本文選題：結(jié)核分枝桿菌 + 抗原蛋白　； 參考：《揚州大學(xué)》2012年碩士論文

【摘要】：結(jié)核病(Tuberculosis, TB)是由結(jié)核分枝桿菌復(fù)合群(Mycobacterium tuberculosis complex, MTC)所引起的以呼吸系統(tǒng)感染為主的慢性傳染病,它是現(xiàn)今世界范圍內(nèi)最主要的疾病之一。據(jù)WHO估計,當前全世界約有1/3人口感染了結(jié)核分枝桿菌(Mycobacterium tuberculosis, MTB),我國的結(jié)核病患者數(shù)量居世界第二位,感染人數(shù)已達到4億。而這部分人中只有10%可能最終發(fā)展為活動性結(jié)核,而絕大多數(shù)為潛伏感染�？ń槊�(Bacille Calmette and Guerin, BCG)是牛結(jié)核分枝桿菌的減毒株,是目前唯一用于TB預(yù)防的疫苗,但其對潛伏感染者無效,并且存在著保護期短、免疫應(yīng)答較弱等缺陷。因此,為了減輕結(jié)核病造成的負擔,新型抗原的篩選和新型疫苗的研制迫在眉睫。 有國外學(xué)者利用生物信息學(xué)的方法,預(yù)測出抗原候選分子,本研究從這些候選分子中選取了3個評分最高的蛋白,對其進行原核表達和純化,并對其免疫應(yīng)答特性進行分析。 1.結(jié)核分枝桿菌Rv1738、TB31.7及RPFE蛋白的表達純化和鑒定 以MTB蛋白Rv1738、TB31.7和RPFE為研究對象,首先從H37Rv基因組DNA上PCR擴增其相應(yīng)的編碼基因,克隆篩選和序列鑒定正確以后,利用pET30a或pET32a構(gòu)建原核表達質(zhì)粒。將鑒定正確的重組表達質(zhì)粒轉(zhuǎn)化BL21(DE3),誘導(dǎo)表達。對融合蛋白進行親和層析純化,并通過Western blotting實驗對蛋白的免疫反應(yīng)性進行分析。 誘導(dǎo)表達后,SDS-PAGE結(jié)果顯示,各目的蛋白都得到成功表達,其中Rv1738和RPFE主要以可溶形式存在,而TB31.7主要以包涵體形式表達。對于包涵體蛋白我們采用了緩慢變復(fù)性的方法使其變?yōu)榭扇苄孕问酱嬖�。各融合蛋白與兔抗H37Rv多抗血清的Western blotting結(jié)果都顯示出了特異性目的條帶,顯示出良好的免疫反應(yīng)性。 2.結(jié)核分枝桿菌Rv1738、TB31.7及RPFE蛋白免疫特性分析 將Rv1738、TB31.7和RPFE三種融合蛋白與DDA佐劑等體積充分乳化后皮下接種免疫6周齡雌性C57BL/6小鼠,三免兩周后摘眼球處死小鼠,采血清和制備淋巴細胞分別進行抗體效價和細胞因子測定�？贵w檢測結(jié)果顯示,在三種融合蛋白免疫小鼠的血清中均可以檢測到高水平的特異性抗體。夾心ELISA分析三種蛋白作為刺激劑活化淋巴細胞分泌IFN-γ、IL-2和IL-4的差異,結(jié)果顯示,三種蛋白刺激細胞產(chǎn)生較高水平的IFN-γ和IL-2,其中IFN-γ的分泌水平明顯高于IL-2。三種蛋白刺激細胞產(chǎn)生IL-4的水平很低。結(jié)果表明,三種蛋白具有優(yōu)良的免疫原性,它們所誘導(dǎo)的免疫應(yīng)答趨向于Thl型,這為進一步的研究提供了重要的信息
[Abstract]:Tuberculosism (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis complex. It is one of the most important diseases in the world. According to WHO estimates, about one third of the world's population has been infected with Mycobacterium tuberculosism (MTBN). The number of tuberculosis patients in China ranks second in the world, and the number of infected people has reached 400 million. Only 10% of these people may eventually develop active TB, while the vast majority are latent infections. Bacille Calmette and Guerin (BCG) is the attenuated strain of Mycobacterium bovis and the only vaccine used for TB prevention at present, but it is ineffective against latent infection and has some defects such as short protection period and weak immune response. Therefore, in order to reduce the burden caused by tuberculosis, the screening of new antigens and the development of new vaccines are urgent. Some foreign scholars used bioinformatics to predict antigen candidate molecules. In this study, three proteins with the highest score were selected from these candidate molecules for prokaryotic expression and purification. And its immune response characteristics were analyzed. 1. The expression, purification and identification of Rv1738 TB31.7 and RPFE proteins of Mycobacterium tuberculosis Rv1738 TB31.7 and RPFE were studied. The corresponding coding genes were amplified by PCR from the genomic DNA of H37Rv, then cloned and sequenced. The prokaryotic expression plasmid was constructed by using pET30a or pET32a. The identified recombinant plasmid was transformed into BL21 and DE3 to induce expression. The fusion protein was purified by affinity chromatography and its immunoreactivity was analyzed by Western blotting assay. The results of SDS-PAGE showed that all the target proteins were successfully expressed, Rv1738 and RPFE were mainly expressed in soluble form, while TB31.7 was mainly expressed as inclusion body. For inclusion body proteins, we use a slow renaturation method to make them soluble. The results of Western blotting of each fusion protein and rabbit anti-H37Rv polyantibody showed specific target bands and showed good immunoreactivity. 2. Immune characteristics of Mycobacterium tuberculosis Rv1738TB31.7 and RPFE protein the fusion proteins Rv1738TB31.7 and RPFE were fully emulsified with DDA adjuvant and subcutaneously inoculated to 6-week-old female C57BL / 6 mice. The antibody titers and cytokines of blood samples and lymphocytes were measured. The results of antibody detection showed that a high level of specific antibody could be detected in the sera of mice immunized with three fusion proteins. Sandwich Elisa was used to analyze the difference between the secretion of IL-2 and IL-4 by lymphocytes activated by three proteins. The results showed that the three proteins stimulated the cells to produce higher levels of IFN- 緯 and IL-2, in which the secretion of IFN- 緯 was significantly higher than that of IL-2. The level of IL-4 produced by the three proteins was very low. The results show that the three proteins have excellent immunogenicity, and the immune response induced by them tends to be Thl type, which provides important information for further research.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R378.911

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