稳定表达结核分枝杆菌ESAT-6蛋白的RAW264.7细胞系的建立

Establishment of RAW264.7 cell line stably expressing Mycobacterium tuberculosis protein ESAT-6

为研究结核分枝杆菌Mycobacterium tuberculosis分泌蛋白ESAT-6(Early secreted antigenic target of 6 kDa)对巨噬细胞相关功能的影响,将正确构建的重组质粒pEGFP-C1-ESAT-6和空载体pEGFP-C1以脂质体介导的方法转染至小鼠巨噬细胞RAW264.7中,经过G418筛选后建立稳定表达EGFP-ESAT6融合蛋白以及EGFP的细胞系,并通过RT-PCR、荧光显微镜及Western blotting方法,在基因和蛋白两个水平对所建立的稳转细胞系进行鉴定。结果证实EGFP-ESAT6融合基因成功整合入RAW264.7细胞基因组并能够稳定表达,为后续的ESAT-6调控巨噬细胞机理研究提供了平台。

For studying the effects of Mycobacterium tuberculosis secretory protein ESAT-6 on the related functions of macrophages,RAW264.7 cells were transfected with pEGFP-C1-ESAT-6 and pEGFP-C1 by liposome respectively.After screening with a high level of G418,the macrophage cell lines that stably expressed EGFP-ESAT-6 fusion protein or EGFP were established.The gene and protein expression levels were further analyzed by RT-PCR,fluorescence microscopy and Western blotting.The results indicated that the EGFP-ESAT6 fusion gene was integrated into the chromosome and the protein could be stably expressed in the selected macrophage cell line.These results gave us a tool for the future study in the mechanisms of ESAT-6 protein in modulating the macrophage cells.