结核分枝杆菌分泌蛋白EspB 在RAW264.7细胞中的稳定表达

目的]建立能够稳定表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分泌蛋白EspB(Rv3881c)的RAW264.7细胞系,为研究EspB蛋白在调控巨噬细胞功能中所起的作用提供科学依据.方法]首先成功构建的重组质粒pEGFP-C1-EspB,然后将重组载体pEGFP-C1-EspB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-EspB融合蛋白以及EGFP的细胞系,并通过RT-PCR、荧光显微镜及Western blot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]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,成功获得了能够稳定表达EGFP-EspB融合蛋白以及EGFP的细胞系.结论]本试验利用脂质体介导的方法,将构建的重组载体pEGFP-C1-EspB和空载体pEGFP-C1转染至Raw264.7细胞系中,获得了稳定表达EGFP-EspB融合蛋白的巨噬细胞系,为阐明EspB分泌蛋白在调控巨噬细胞功能中所起的作用以及EspB与巨噬细胞蛋白之间的相互作用提供了研究平台.

Stable expression of Mycobacterium tuberculosis protein EspB in RAW264. 7

Objective] We established a cell line stably expressing Mycobacterium tuberculosis secretory protein EspB in order to provide evidences for studying EspB in modulating the functions of macrophage.Methods] The recombinant plasmid pEGFP-C1-EspB was first constructed,then RAW264.7 cell was transfected with pEGFP-C1-EspB and pEGFP-C1 by liposome respectively.After screening with a high level of G418,the macrophage cell lines that stably expressed EGFP-EspB fusion protein or EGFP were established.The gene and ...