炭疽芽胞杆菌BslA蛋白粘附功能研究

发布时间：2018-06-07 15:34

本文选题：炭疽芽胞杆菌 + BslA蛋白　；参考：《安徽大学》2012年硕士论文

【摘要】：炭疽病是由炭疽芽胞杆菌(Bacillus anthracis)引起的一种人畜共患烈性传染病,主要通过皮肤、呼吸道和消化道感染动物和人,严重危害人类和家畜的生命健康。大量研究表明,单一的PA组分疫苗并不能够提供完全的免疫保护,以PA为主,辅以其它来自菌体或芽胞的保护性组份是一个比较理想的炭疽疫苗研究策略,因此研究者一直致力于寻找和鉴定新的炭疽芽胞杆菌保护性抗原。炭疽芽孢杆菌的细胞外膜由一层二维晶状结构S层蛋白包裹,其生物学功能主要是作为保护性的外膜、分子泵和细菌粘附等。BslA蛋白(B. anthracis S-layer protein A)由炭疽芽胞杆菌pXO1致病岛区域上的bslA(pXO1-90)基因编码,包含652个氨基酸残基。BslA蛋白N端1-259位氨基酸残基中含有三个保守的SLH基序(S-layer-homologous motifs),是一种典型的S层蛋白。BslA蛋白炭疽芽胞杆菌感染宿主后有稳定的表达,天然表达的BslA与其它S层蛋白共同展示于菌体表面。研究表明BslA蛋白在炭疽芽胞杆菌致病过程中起着重要作用。本研究拟对BslA蛋白功能及其核心结构域方面进行研究,具体如下：第一部分工作利用PCR的技术扩增bslA基因片段,进行原核表达载体构建、重组蛋白分离纯化、多克隆抗体制备,并且通过流式细胞、细菌粘附抑制实验、免疫荧光等实验方法鉴定BslA蛋白的功能。从实验结果来看,成功构建重组菌株pET28a-BslA/Rosetta((DE3)并对BslA实现了可溶性表达。通过ProBondTM Purification system得到纯度在87%以上的BslA蛋白,流式细胞与免疫荧光的结果表明BslA蛋白具有黏附功能。细菌粘附抑制实验计算出BslA重组蛋白及其抗血清能够抑制约为98%的炭疽杆菌黏附,表明BslA蛋白可能是一种潜在的保护性抗原。第二部分工作中,本实验通过蛋白结构预测软件,确定核心功能结构域可能存在的位置,从而进行分段研究。为了更好、更方便的检测,我们将蛋白片段与EGFP蛋白融合表达,使EGFP成为标签。再通过免疫荧光等方法检测(?)BslA蛋白片段功能。结果表明,成功构建了pET28a-EGFP/Rosetta(DE3)和实现了可溶性表达,并对其进行了纯化。表达分析和流式细胞检测结果表明,构建的EGFP蛋白有很好的荧光性。另外,根据蛋白结构软件的预测我们对BslA蛋白进行了首次分段,成功构建了pET28a-EGFP-BslA(260-500)/Rosetta(DE3)、pET28a-EGFP-BslA(411-560)/Rosetta(DE3)、pET28a-EGFP-BslA(511-652)/Rosetta(DE3),进行了截短BslA的表达与纯化。经荧光电镜检测结果表明,BslA(260-500)、BslA(411-560)具有黏附功能,以此推断核心功能结构域位于BslA蛋白的411-500位氨基酸。根据此结果,进行了第二次分段,成功构建pET28a-EGFP-BslA(411-455)/Rosetta(DE3)、pET28a-EGFP-BslA(431-485)/Rosetta(DE3)、pET28a-EGFP-BslA(456-500/Rosetta(DE3)重组菌,对表达的蛋白进行了纯化。免疫荧光检测的结果表明BslA(411-455)、BslA(431-485)片段具有粘附功能,进一步确定了BslA蛋白的核心功能结构域位于431455位点。综上,BslA是一种重要的保护性抗原,可能在新一代炭疽疫苗的研究和开发中扮演重要的角色。BslA蛋白是具有细胞粘附功能,其核心功能结构域的确定为进一步研究炭疽芽胞杆菌的致病机制与免疫预防打下了良好的基础。
[Abstract]:Anthrax is a kind of zoonotic and zoonotic infectious disease caused by Bacillus anthracis (Bacillus anthracis), which mainly infect animals and people through skin, respiratory and digestive tract, and seriously endangers the life and health of human and domestic animals. A large number of studies have shown that a single PA vaccine can not provide complete immune protection, which is based on PA, supplemented by the vaccine. Other protective components from the bacteria or spore are an ideal research strategy for the anthrax vaccine, so researchers have been working to identify and identify new protective antigens of Bacillus anthracis.
The outer membrane of Bacillus anthracis is encapsulated by a two-dimensional crystalline structure of S layer protein. Its biological function is mainly as a protective outer membrane, and the.BslA protein (B. anthracis S-layer protein A) is encoded by the bslA (pXO1-90) gene on the region of Bacillus anthracis pXO1 pathogenicity island, including 652 amino acid residues. The 1-259 amino acid residues in the N terminal of the BslA protein contain three conservative SLH sequences (S-layer-homologous motifs). It is a typical S layer protein.BslA protein, Bacillus anthracis infected with the host, and has a stable expression. The natural BslA and other S layer proteins are displayed on the surface of the bacteria. The study shows that the BslA protein is in Bacillus anthracis. The purpose of this study is to study the function of BslA protein and its core domain, as follows:
In the first part, the bslA gene fragment was amplified by PCR technique, the prokaryotic expression vector was constructed, the recombinant protein was isolated and purified, and the polyclonal antibody was prepared. The function of BslA protein was identified by flow cytometry, bacterial adhesion inhibition experiment and immunofluorescence. From the experimental results, the recombinant strain pET28a-BslA/R was successfully constructed. Osetta (DE3) and the soluble expression of BslA were achieved. The purity of BslA protein above 87% was obtained by ProBondTM Purification system. The results of flow cytometry and immunofluorescence showed that BslA protein had adhesion function. The bacterial adhesion inhibition experiment calculated that the BslA recombinant protein and its antiserum could inhibit the adhesion of anthrax of about 98%. It is suggested that BslA protein may be a potential protective antigen.
In the second part, in this experiment, the possible location of the core functional domain was determined by the protein structure prediction software. In order to better, more convenient detection, we fused the protein fragment with EGFP protein to make EGFP a label. Then the function of BslA protein fragment was detected through immunofluorescence and other methods. The results showed that the soluble expression of pET28a-EGFP/Rosetta (DE3) was successfully constructed and purified. The results of expression analysis and flow cytometry showed that the constructed EGFP protein had good fluorescence. In addition, we successfully constructed the pET28a-EGFP based on the prediction of the protein structure software for the first time according to the protein structure software. The expression and purification of -BslA (260-500) (260-500) /Rosetta (DE3), pET28a-EGFP-BslA (411-560) /Rosetta (DE3), pET28a-EGFP-BslA (511-652) /Rosetta (DE3) were carried out. The results of fluorescence electron microscopy showed that BslA (260-500) and BslA (411-560) had adhesion function, so that the core functional domain was located in the 411-500 amino acids of the protein. According to the results, second subsections were carried out to successfully construct pET28a-EGFP-BslA (411-455) /Rosetta (DE3), pET28a-EGFP-BslA (431-485) /Rosetta (DE3) and pET28a-EGFP-BslA (456-500/Rosetta (DE3) recombinant bacteria to purify the expressed protein. The results of immunofluorescence detection showed that BslA (411-455), BslA (431-485) fragment had adhesion function. The core functional domain of BslA protein was located at the 431455 locus.
In conclusion, BslA is an important protective antigen. It may play an important role in the research and development of the new anthrax vaccine..BslA protein is a cell adhesion function. The determination of its core functional domain has laid a good foundation for further research on the pathogenesis and immune prevention of Bacillus anthracis.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378;S855.12

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