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

作　　者：李浩[1] 殷瑛[1] 董大勇[1] 张军[1] 付玲[1] 蔡晨光[1] 王猛[1] 徐俊杰[1] 陈薇[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物工程学报》2011年第9期1390-1396,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30972772)资助~~

摘　　要：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.

关 键 词：ESAT-6 RAW264.7 稳定转染 表达 

分 类 号：Q78[生物学—分子生物学]