结核分枝杆菌Rv1738、TB31.7及RPFE的表达纯化及免疫特性分析

发布时间：2018-06-21 02:17

本文选题：结核分枝杆菌 + 抗原蛋白　；参考：《扬州大学》2012年硕士论文

【摘要】：结核病(Tuberculosis, TB)是由结核分枝杆菌复合群(Mycobacterium tuberculosis complex, MTC)所引起的以呼吸系统感染为主的慢性传染病,它是现今世界范围内最主要的疾病之一。据WHO估计,当前全世界约有1/3人口感染了结核分枝杆菌(Mycobacterium tuberculosis, MTB),我国的结核病患者数量居世界第二位,感染人数已达到4亿。而这部分人中只有10%可能最终发展为活动性结核,而绝大多数为潜伏感染。卡介苗(Bacille Calmette and Guerin, BCG)是牛结核分枝杆菌的减毒株,是目前唯一用于TB预防的疫苗,但其对潜伏感染者无效,并且存在着保护期短、免疫应答较弱等缺陷。因此,为了减轻结核病造成的负担,新型抗原的筛选和新型疫苗的研制迫在眉睫。有国外学者利用生物信息学的方法,预测出抗原候选分子,本研究从这些候选分子中选取了3个评分最高的蛋白,对其进行原核表达和纯化,并对其免疫应答特性进行分析。 1.结核分枝杆菌Rv1738、TB31.7及RPFE蛋白的表达纯化和鉴定以MTB蛋白Rv1738、TB31.7和RPFE为研究对象,首先从H37Rv基因组DNA上PCR扩增其相应的编码基因,克隆筛选和序列鉴定正确以后,利用pET30a或pET32a构建原核表达质粒。将鉴定正确的重组表达质粒转化BL21(DE3),诱导表达。对融合蛋白进行亲和层析纯化,并通过Western blotting实验对蛋白的免疫反应性进行分析。诱导表达后,SDS-PAGE结果显示,各目的蛋白都得到成功表达,其中Rv1738和RPFE主要以可溶形式存在,而TB31.7主要以包涵体形式表达。对于包涵体蛋白我们采用了缓慢变复性的方法使其变为可溶性形式存在。各融合蛋白与兔抗H37Rv多抗血清的Western blotting结果都显示出了特异性目的条带,显示出良好的免疫反应性。 2.结核分枝杆菌Rv1738、TB31.7及RPFE蛋白免疫特性分析将Rv1738、TB31.7和RPFE三种融合蛋白与DDA佐剂等体积充分乳化后皮下接种免疫6周龄雌性C57BL/6小鼠,三免两周后摘眼球处死小鼠,采血清和制备淋巴细胞分别进行抗体效价和细胞因子测定。抗体检测结果显示,在三种融合蛋白免疫小鼠的血清中均可以检测到高水平的特异性抗体。夹心ELISA分析三种蛋白作为刺激剂活化淋巴细胞分泌IFN-γ、IL-2和IL-4的差异,结果显示,三种蛋白刺激细胞产生较高水平的IFN-γ和IL-2,其中IFN-γ的分泌水平明显高于IL-2。三种蛋白刺激细胞产生IL-4的水平很低。结果表明,三种蛋白具有优良的免疫原性,它们所诱导的免疫应答趋向于Thl型,这为进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- 纬 and IL-2, in which the secretion of IFN- 纬 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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378.911

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