编码人B7-2胞外区cDNA的克隆、表达及生物活性鉴定  

Cloning, Expression and Biological Activity Identification of a cDNA Encoding the Extracellular Region of Human B7-2

作　　者：袁志宏[1] 奚永志[1] 孔繁华[1] 张惠丽[1] 刘楠[1] 梁飞[1] 

机构地区：[1]军事医学科学院附属医院免疫学研究室及国家生物医学分析中心免疫分析实验室,北京100039

出　　处：《中国实验血液学杂志》2002年第6期508-511,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目编号3990 0 187与 30 2 0 0 111;军队"九五"医学卫生科研基金资助项目编号 0 985

摘　　要：B7 2分子是共刺激信号系统B7/CD2 8/CTLA 4中的重要成员之一 ,为了更深入地探讨B7 2分子在调控T细胞增殖、分化及相关信号传导过程中的作用 ,本研究通过聚合酶链式反应 (PCR)扩增编码人B7 2分子胞外区的cDNA ,测序后定向插入多个原核表达载体并诱导目的蛋白表达 ,用Western印迹和MTT鉴定生物活性。结果表明 :通过pGEX 4T 2载体实现了在宿主菌BL 2 1 (DE3) codenplus RIL中较高水平的表达 ,蛋白表达量为 2 0 %。经Western印迹和MTT鉴定 ,所表达及初步纯化的B7 2胞外区重组蛋白能与抗人B7 2单抗发生特异性结合 ;在抗人CD3单抗的协同下 ,B7 2胞外区重组蛋白能有效地促进外周血单核细胞的增殖。结论 :B7 2胞外区重组蛋白的获得为进一步研究B7s As one important member of B7/CD28/CTLA 4 costimulatory signal pathway, B7 2 molecule plays a critical role in regulating T cell response. In order to further explore its effects on regulation of T cell activation, proliferation and associated signal pathways, the cDNA encoding extracellular region of human B7 2 was amplified via PCR and subcloned into some prokaryotic expression vectors to express target protein in host strains. The expressed protein was identified with Western blot and MTT. Results showed that after screening, the expression level of the protein of interest attained the yield of over 20% total bacterial protein by using pGEX 4T 2 vector and E.coli BL21(DE3) CodonPlus RIL host cells. The recombinant protein could specially react with B7 2 McAb and could stimulate T cell proliferation combined with anti CD3 antibody. In conclusion, the recombinant protein was bioactive,therefore the study will make it possible for the research of relationship between B7 2 structure and its function.

关 键 词：编码 人B7-2胞外区 CDNA 生物活性 鉴定 共刺激信号系统 原核表达 基因克隆 

分 类 号：Q753[生物学—分子生物学] Q785