结核分枝杆菌Hsp65与IL-2融合蛋白稳定表达细胞系的建立

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。

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

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 technique(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.