7型庚型肝炎病毒包膜糖蛋白E2基因的克隆及原核表达分析  

Cloning,Sequence Analysis and Expression of Recombinant E2 Protein of GB Virus C Genotype 7

作　　者：杨晓钰[1] 赵跃[1] 冯悦[1] 夏雪山[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500

出　　处：《病毒学报》2016年第5期545-550,共6页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:81360247)

摘　　要：为了探究7型庚型肝炎病毒E2基因编码蛋白作为ELISA试剂盒研发所需检测抗原的可能性,建立更为可靠的GBV-C检测方法,本研究应用在线软件对GBV-C E2基因序列的编码区进行生物信息学分析,预测了E2基因编码蛋白的抗原表位、空间结构及线性B细胞表位等;通过逆转录PCR从7型GBV-C病毒中克隆出E2基因片段,将其克隆到pET-32a载体上,重组载体pET-32a-E2转化大肠杆菌BL21后诱导表达,用12%SDS-PAGE检测,结果重组蛋白主要以包涵体形式存在,其分子量大小约为55kD,利用His标签抗体对重组蛋白进行Western-blotting验证。结果表明GBV-C E2蛋白有多个抗原表位点,克隆的E2基因序列长度为945bp,重组蛋白以包涵体形式表达,其分子量大小与预期一致,此研究为GBV-C检测试剂盒的研制工作奠定了基础。The purpose of this study was to explore the potential of the GB virus C（GBV-C）genotype 7E2 protein as a detection antigen for ELISA kit development.In this study,analyses of antigen epitopes,space structures and the linear B cell epitopes from the GBV-C genotype 7E2 protein were performed using an online analysis program.To establish a more reliable detection method for GBV-C studies,a 945 bp gene fragment from GBV-C E2 was amplified by RT-PCR and ligated into the pET-32 aprokaryotic expression vector,which was then transformed into E.coli BL21 cells for protein expression.A protein with a molecular weight of 55 kDa was detected by 12% SDS-PAGE.The protein was found in inclusion bodies,and the His-tagged protein was detected by western blotting.The results showed that the cloned E2 gene sequence was 945 bp,and that the GBV-C E2 protein sequence had multiple antigenic epitopes.The recombinant protein formed inclusion bodies,which was consistent with expectations.These findings may provide the foundation for the development of a GBV-C detection kit.

关 键 词：庚型肝炎病毒 包膜糖蛋白 克隆 表达分析 

分 类 号：R373.2[医药卫生—病原生物学]