| Objective:1. To screen anti-DENV viral targets by artificial microRNAs (amiRNA) targeting its conserved genome sequences;2. To screen anti-HBV host targets by amiRNA targeting potential HBV-ralated host molecules.Methods:1. Anti-DENV viral targets:(1) To construct vectors expressing amiRNAs;(2) To observe cell conditions infected by DENV;(3) To detect the cell survival rate by CCK-8;(4) To detect the DENV envelope E protein expression by indirect immunofluorescence;(5) To detect the titer of DENV in cell supernatant by virus plaque assay;(6) To detect the mRNA levels of DENV and interferon genes by semi-quantitative RT-PCR.2. Anti-HBV host targets:(1) To construct lentiviral vectors expressing amiRNAs;(2) To detect HBsAg and HBeAg in cell supernatant by ELISA;(3) To detect HBV DNA in cell supernatant by quantitative PCR;(4) To select stably transfected HepG2cells by puromycin;(5) To detect AFP mRNA level in stably transfected HepG2cells by RT-PCR;(6) To detect AFP protein level in stably transfected HepG2cells by indirect immunofluorescence;(7) To quantify AFP in supernatant of the stably transfected HepG2cells by ELISA.Results:1. The targets of anti-DENV:21amiRNAs were designed to target the conservative regions of DENV, and6amiRNAs were identified for their protective effects on DENV infected cells. The cell survival rate of DENV-128was90%which was1.8-fold of the virus infection control. This result was generally consistent with the indirect immunofluorescence assay for DENV E (envelope) protein expressed in BHK-21cells48hours post-infection. Then miRNA-like polycistrons were constructed containing DENV-128and another amiRNA with inhibition effect on DENV replication by primary experiment test, and DENV128-1382was the most effective sequence after observation of CPE, CCK-8and indirect immunofluorescence. The cell survival rate was98%in DENV128-1382group, which was1.6times higher than the DENV infection group, and the expression of viral envelope E proteion was also greatly reduced. Finally, the lentivectors were constructed to stably and serially express DENV-128and DENV128-1382, and the results of CCK-8, indirect immunofluorescence, semi-quantitative RT-PCR, virus plaque assay and interferon induction assay showed that DENV replication was substantially inhibited by amiRNAs in a sequence-specific way.2. The host targets of anti-HBV:amiRNAs were designed to target33host moleculars related to HBV infection and replication.3host genes were obtained by detection of HBsAg and HBeAg levels in cell supernatant as evaluation index (efficiency>30%). Alpha-fetoprotein (AFP) gene was selected for further research, and5amiRNAs were designed to target AFP. Among them, AFP-559and AFP-1621were indentified to inhibit HBV replication efficiently from the results of HBsAg and HBeAg and HBV DNA by transient transfection. The inhibition rate for HBsAg was59%(96h), for HBeAg44%(96h), and for HBV DNA40%in AFP-559group. The inhibition rate for HBsAg was49%(96h), for HBeAg33%(96h), and for HBV DNA37%in AFP-1621group. Lentiviral amiRNA expression plasmids targeted AFP were transfected into HEK293T cells and lentiviruses were packaged to infect HepG2cells. The stably transduct cell lines were screened by puromycin, and the effiency of GFP expression was>99%. The results of RT-PCR, indirect immunofluorescense assay and ELISA demonstrated that the expression of AFP was remarkably reduced both in mRNA and protein levels in AFP-amiRNA stably transducted HepG2cell lines.Conclusions:1. Six amiRNAs and one amiRNA polycistron targeting the conservative regions of DENV were screened and validated for their anti-DENV activity. DENV-128and DENV128-1382efficiently inhibited DENV-2replication and the cell survival rates were also enhanced.2. Three host targets with potential anti-HBV activity were found, and it was also initially validated that AFP-amiRNA could efficiently inhibit HBV replication. For further study of AFP function in the HBV life cycle, AFP-amiRNA stably transfected HepG2cell lines were established successfully. |
PDF Full Text Request