基于核酶自剪切机制稳定分泌丙型肝炎病毒的细胞模型  

Construction of HCV-producing cell model based on self-cleaving ribozyme

作　　者：王盛[1] 安小平[1] 米志强[1] 刘大斌[1] 张宝中[1] 闾军[2] 周育森[1] 童贻刚[1] 

机构地区：[1]北京军事医学科学院微生物流行病研究所病原微生物国家重点实验室,100071 [2]首都医科大学附属北京佑安医院

出　　处：《中华肝脏病杂志》2010年第6期437-439,共3页Chinese Journal of Hepatology

基　　金：基金项目：国家高科技研究发展计划（863计划）（2007AA02Z151,2009AA02Z111）

摘　　要：目的 利用核酶自剪切机制建立稳定分泌HCV的细胞膜型.方法 以HCV感染性克隆的全基因组cDNA重组质粒为基础,在基因组两侧引入核酶序列,构建重组质粒pJFH1-核酶,并转染入HepG2细胞,加入G418溶液筛选整合有该质粒的细胞克隆.用荧光定量PCR、免疫荧光、Westrn blot、透射电镜等技术挑选稳定分泌HCV的单细胞克隆.结果 成功筛选到稳定分泌HCV的单细胞克隆,该细胞克隆能够有效产生HCV相关蛋白,上清液中HCV滴度达到1×107拷贝/ml,电子显微镜观察HCV直径为55 nm,上清液中病毒滴度随干扰素α浓度的升高而降低. 结论 利用核酶自剪切机制可建立稳定分泌HCV的细胞模型,该模型可用于抗HCV药物的筛选.Abstract：Objective To construct a stable HCV-producing cell model for anti-HCV drug research. Methods The HCV-ribozyme recombinant plasmid pJFHl-Rbz was constructed to generate the exact 5＇ and 3＇ ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy. Results HCV core protein was detected in die screened cell clone, and the level of HCV RNA was up to 1 ×107 in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN α. Conclusions We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.

关 键 词：肝炎病毒 丙型 基因 转染 

分 类 号：R3[医药卫生—基础医学]