【作者】 李浩；

【导师】 陈薇； 徐俊杰；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2011， 硕士

【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。更多还原

【Abstract】 ESX-1 secretion system is a special protein secretion system in M. tuberculosis and critical for its virulence. ESX-1 secretion system is also named Type VII secretion system. More than five proteins have been confirmed to be secreted by ESX-1 secretion system including ESAT-6、CFP-10、EspA、EspB and EspR. ESAT-6 is one of secreted proteins found earliest, however, there is still controversy about its functions.Firstly, it is an important T-cell antigen and has protective effects on animal model infected by Mtb. Moreover, it is an important compotent of candidate vaccines and is extensively applied to the design of new Mtb vaccines and diagnostic antigen of Mtb. Secondly, ESAT-6 can affect the functions of macrophage and dentritic cell and is related to the virulence of Mtb. There is another new protein named EspB which can be co-secreted with ESAT-6 and CFP-10. The co-secretion of EspB and ESAT-6 is important for Mtb intracellular growth in macrophages and is required for inhibiting phagosome maturation. In this study, we make full use of the techniques and methods of molecular biology, Cellular Biology and Immunology to preliminaryly investigate ESX-1 system secreted proteins ESAT-6 and EspB in modulating macrophage functions.In this research, ESAT-6 and EspB genes were first seperately amplified by PCR from the genome of Mycobacterium tuberculosis H37Rv, and then cloned into pET21a(+) plasmid. The recombinant plasmids pET21a(+)-ESAT-6 and pET21a(+)-EspB that were successfully constructed were individually transformed into E.coli BL21(DE3). After induced with IPTG, the expressed recombinant proteins were confirmed by SDS-PAGE and Western blot. This vectors yielded satisfactory levels of recombinant proteins expressed as soluble proteins in E. coli. After ultrasonication, the recombinant ESAT-6 protein or EspB protein was firstly purified by a column packed with Ni-NTA Resin and then a column packed with DEAE-SepharoseTM Fast Flow matrix. The purity of the purified proteins were about 95%. The purified ESAT-6 protein was incubated with RAW264.7, and the result got by Immunofluorescence showed that ESAT-6 could directly bind to the macrophage membrane. In order to faciliate the following research work, The deleted C terminus mutant of EspB was also expressed and purified. The purified proteins ESAT-6 and EspB were seperately used to immunize BALB/c mice. Twelve stably anti-ESAT-6 specific mAb cell strains and eleven stably anti-EspB apecific mAb cell strains were obtained by hybridoma cell fusion technique.The five strains of anti-ESAT-6 cell strains and five strains of anti-EspB cell strains were seperately identified and used to prepare the ascite fluids.And the monocolonal antibodies were purified by Protein G.For studying the effects of ESAT-6 and EspB on the related functions of macrophages, RAW264.7 cell was transfected with pEGFP-C1-ESAT-6、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-ESAT-6、EGFP-EspB or EGFP were established. The gene and protein expression levels were further analyzed by RT-PCR, fluorescence microscopy and Western Blot. The results indicated that the EGFP-ESAT6 or EGFP-EspB fusion gene was integrated into the chromosome and the proteins can be stably expressed in the selected macrophage cell lines. These results gave us a tool for the future study in the mechanisms of ESAT-6 or EspB protein in modulating the macrophage cells.In order to investigate ESAT-6 and EspB in modulating macrophage phagocytosis, the macrophage cells were incubated with fluorescent beads for 2h at 5% CO2、37℃,then the samples were detected by flow cytometry. The results of data analysis indicated that cellularly-expressed ESAT-6 can remarkably enhance the macrophage phagocytosis. On the contrary, cellularly-expressed EspB have no effect on macrophage phagocytosis. The results got by the technique of confocal microscopy was consistent with the results of flow cytometry which further proved the results. In this research, the situation of cell apoptosis was also researched after cultivation for 48 h.The results got by flow cytometry showed that cellularly-expressed ESAT-6 can significantly increase macrophage apoptosis.In this reseach, we expressed and purified ESAT-6 and EspB recombinant proteins in E.coli expression system. The result got by immunofluorescence showed that ESAT-6 could directly bind to the macrophage membrane. Using the purified proteins ESAT-6 and EspB seperately to immunize BALB/c mice, several ESAT-6 and EspB monoclonal antibodies were produced; The macrophage cell lines that stably expressed EGFP-ESAT-6 or EGFP-EspB fusion protein were established; At the basis of establishment of macrophage cell lines, we have demonstrated that ESAT-6 protein can significantly enhance the phagocytosis ability of the macrophage, however, EspB have no effect on macrophage phagocytosis. Moreover, cellularly-expressed ESAT-6 can induce the macrophage apoptosis. And these results gave us tools for the future study in the interaction of the M.tuberculosis and the host and the molecular mechanisms of secreted proteins in modulating macrophage cells.更多还原