Targeted ribonuclease can inhibit replication of hepatitis B virus

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.

Effect of vector-expressed siRNA on HBV replication in hepatoblastoma cells

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.

Effect of vector-expressed shRNAs on hTERT expression

AIM: To study the effect of short hairpin RNAs (shRNAs)expressed from DNA vector on hTERT expression.METHODS: Oligonucleotides coding for four shRNAs against hTERT were cloned into a mammalian shRNA expressionvector pUC18U6 to form pUC18U6ht1-4, which were thenintroduced into HepG2 cells by using liposome-mediated transfection. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir, which expressed shRNA against green fluorescent protein (GFP), were used as controls. hTERT mRNA in the transfected cells were quantified by using real-time fluorescent RT-PCR.RESULTS: Among the four shRNAs against hTERT, two decreased the hTERT mRNA level. Compared with the controls, pUC18U6ht which expressed the two shRNAs reduced hTERT mRNA by 39% and 49% (P＜0.05).CONCLUSION: hTERT expression is inhibited by the shRNAs expressed from the DNA vector.