1.1倍体全长HBV基因组克隆转染细胞系的建立.docVIP

1.1倍体全长HBV基因组克隆转染细胞系的建立 罗强1，苏怀彬1，梁盼盼1，袁作为1，张文露1，黄爱龙1，胡接力1(（1重庆医科大学感染性疾病分子生物学教育部重点实验室，400016 重庆） 摘 要：目的 构建pneo-CH9/3091真核表达质粒，并建立其稳定表达的HepG2细胞系。 方法 质粒pCH9/3091包含HBV1.1倍体和CMV启动子，将PCR扩增出的带有新霉素（neo）抗性的基因片段插入pCH9/3091构建成重组质粒pneo-CH9/3091。重组质粒经酶切，测序验证正确后，通过脂质体介导转入人肝癌细胞HepG2。G418筛选后，利用酶联免疫吸附法（ELISA）检测所得单克隆细胞培养上清液的HBV表面抗原（HBsAg）和e抗原（HBeAg），PCR检测细胞上清液的病毒颗粒，Western blotting检测细胞内HBV核心蛋白（HBcAg）的表达，以及Southern blotting检测细胞内核心颗粒HBV DNA的复制情况。 结果 有3株细胞系培养上清HBsAg和HBeAg检测呈现阳性，且HBV C区片段可通过PCR扩增出来，Western botting结果提示只有细胞株HepG2-H7可以表达HBV核心蛋白。Southern blotting结果说明此细胞株基因组中有HBV基因组的稳定整合，并且能在细胞内检测出HBV DNA复制中间体。 结论 成功构建一株HBV稳定整合细胞株HepG2-H7，能持续产生HBV病毒颗粒。 关键词：乙肝病毒；HepG2细胞；稳定细胞株 Eestablishment of the stable cell line with stably transfected 1.1-unit-length HBV genome Luo Qiang，Su Huaibin，Liang Panpan，Yuan Zuowei，Zhang Wenlu，Huang Ailong，Hu Jieli．（Key laboratory of Molecular Biology on Infectious Diseases，ministry of education，Chongqing Medical University，Chongqing 40016，China） Objective To construct the eukaryotic expression plasmid pneo-CH9/3091 and establish a stable cell line based on HepG2 cells with the plasmid. Methods Plasmid pCH9/3091, which contains an HBV 1.1-unit-length genome and CMV promoter, was used to construct recombinant plasmid pneo-CH9/3091 by the insertion of fragment carrying with neo resistance gene obtained by PCR. The recombinant plasmid was stably transfected into HepG2 cells. Following G418 screening, HBsAg and HBeAg secretion in the supernatant of the cell lines were tested by ELISA; HBV particles in the cell medium were determined by PCR; Western blotting was employed to analyze the expression of HBV core protein in the cell line，and HBV replication was checked by Southern blotting． Result Three cell lines can secret HBsAg and HBeAg, and all their culture mediums gave a effective amplification of HBV core gene by PCR. Western blotting showed HBV core protein was expressed exclusively in the HepG2-H7 cell line. Furthermore, Southern blotting indicated that HBV genom