HBV-TRL重组腺病毒载体的抗病毒活性

目的:观察有linker插入的乙肝病毒靶向核糖核酸酶(HBV-TRL)重组腺病毒载体(RAd)对HBV复制的抑制作用.方法:以第四军医大学病原生物学教研室已构建的pc DNA3.1(-)/TRL,TR,TRmut,HBVc和hEDN为基础,分别将目的片段TRL,TR,TRmut,HBVc和hEDN插入腺病毒穿梭质粒pDC316,与辅助质粒共转染HEK293细胞,重组产生含各目的片段的复制缺陷型腺病毒,即RAd/TRL,TR,HBVc和hEDN,经鉴定、扩增及滴度测定.其中RAd/TRL,TR为实验组,其余重组腺病毒为对照组.将获得的重组腺病毒感染HepG2.2.15细胞后通过间接免疫荧光检测重组腺病毒在细胞中的表达,并应用荧光定量PCR法检测细胞上清中HBVDNA含量.同时,应用MTT比色法检测细胞的代谢活性.结果:成功获得了含TRL,TR,HBVc和hEDN等不同目的片段的复制缺陷型腺病毒载体.RAd/TRL在HepG2.2.15细胞中得到有效表达,并且明显降低了细胞上清HBV-DNA含量,与RAd/TR相比(P=0.0266,P＜0.05),与其他对照组相比(P＜0.01).MTT比色分析表明细胞代谢活性未受到影响(P＞0.05).结论:成功地构建了RAd/TRL,其有效表达成功地抑制了HBV的复制.

Anti-virus activity of recombinant adenoviral vector HBV-TRL

AIM: To investigate the inhibitive effect of HBV-TRL on the HBV replication. METHODS: Based on previously constructed pcDNA3.1(-)/TRL, TR, TRmut, HBVc and hEDN, we inserted interest gene sequences TRL, TR, HBVc and hEDN respectively into adenovirus shuttle plasmid pDC316 and cotransfected HEK293 cells with rescue plasmid pBHGlox (delta)E1,3Cre to acquire RAd/TRL, TR, HBVc and hEDN. RAds were then identified, amplified and the titers were determined. RAd/TRL and TR were taken as the experimental groups and others were as control. After infecting HepG2.2.15 cells, RAd/TRL expression was identified by indirect immunofluorescence staining. The supernatant HBV-DNA content was determined by fluorescent quantification PCR (FQ-PCR). The 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 with that of RAd/TR (P=0.0266, P<0.05) and other control groups (P<0.01). MTT assay suggested that there were no significant differences between the groups (P>0.05). CONCLUSION: RAd/TRL is successfully constructed and its effective expression inhibits the HBV replication.