg mutation wasintroduced by sequential PCR to eliminate the ribonucleaseactivity of hEDN, were also constructed. Liposome-mediatedtransfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection wasperformed. After that, RT-PCR was used to verify thetransgene expression. Morphology of the transfected cellswas observed and MTT assay was performed to detect thecytotoxicity of transgene expression. Concentration of HBsAgin the supernatant of the transfected cells was measuredusing solid-phase radioimmunoassay.RESULTS: Transgenes were successfully expressed in 2.2.15cells. No obvious cytotoxic effect of transgene expressionon 2.2.15 cells was found. The HBsAg concentration in thep/TN transfected cells was reduced by 58 % compared withthat of mock transfected cells. No such an effect was foundin all other controls.CONCLUSION: The targeted ribonuclease can inhibit HBVreplication in vitro while it has no cytotoxicity on host cells.The targeted ribonuclease may be used as a novel antiviralagent for human HBV infection.
基金Supported by National Natural Science Foundation of China,No.30100157Medical Research Fund of Chinese PLA,No.01MA184and Innovation Project of FMMU,No.CX99005
文摘AIM: To study the effect of siRNA expressed from DNA vector on HBV replication.METHODS: Human U6 promoter was amplified from genomic DNA and cloned into plasmid pUC18 to construct a mammalian siRNA expression vector pUC18U6. Then oligonucleotides coding for a short hairpin RNA against HBV were cloned into pUC18U6 to form pUC18U6HBVsir which was introduced into 2.2.15 cells by using liposome-mediated transfection.2.2.15 cells transfected by pUC18U6 and pUC18U6GFPsir which expressed siRNA against green fluorescent protein and mock-transfected 2.2.15 cells were used as controls.Concentration of HBsAg in the supernatant of the transfected cells was measured by using solid-phase radioimmunoassay.RESULTS: A mammalian siRNA expression vector pUC18U6 was constructed successfully. Compared with controls,pUC18U6HBVsir which expressed siRNA against HBV decreased concentration of HBsAg significantly by 44%(P<0.05).CONCLUSION: HBV replication in 2.2.15 cells is inhibited by siRNA expressed from the DNA vector.
