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
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.
