稳定表达乙型肝炎病毒X基因肝细胞株的构建

Establishment of hepatitis B virus X gene transferred hepatocellular cell line

目的构建稳定表达乙型肝炎病毒X基因(HBVx)的正常人肝细胞株。方法用PCR法扩增含EcoRⅠ和H indⅢ酶切位点的X基因序列,构建HBVx基因真核表达载体pcDNA3.1(+)-HBVx,转化大肠杆菌DH5α,筛选阳性克隆并对其进行双酶切和测序鉴定;用脂质体转染法将HBVx基因导入Chang细胞系,G418筛选,RT-PCR和W estern b lot鉴定。结果X基因亚克隆入pcDNA3.1(+),含有完整的X基因片段,转染后Chang细胞有HBVxmRNA表达,Chang/HBVx有HBVx蛋白的表达。结论构建了稳定表达HBVx基因的肝细胞株Chang/HBVx。

Objective To establish a hepatitis B virus X gent transferred hepatocellular cell line. Methods X gene with EcoR I and Hind Ⅲ endoenzyme sites was obtained by PCR,and subcloned into pcDNA3.1（ ＋ ）vector. After positive clones were picked out,ambi-mstriction cndonucleascs digest analysis and sequence analysis were used to identify the recomhinants;The reeorabinant plasmid was transferred into the Chang cell line by lipofection method. The gene transferred cells were selectively cultured with G418 and identified by PCR and Western blot techniques. Results X gene was sub- cloned into peDNA.3.1 （ ＋ ） and contained a complete X gene fragment, a steady expression of HBVx mRNA and protein were detected in the Chang/HBVx cells. Conclusion HepatoeeUular cell line Chang/ HBVx which express HBVx gene stably is established successfully.