结核分枝杆菌Hsp65与IL-2融合蛋白稳定表达细胞系的建立  

Establishment of a stable cell line expressing the fusion protein of Mycobacterium tuberculosis Hsp65 and IL-2

作　　者：师长宏[1] 张海[1] 席丽[1] 王晓武[1] 王丽梅[2] 赵勇[1] 

机构地区：[1]第四军医大学实验动物研究中心,西安710032 [2]第四军医大学基础部微生物学教研室,西安710032

出　　处：《中国人兽共患病学报》2008年第8期718-721,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(30400381);陕西省自然科学基金项目(2006C218)

摘　　要：目的构建了表达结核分枝杆菌Hsp65与IL-2融合蛋白的真核表达载体;获得重组质粒稳定转染的P815细胞系。方法将Hsp65与IL-2基因分别克隆至真核表达载体pcDNA3.1,转化DH5α菌株感受态细胞,获得阳性重组质粒Hsp65-IL-2-pcDNA3.1。在阳离子聚合物作用下,重组质粒转染与BALB/c遗传背景一致的P815(H-2d)细胞。G418筛选抗性克隆,免疫荧光检测融合蛋白的表达。结果真核质粒转染的阳性克隆细胞,经RT-PCR检测到Hsp65与IL-2融合蛋白mRNA水平的表达,分别用抗Hsp65和IL-2的单抗进行间接免疫荧光检测,可在转染重组质粒的P815细胞膜上观察到较强的绿色荧光。结论获得表达Hsp65与IL-2融合蛋白的稳定细胞系,为其CTL研究提供了合适的靶细胞。The genes encoding Mycobacterium tuberculosis Hsp65 and IL-2 were cloned into eukaryotic expression vector pcDNA3. 1 and transformed to E. coli DH5a. Then,the positive recombinant plasmid Hsp65-IL-2-pcDNA 3. 1 was selected and transfected to P815 cells（H-2^d）whose genetic background was identical to that of BALB/c mice, In this way,one positive cell clone was selected by G418, and the specific mRNA of the fusion protein was detected by RT-PCR. By immunofluorescence teehnique（IFT）the P815 cells transfected with recombinant plasmid showed strong fluorescence,indicating the expression of fusion protein in this cell. It is apparent that a stable cell line expressing M. tuberculosis Hsp65 and IL-2 proteins in P915 cells is obtained,which may be used as the optimal target cell for CTL experiment.

关 键 词：结核分枝杆菌 真核表达 热休克蛋白65 白细胞介素2 融合蛋白 

分 类 号：R378.91[医药卫生—病原生物学]