中国人共刺激信号抑制配体表达载体的构建及在大肠杆菌DH5α中的表达  

Construction of the suppressor ligand expression vector of Chinese costimulatory signal and its expression in E. coli DH5α

作　　者：朱乃硕[1] 陆敏依[1] 于善谦[1] 朱亚军[1] 李勇[1] 

机构地区：[1]复旦大学生命科学院病毒及分子免疫实验室,上海200433

出　　处：《中国免疫学杂志》2001年第10期559-562,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金;上海市科委重点项目资助

摘　　要：目的 :构建CTLA 4基因表达载体并在大肠杆菌DH5α中表达CTLA 4蛋白。方法 :用PCR方法获得中国人CTLA 4基因第二外显子 ,构建重组表达质粒pPBCTLA ,转化大肠杆菌DH5α ,以PCR和RFLP方法筛选阳性克隆 ,以免疫印迹技术检测表达产物。结果 :阳性重组子在DH5α中经温度诱导可表达CTLA 4蛋白 ,分子量与理论值相符 (约 12kD)。进一步分析表明 ,CTLA 4蛋白主要以包涵体形式存在。Western blot结果证实 ,12kD的表达条带可与标准羊抗人CTLA 4IgG起特异反应。淋巴细胞转化实验结果表明该蛋白具有明显的免疫抑制作用。结论 :用基因工程技术获得中国人CTLA 4基因表达蛋白 ,为进一步研究CTLA 4的生物学功能及研制抗CTLA 4抗体奠定了基础。Objective:To construct CTLA 4 DNA expression vector and express CTLA 4 protein in E coli DH5α Methods:The gene coding the second domain of cytotoxic T lymphocyte associated antigen 4(CTLA 4) was amplified by PCR using human genomic DNA as template Then amplified CTLA 4 gene that coding outer membrane soluble fragment was inserted into pBV220 to construct expression vector pPBCTLA E coli DH5α was transformed by the recombinant plasmid Results:The expression of CTLA 4 protein was obtained by heat induction Tricine SDS PAGE analysis showed that the recombinant E coli could express a 12 kD protein Western blot analysis showed that anti CTLA 4 McAb specifically bound to the expression product of 12 kD band The functional analysis of the protein showed that the purified protein had the effect on immune inhibition significantly Conclusion:The recombinant human CTLA 4 was obtained and it was useful for the study of biological functions of CTLA 4 and for further producing anti CTLA 4 antibody

关 键 词：CTLA-4 基因表达 淋巴细胞转化实验 

分 类 号：R392.2[医药卫生—免疫学]