结核分枝杆菌分泌蛋白EspB在RAW264.7细胞中的稳定表达  

Stable expression of Mycobacterium tuberculosis protein EspB in RAW264.7

作　　者：李浩[1] 殷瑛[1] 董大勇[1] 张军[1] 付玲[1] 任军[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：《微生物学报》2011年第7期984-990,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30972772;81025018)~~

摘　　要：【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。[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 protein expression levels were further analyzed by RT-PCR,fluorescence microscopy and western blot.[Results] The EGFP-EspB fusion gene was integrated into the chromosome and the protein was stably expressed in the selected macrophage cell line.The macrophage cell lines that stably expressed EGFP-EspB fusion protein or EGFP were established.[Conclusion] These results gave us a tool for the future study in the effects of EspB protein in modulating the functions of macrophage and its interaction with other molecules of macrophage.

关 键 词：EspB EGFP RAW264.7 稳定转染 表达 

分 类 号：R378[医药卫生—病原生物学]