AIM:To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1 (-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequenc...AIM:To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1 (-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGIox(delta)El,3Cre to acquire RAd/TRL, TR, HBVc and hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry. RESULTS: RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63±0.14 vs1.60±0.47, P= 0.0266, <0.05) and other control groups (0.63±0.14 vs8.50±2.78,8.25±2.26, 8.25±2.29, 8.50±1.51, 8.57±1.63, P<0.01). MTT assay suggested that there were no significant differences in cell metabolic activity between groups (P>0.05). CONCLUSION: The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.展开更多
基金Supported by the National Natural Scientific Foundation, No.30400380 Shaanxi Natural Scientific Foundation, No. 22C2-28
文摘AIM:To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1 (-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGIox(delta)El,3Cre to acquire RAd/TRL, TR, HBVc and hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry. RESULTS: RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63±0.14 vs1.60±0.47, P= 0.0266, <0.05) and other control groups (0.63±0.14 vs8.50±2.78,8.25±2.26, 8.25±2.29, 8.50±1.51, 8.57±1.63, P<0.01). MTT assay suggested that there were no significant differences in cell metabolic activity between groups (P>0.05). CONCLUSION: The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.
