猪脑心肌炎病毒非结构蛋白3AB的原核表达及其单克隆抗体的研制  

Expression of NSP 3AB Gene of Encephalomyocarditis Virus(EMCV)in E.coli and Development of Monoclonal Antibodies Against 3AB Protein

作　　者：沈芳[1] 姜平[1] 李玉峰[1] 李军星[1] 王海燕[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,南京210095

出　　处：《中国生物工程杂志》2008年第9期27-31,共5页China Biotechnology

基　　金：国家"863"计划(2006AA10A203);教育部重点项目(104101)资助项目

摘　　要：目的:利用原核表达系统表达猪脑心肌炎病毒(EMCV)非结构蛋白3AB,并通过杂交瘤细胞技术制备其单克隆抗体,为相关研究工作奠定基础。方法:利用大肠杆菌系统表达具有良好抗原性的重组3AB蛋白,经包涵体纯化后免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,间接ELISA筛选阳性的杂交瘤细胞,并结合免疫荧光(IFA)和Western blot对抗体的特异性进行鉴定。结果:经间接ELISA筛选阳性的杂交瘤细胞,获得1株能稳定分泌抗3AB蛋白抗体的杂交瘤细胞株,将其命名为2D12,其亚类测定为IgG1/κ。Westernblot和间接免疫荧光试验证明该单抗能特异性识别3AB蛋白。结论:成功获得了针对EMCV-3AB的特异性单抗,为进一步研究猪脑心肌炎病毒非结构蛋白3AB的结构与功能及临床诊断试剂的研发奠定必要的物质基础。Objective： To express the EMCV 3AB gene by prokaryotic expression systerm, and prepare monoclonal antibodies against it. Method： NSP 3AB gene of Encephalomyocarditis virus （EMCV） was amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and a recombinant protein 3AB with high antigenicity was expressed in E. coli. Balb/c mice were immunized by purified recombinant 3AB protein of inclusion-body, and the splenocytes of the immunized mice were fused with murine myeloma cells to produce hybridoma cell line. Results： After subcloning by 3 times, one strain of hybridoma cell line steadily secreting antibodies of 3AB protein was obtained, named 2D12. The McAb belongs to IgG1/K. The McAb and was confirmed by indirect immunofluoreseent assay （IFA） and Western blot. Conclusion： These results can provide a potential value for structural and functional studies of EMCV-3AB and early diagnosis of Encephalomyocarditis virus infection.

关 键 词：脑心肌炎病毒 非结构蛋白 原核表达 单克隆抗体 

分 类 号：Q789[生物学—分子生物学]