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【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】 To establish a RAW264.7 cell line capable of stably expressing EspB (Mycobacterium tuberculosis) secreted protein EspB (Rv3881c), and provide a scientific basis for studying the role of EspB in the regulation of macrophage function. 【Method】 The recombinant plasmid pEGFP-C1-EspB was successfully constructed. The recombinant vector pEGFP-C1-EspB and empty vector pEGFP-C1 were transfected into mouse macrophage RAW264.7 by liposome-mediated method After G418 screening, we established a cell line stably expressing EGFP-EspB fusion protein and EGFP. The established stable cell lines were identified by RT-PCR, fluorescence microscopy and Western blot. 【Results】 EGFP-ESAT6 fusion gene was successfully integrated into the genome of RAW264.7 cells and stably expressed. The cell line stably expressing EGFP-EspB fusion protein and EGFP was successfully obtained. 【Conclusion】 In this study, the recombinant plasmid pEGFP-C1-EspB and empty vector pEGFP-C1 were transfected into Raw264.7 cell line by liposome-mediated method. The results showed that EGFP-EspB fusion protein Macrophages provide a research platform to elucidate the role of EspB secreted proteins in the regulation of macrophage function and the interaction between EspB and macrophage proteins.