本文選題：結(jié)核分枝桿菌 + 表達(dá)��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年碩士論文

【摘要】：ESX-1分泌系統(tǒng)是結(jié)核分枝桿菌中存在的一種特殊的蛋白分泌系統(tǒng),對細(xì)菌的毒力非常重要,又被稱為VII型分泌系統(tǒng)。已有至少5種蛋白被證實(shí)由ESX-1分泌系統(tǒng)所分泌,即ESAT-6、CFP-10、EspA、EspB和EspR。ESAT-6是最早發(fā)現(xiàn)的ESX-1分泌蛋白之一,但是對它的功能還存在爭論:一方面,它是重要的T細(xì)胞抗原,是候選疫苗的重要組分,被廣泛用于新型結(jié)核疫苗的設(shè)計(jì)以及結(jié)核的診斷抗原;另一方面,許多研究顯示它會影響巨噬細(xì)胞和樹突狀細(xì)胞的功能,與細(xì)菌的毒力相關(guān)。另一種新發(fā)現(xiàn)的分泌蛋白EspB能夠和ESAT-6、CFP-10共分泌,這種共分泌機(jī)制對結(jié)核菌在巨噬細(xì)胞內(nèi)的生長非常重要,并且能抑制巨噬細(xì)胞吞噬體的成熟,但EspB本身對巨噬細(xì)胞是否存在直接的調(diào)控還不清楚。本研究運(yùn)用分子生物學(xué)、細(xì)胞生物學(xué)、免疫學(xué)等技術(shù)方法,對ESX-1分泌蛋白ESAT-6和EspB對巨噬細(xì)胞功能的調(diào)控作用進(jìn)行了初步研究。 本研究首先從結(jié)核分枝桿菌H37Rv基因組中擴(kuò)增出ESAT-6(Rv3875)與EspB(Rv3881c)基因,克隆入pET21a(+)載體。分別將成功構(gòu)建的pET21a(+)-ESAT-6以及pET21a(+)-EspB重組質(zhì)粒轉(zhuǎn)化入感受態(tài)BL21(DE3)中,經(jīng)誘導(dǎo)表達(dá)后,使用SDS-PAGE和Western Blot鑒定。超聲破碎后發(fā)現(xiàn)目的蛋白以大量可溶性形式存在,經(jīng)過Ni-NTA柱和DEAE-Sepharose柱純化,獲得純度約95%的重組ESAT-6與EspB蛋白。將重組蛋白和RAW264.7細(xì)胞共孵育后,使用免疫熒光技術(shù)發(fā)現(xiàn)ESAT-6能夠直接和RAW264.7的細(xì)胞膜結(jié)合。為了方便后續(xù)研究,本實(shí)驗(yàn)同時完成了對EspB蛋白C端缺失突變體(EspB-N)的表達(dá)和純化。將純化的ESAT-6蛋白和EspB蛋白分別免疫小鼠。采用雜交瘤技術(shù),獲得了12株針對ESAT-6以及11株針對EspB的鼠mAb雜交瘤細(xì)胞株,分別對其中5株針對ESAT-6抗原的雜交瘤細(xì)胞株和5株針對EspB抗原的雜交瘤細(xì)胞株進(jìn)行了小鼠腹水的制備及相關(guān)鑒定,并用Protein G親和層析株進(jìn)行了純化。 為研究ESAT-6以及EspB蛋白對巨噬細(xì)胞相關(guān)功能的影響,將正確構(gòu)建的重組質(zhì)粒pEGFP-C1-ESAT-6、pEGFP-C1-EspB以及空載體pEGFP-C1以脂質(zhì)體介導(dǎo)的方法轉(zhuǎn)染至小鼠巨噬細(xì)胞RAW264.7中,經(jīng)過G418篩選和克隆化后分別建立了穩(wěn)定表達(dá)EGFP-ESAT6融合蛋白、EGFP-EspB融合蛋白以及EGFP的細(xì)胞系,并通過RT-PCR、熒光顯微鏡及Western Blot方法,從基因和蛋白兩個水平對所建立的穩(wěn)轉(zhuǎn)細(xì)胞系進(jìn)行鑒定。結(jié)果表明EGFP-ESAT-6以及EGFP-EspB融合基因成功整合入RAW264.7細(xì)胞基因組并能夠穩(wěn)定表達(dá),為后續(xù)的ESAT-6以及EspB調(diào)控巨噬細(xì)胞機(jī)理研究提供了平臺。 為探索ESAT-6蛋白與EspB蛋白對巨噬細(xì)胞吞噬功能的影響,將熒光微球和RAW264.7細(xì)胞在37℃,5%CO2條件下孵育2 h后,使用流式細(xì)胞儀進(jìn)行檢測。數(shù)據(jù)分析結(jié)果顯示細(xì)胞內(nèi)表達(dá)的ESAT-6能夠顯著增強(qiáng)RAW264.7巨噬細(xì)胞的吞噬能力。相反,未觀察到細(xì)胞內(nèi)表達(dá)的EspB蛋白對巨噬細(xì)胞的吞噬能力的影響。為了驗(yàn)證流式分析結(jié)果,采用共聚焦顯微鏡技術(shù)觀察巨噬細(xì)胞吞噬能力改變并進(jìn)行了定量分析,所獲得的結(jié)論與流式分析結(jié)果一致。本實(shí)驗(yàn)還觀察了培養(yǎng)48 h后的穩(wěn)定表達(dá)ESAT-6蛋白的巨噬細(xì)胞系的凋亡狀況,流式結(jié)果分析發(fā)現(xiàn)細(xì)胞內(nèi)表達(dá)ESAT-6蛋白能夠顯著的增加巨噬細(xì)胞的凋亡。 綜上所述,本研究成功表達(dá)和純化了結(jié)核分枝桿菌ESAT-6和EspB重組蛋白,使用免疫熒光技術(shù)發(fā)現(xiàn)ESAT-6蛋白能夠直接和RAW264.7的細(xì)胞膜結(jié)合;將純化的ESAT-6蛋白和EspB蛋白用于抗體制備,獲得了多株針對ESAT-6蛋白的單克隆抗體和針對EspB的單克隆抗體;分別建立了能夠穩(wěn)定表達(dá)ESAT-6和EspB蛋白的巨噬細(xì)胞系;在所建立細(xì)胞系的基礎(chǔ)上,發(fā)現(xiàn)ESAT-6能夠顯著增強(qiáng)巨噬細(xì)胞的吞噬能力,EspB蛋白對巨噬細(xì)胞的吞噬能力沒有影響,同時ESAT-6能夠誘導(dǎo)巨噬細(xì)胞的凋亡;這些結(jié)果的獲得進(jìn)一步研究結(jié)核分枝桿菌和宿主之間的相互作用以及分泌蛋白調(diào)控巨噬細(xì)胞的分子機(jī)理提供了條件。
[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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378

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本文編號：1879149