不同基因型HBV真核表达载体的构建和表达  

Construction of eukaryotic expression vectors with different HBV genotypes and their expression

作　　者：王天真[1] 赵然[1] 韩昌松[1] 张玉华[1] 叶盛前[1] 

机构地区：[1]哈尔滨医科大学病理学教研室,黑龙江哈尔滨150081

出　　处：《哈尔滨医科大学学报》2014年第4期263-267,共5页Journal of Harbin Medical University

基　　金：黑龙江省教育厅科学技术研究项目(12521221)

摘　　要：目的构建A、B、C和D四种基因型HBV克隆载体,分析和比较它们在HepG2和Huh7细胞中的表达和复制。方法利用分子生物学技术将连接在pUC19上的A、B、C和D四种基因型HBV全序列连接到改建的pIRES2-EGFP真核表达载体中;脂质体转染法将构建的重组载体瞬时转染入HepG2和Huh7细胞中;利用载体所携带的绿色荧光蛋白在显微镜下观察转染效率,并利用ELISA和荧光定量PCR技术检测细胞培养上清中HBsAg、HBeAg和HBV DNA的表达水平。结果经鉴定,重组HBV真核表达载体构建成功,在HepG2和Huh7细胞中转染效率在50%～60%之间,四种基因型HBV在宿主细胞中均有稳定的复制和表达,但不同基因型在表达水平上存在差异。结论成功构建A、B、C和D四种基因型HBV真核表达载体,各基因型在HepG2和Huh7肝癌细胞系中均表现出较高的转染效率,为后续HBV及其基因型研究提供了实验载体。Objective To construct A,B,C and D four kinds of HBV genotypes cloning vectors,and to analyze and compare their expression and replication in HepG2 and Huh7 cells.Methods Whole sequences of A,B,C and D genotype HBV in pUC19 were connected with alternative pIRES2-EGFP eukaryotic vector using molecular biology techniques,respectively.The recombinant vectors were transiently transfected into HepG2 and Huh7 cells by liposome transfection. In order to determine the transfection efficiency,green fluorescent protein was observed under the microscope. Meanwhile,ELISA and fluorescence quantitative PCR were used to detect the expression levels of HBsAg,HBeAg and HBV DNA in the supernatant. Results HBV recombinant eukaryotic expression vector was successfully constructed,and the transfected efficiency was about 50% ～ 60% in HepG2 and Huh7 cells. Four kinds of HBV genotypes replicated and expressed stablely in the host cells,but there was difference on the expression levels of different genotypes. Conclusion Eukaryotic expression vector with A,B,C and D four kinds of HBV genotypes are successfully constructed,and show high transfection efficiency in HepG2 and Huh7 liver cancer cells. The results provide experimental support for the subsequent HBV and genotype research.

关 键 词：肝癌 HBV 基因型 载体 

分 类 号：R373.21[医药卫生—病原生物学]