结核分枝杆菌ESAT-6与EspB蛋白调控巨噬细胞功能的初步研究

发布时间：2018-05-12 15:32

本文选题：结核分枝杆菌 + 表达　；参考：《中国人民解放军军事医学科学院》2011年硕士论文

【摘要】：ESX-1分泌系统是结核分枝杆菌中存在的一种特殊的蛋白分泌系统,对细菌的毒力非常重要,又被称为VII型分泌系统。已有至少5种蛋白被证实由ESX-1分泌系统所分泌,即ESAT-6、CFP-10、EspA、EspB和EspR。ESAT-6是最早发现的ESX-1分泌蛋白之一,但是对它的功能还存在争论:一方面,它是重要的T细胞抗原,是候选疫苗的重要组分,被广泛用于新型结核疫苗的设计以及结核的诊断抗原;另一方面,许多研究显示它会影响巨噬细胞和树突状细胞的功能,与细菌的毒力相关。另一种新发现的分泌蛋白EspB能够和ESAT-6、CFP-10共分泌,这种共分泌机制对结核菌在巨噬细胞内的生长非常重要,并且能抑制巨噬细胞吞噬体的成熟,但EspB本身对巨噬细胞是否存在直接的调控还不清楚。本研究运用分子生物学、细胞生物学、免疫学等技术方法,对ESX-1分泌蛋白ESAT-6和EspB对巨噬细胞功能的调控作用进行了初步研究。本研究首先从结核分枝杆菌H37Rv基因组中扩增出ESAT-6(Rv3875)与EspB(Rv3881c)基因,克隆入pET21a(+)载体。分别将成功构建的pET21a(+)-ESAT-6以及pET21a(+)-EspB重组质粒转化入感受态BL21(DE3)中,经诱导表达后,使用SDS-PAGE和Western Blot鉴定。超声破碎后发现目的蛋白以大量可溶性形式存在,经过Ni-NTA柱和DEAE-Sepharose柱纯化,获得纯度约95%的重组ESAT-6与EspB蛋白。将重组蛋白和RAW264.7细胞共孵育后,使用免疫荧光技术发现ESAT-6能够直接和RAW264.7的细胞膜结合。为了方便后续研究,本实验同时完成了对EspB蛋白C端缺失突变体(EspB-N)的表达和纯化。将纯化的ESAT-6蛋白和EspB蛋白分别免疫小鼠。采用杂交瘤技术,获得了12株针对ESAT-6以及11株针对EspB的鼠mAb杂交瘤细胞株,分别对其中5株针对ESAT-6抗原的杂交瘤细胞株和5株针对EspB抗原的杂交瘤细胞株进行了小鼠腹水的制备及相关鉴定,并用Protein G亲和层析株进行了纯化。为研究ESAT-6以及EspB蛋白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、pEGFP-C1-EspB以及空载体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筛选和克隆化后分别建立了稳定表达EGFP-ESAT6融合蛋白、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,从基因和蛋白两个水平对所建立的稳转细胞系进行鉴定。结果表明EGFP-ESAT-6以及EGFP-EspB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以及EspB调控巨噬细胞机理研究提供了平台。为探索ESAT-6蛋白与EspB蛋白对巨噬细胞吞噬功能的影响,将荧光微球和RAW264.7细胞在37℃,5%CO2条件下孵育2 h后,使用流式细胞仪进行检测。数据分析结果显示细胞内表达的ESAT-6能够显著增强RAW264.7巨噬细胞的吞噬能力。相反,未观察到细胞内表达的EspB蛋白对巨噬细胞的吞噬能力的影响。为了验证流式分析结果,采用共聚焦显微镜技术观察巨噬细胞吞噬能力改变并进行了定量分析,所获得的结论与流式分析结果一致。本实验还观察了培养48 h后的稳定表达ESAT-6蛋白的巨噬细胞系的凋亡状况,流式结果分析发现细胞内表达ESAT-6蛋白能够显著的增加巨噬细胞的凋亡。综上所述,本研究成功表达和纯化了结核分枝杆菌ESAT-6和EspB重组蛋白,使用免疫荧光技术发现ESAT-6蛋白能够直接和RAW264.7的细胞膜结合;将纯化的ESAT-6蛋白和EspB蛋白用于抗体制备,获得了多株针对ESAT-6蛋白的单克隆抗体和针对EspB的单克隆抗体;分别建立了能够稳定表达ESAT-6和EspB蛋白的巨噬细胞系;在所建立细胞系的基础上,发现ESAT-6能够显著增强巨噬细胞的吞噬能力,EspB蛋白对巨噬细胞的吞噬能力没有影响,同时ESAT-6能够诱导巨噬细胞的凋亡;这些结果的获得进一步研究结核分枝杆菌和宿主之间的相互作用以及分泌蛋白调控巨噬细胞的分子机理提供了条件。
[Abstract]:The ESX-1 secretory system is a special protein secreting system in Mycobacterium tuberculosis. It is also known as the VII secretory system. At least 5 proteins have been secreted by the ESX-1 secretory system. That is, ESAT-6, CFP-10, EspA, EspB and EspR.ESAT-6 are one of the earliest found ESX-1 secreting proteins. Its function is still controversial: on the one hand, it is an important T cell antigen, an important component of the candidate vaccine, widely used in the design of a new type of tuberculosis vaccine and the diagnostic antigen of tuberculosis; on the other hand, many studies have shown that it affects the function of macrophages and dendritic cells and is associated with the virulence of bacteria. Another new discovery The secretory protein EspB is co secreted with ESAT-6 and CFP-10, which is very important for the growth of Mycobacterium tuberculosis in macrophages and can inhibit the maturation of macrophage phagocytes. But it is not clear whether EspB itself has direct regulation of macrophages. This study uses molecular biology, cell biology, immunology and other techniques. The regulation of ESX-1 secreted protein ESAT-6 and EspB on macrophage function was preliminarily studied.
In this study, ESAT-6 (Rv3875) and EspB (Rv3881c) genes were amplified from the H37Rv genome of Mycobacterium tuberculosis and were cloned into pET21a (+) carriers. The recombinant plasmid of pET21a (+) -ESAT-6 and pET21a (+) -EspB was transformed into BL21 (DE3) respectively. It was found that the target protein existed in a large amount of soluble form and purified by Ni-NTA column and DEAE-Sepharose column to obtain the recombinant ESAT-6 and EspB protein with a purity of about 95%. After reincubating the recombinant protein and RAW264.7 cells, the immunofluorescence technique was used to find that ESAT-6 could be directly associated with the cell membrane of RAW264.7. In order to facilitate follow-up study, the experiment was the same. The expression and purification of the C terminal deletion mutant (EspB-N) of EspB protein was completed. The purified ESAT-6 protein and EspB protein were immunized in mice respectively. By hybridoma, 12 mouse mAb hybridoma cell lines against ESAT-6 and 11 strains of EspB were obtained, and 5 of these hybridoma cells and 5 strains of ESAT-6 antigen were targeted to EspB respectively. The hybridoma cell lines of antigen were prepared and identified by mouse ascites and purified by Protein G affinity chromatography.
In order to study the effect of ESAT-6 and EspB protein on macrophage related functions, the recombinant plasmid pEGFP-C1-ESAT-6, pEGFP-C1-EspB and pEGFP-C1 were transfected into RAW264.7 of mouse macrophage in the liposome. After G418 screening and cloning, the stable expression of EGFP-ESAT6 fusion protein was established, EG, respectively. FP-EspB fusion protein and EGFP cell lines were identified by RT-PCR, fluorescence microscopy and Western Blot methods. The results showed that the EGFP-ESAT-6 and EGFP-EspB fusion genes were successfully integrated into the genome of RAW264.7 cells and could be expressed steadily, for the subsequent ESAT-6. And EspB provides a platform for regulating the mechanism of macrophages.
In order to explore the effect of ESAT-6 protein and EspB protein on phagocytosis of macrophages, fluorescence microspheres and RAW264.7 cells were incubated for 2 h at 37 and 5%CO2, and flow cytometry was used to detect the phagocytosis. The results of data analysis showed that the ESAT-6 expressed in the cells could significantly enhance the phagocytosis of RAW264.7 macrophages. On the contrary, no fine observation was observed. The effect of EspB protein expressed in the cell on phagocytosis of macrophages. In order to verify the results of flow analysis, confocal microscopy was used to observe the change of macrophage phagocytosis and quantitative analysis. The results obtained were in accordance with the results of flow analysis. The stable expression of ESAT-6 protein after 48 h was also observed. The apoptotic status of macrophages was analyzed by flow cytometry. It was found that the expression of ESAT-6 protein in the cells could significantly increase the apoptosis of macrophages.
To sum up, the recombinant protein of Mycobacterium tuberculosis ESAT-6 and EspB was successfully expressed and purified. Using immunofluorescence technique, it was found that ESAT-6 protein could be directly combined with the cell membrane of RAW264.7, and the purified ESAT-6 protein and EspB protein were used for antibody preparation, and a number of monoclonal antibodies against ESAT-6 protein and EspB were obtained. A monoclonal antibody was used to establish a macrophage system capable of stably expressing ESAT-6 and EspB protein. On the basis of the established cell lines, it was found that ESAT-6 could significantly enhance macrophage phagocytosis. EspB protein had no effect on macrophage phagocytosis, and ESAT-6 could induce apoptosis of macrophages; these results were obtained. Further studies on the interaction between Mycobacterium tuberculosis and host, as well as the molecular mechanism of secreting proteins regulating macrophages, provide the conditions.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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