丙型肝炎病毒NS5A基因的克隆以及在原核和昆虫细胞中表达的研究  

Study on the cloning of HCV NS5A gene and its expression in prokaryotic and insect cells

作　　者：梁昌镛[1] He Bin 胡志红[1] 王汉中[1] 陈新文[1] 

机构地区：[1]中国科学院,武汉病毒研究所,无脊椎动物病毒学联合开放实验室,武汉,430071 [2]美国伊利诺斯大学芝加哥医学院,微生物及免疫系,芝加哥美国

出　　处：《华中师范大学学报（自然科学版）》2003年第2期236-241,共6页Journal of Central China Normal University：Natural Sciences

基　　金：中国科学院知识创新工程项目(kscx2-sw-302);武汉市科技计划项目(20027006144).

摘　　要：克隆了丙型肝炎病毒的NS5A基因,并在细菌和昆虫两种不同的系统中成功地进行了该基因的表达.利用PCR方法获得了NS5A完整的编码区并克隆到原核表达载体pGEX-KG上,在大肠杆菌中诱导表达了78kDa的NS5A与GST的融合蛋白.同时发现,融合蛋白被部分切割为50kDa的NS5A和28kDa的GST.在原核细胞中表达的78kDa和50kDa两种蛋白均具有较高的免疫原性,通过免疫家兔获得了高特异性的兔抗血清.另外,利用两种昆虫表达系统,即AcMN-PV和HaSNPV的Bac-to-Bac系统对NS5A进行了表达,表达产物为58kDa.NS5A gene of hepatitis C virus is very important to HCV infection and its function has not been completely revealed. In this study, the open reading frame of HCV NS5A was amplified by PCR, cloned and expressed both in prokaryotic and insect cells. In E.coli, by using pGEXKG, NS5A was produced mainly as a GST fusion protein with the size of 78 kDa, while two extra protein bands with the sizes of 50 kDa and 28 kDa were also found. The 78 kDa and 50 kDa proteins were purified and specific antibodies were generated. Western blot analysis indicated that the 50 kDa product was NS5A, which was naturally digested from the 78 kDa fusion protein. The NS5A was expressed in two different baculovirus/insect cell expression system: AcMNPV/SF21 and HaSMPV/HzAM1. In both systems, a single band with the size of 58 kDa was detected by western blot analysis using the NS5A antibody.

关 键 词：丙型肝炎病毒 NS5A基因 基因克隆 原核细胞 昆虫细胞 基因表达 免疫原性 

分 类 号：R373.21[医药卫生—病原生物学] R394[医药卫生—基础医学]