本文選題：結核分枝桿菌 + 表達��； 參考：《中國人民解放軍軍事醫(yī)學科學院》2011年碩士論文

【摘要】：ESX-1分泌系統(tǒng)是結核分枝桿菌中存在的一種特殊的蛋白分泌系統(tǒng),對細菌的毒力非常重要,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細胞抗原,是候選疫苗的重要組分,被廣泛用于新型結核疫苗的設計以及結核的診斷抗原;另一方面,許多研究顯示它會影響巨噬細胞和樹突狀細胞的功能,與細菌的毒力相關。另一種新發(fā)現(xiàn)的分泌蛋白EspB能夠和ESAT-6、CFP-10共分泌,這種共分泌機制對結核菌在巨噬細胞內的生長非常重要,并且能抑制巨噬細胞吞噬體的成熟,但EspB本身對巨噬細胞是否存在直接的調控還不清楚。本研究運用分子生物學、細胞生物學、免疫學等技術方法,對ESX-1分泌蛋白ESAT-6和EspB對巨噬細胞功能的調控作用進行了初步研究。 本研究首先從結核分枝桿菌H37Rv基因組中擴增出ESAT-6(Rv3875)與EspB(Rv3881c)基因,克隆入pET21a(+)載體。分別將成功構建的pET21a(+)-ESAT-6以及pET21a(+)-EspB重組質粒轉化入感受態(tài)BL21(DE3)中,經誘導表達后,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細胞共孵育后,使用免疫熒光技術發(fā)現(xiàn)ESAT-6能夠直接和RAW264.7的細胞膜結合。為了方便后續(xù)研究,本實驗同時完成了對EspB蛋白C端缺失突變體(EspB-N)的表達和純化。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,獲得了12株針對ESAT-6以及11株針對EspB的鼠mAb雜交瘤細胞株,分別對其中5株針對ESAT-6抗原的雜交瘤細胞株和5株針對EspB抗原的雜交瘤細胞株進行了小鼠腹水的制備及相關鑒定,并用Protein G親和層析株進行了純化。 為研究ESAT-6以及EspB蛋白對巨噬細胞相關功能的影響,將正確構建的重組質粒pEGFP-C1-ESAT-6、pEGFP-C1-EspB以及空載體pEGFP-C1以脂質體介導的方法轉染至小鼠巨噬細胞RAW264.7中,經過G418篩選和克隆化后分別建立了穩(wěn)定表達EGFP-ESAT6融合蛋白、EGFP-EspB融合蛋白以及EGFP的細胞系,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)轉細胞系進行鑒定。結果表明EGFP-ESAT-6以及EGFP-EspB融合基因成功整合入RAW264.7細胞基因組并能夠穩(wěn)定表達,為后續(xù)的ESAT-6以及EspB調控巨噬細胞機理研究提供了平臺。 為探索ESAT-6蛋白與EspB蛋白對巨噬細胞吞噬功能的影響,將熒光微球和RAW264.7細胞在37℃,5%CO2條件下孵育2 h后,使用流式細胞儀進行檢測。數(shù)據(jù)分析結果顯示細胞內表達的ESAT-6能夠顯著增強RAW264.7巨噬細胞的吞噬能力。相反,未觀察到細胞內表達的EspB蛋白對巨噬細胞的吞噬能力的影響。為了驗證流式分析結果,采用共聚焦顯微鏡技術觀察巨噬細胞吞噬能力改變并進行了定量分析,所獲得的結論與流式分析結果一致。本實驗還觀察了培養(yǎng)48 h后的穩(wěn)定表達ESAT-6蛋白的巨噬細胞系的凋亡狀況,流式結果分析發(fā)現(xiàn)細胞內表達ESAT-6蛋白能夠顯著的增加巨噬細胞的凋亡。 綜上所述,本研究成功表達和純化了結核分枝桿菌ESAT-6和EspB重組蛋白,使用免疫熒光技術發(fā)現(xiàn)ESAT-6蛋白能夠直接和RAW264.7的細胞膜結合;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ESAT-6和EspB蛋白的巨噬細胞系;在所建立細胞系的基礎上,發(fā)現(xiàn)ESAT-6能夠顯著增強巨噬細胞的吞噬能力,EspB蛋白對巨噬細胞的吞噬能力沒有影響,同時ESAT-6能夠誘導巨噬細胞的凋亡;這些結果的獲得進一步研究結核分枝桿菌和宿主之間的相互作用以及分泌蛋白調控巨噬細胞的分子機理提供了條件。
[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.

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

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本文編號：1879149