C基因截短的HBV复制与包装

Replication and encapsidation of HBV mutants with the truncated C gene

目的 探讨C基因截短型HBV变异体的复制与包装。方法 采用分子克隆、人工定点突变等技术构建C基因截短型HBV变异体质粒 ,用脂质体法转染HepG2 细胞 ,提取细胞内及培养上清液中DNA分别进行Southern杂交 ,PCR及实时定量荧光PCR分析。结果 经DNA测序及酶切鉴定证实C基因截短型HBV质粒载体构建成功 ;C基因截短型HBV为复制缺损型 ,与辅助质粒共转染HepG2 细胞 ,可在细胞内及培养上清液中检测到HBV各种DNA构型 ;DNA定量分析提示C基因截短型HBV的包装效率较野生型HBV提高 3～ 4 0倍。结论 C基因截短型HBV变异体为复制缺损型 ,单独转染后不能在肝细胞内包装与复制 ,但在缺失包装信号ε的相应辅助病毒辅助下可有效复制并包装成子代病毒颗粒分泌到胞外 ,且包装效率大大提高。

Objective To evaluate the replication and encapsidation of HBV mutants with the truncated C gene. Methods The HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro . The replication and encapsidation of HBV mutants were investigated by Southern blotting,PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome. Results The C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication-defective, however,all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay. Conlusion The C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely,however,the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-tranfected with the helper construct which lacks part of 5′-proximal HBV RNA packaging signal ε.