本文選題：炭疽芽胞桿菌 + BslA蛋白��； 參考：《安徽大學》2012年碩士論文

【摘要】：炭疽病是由炭疽芽胞桿菌(Bacillus anthracis)引起的一種人畜共患烈性傳染病,主要通過皮膚、呼吸道和消化道感染動物和人,嚴重危害人類和家畜的生命健康。大量研究表明,單一的PA組分疫苗并不能夠提供完全的免疫保護,以PA為主,輔以其它來自菌體或芽胞的保護性組份是一個比較理想的炭疽疫苗研究策略,因此研究者一直致力于尋找和鑒定新的炭疽芽胞桿菌保護性抗原。 炭疽芽孢桿菌的細胞外膜由一層二維晶狀結構S層蛋白包裹,其生物學功能主要是作為保護性的外膜、分子泵和細菌粘附等。BslA蛋白(B. anthracis S-layer protein A)由炭疽芽胞桿菌pXO1致病島區(qū)域上的bslA(pXO1-90)基因編碼,包含652個氨基酸殘基。BslA蛋白N端1-259位氨基酸殘基中含有三個保守的SLH基序(S-layer-homologous motifs),是一種典型的S層蛋白。BslA蛋白炭疽芽胞桿菌感染宿主后有穩(wěn)定的表達,天然表達的BslA與其它S層蛋白共同展示于菌體表面。研究表明BslA蛋白在炭疽芽胞桿菌致病過程中起著重要作用。本研究擬對BslA蛋白功能及其核心結構域方面進行研究,具體如下： 第一部分工作利用PCR的技術擴增bslA基因片段,進行原核表達載體構建、重組蛋白分離純化、多克隆抗體制備,并且通過流式細胞、細菌粘附抑制實驗、免疫熒光等實驗方法鑒定BslA蛋白的功能。從實驗結果來看,成功構建重組菌株pET28a-BslA/Rosetta((DE3)并對BslA實現(xiàn)了可溶性表達。通過ProBondTM Purification system得到純度在87%以上的BslA蛋白,流式細胞與免疫熒光的結果表明BslA蛋白具有黏附功能。細菌粘附抑制實驗計算出BslA重組蛋白及其抗血清能夠抑制約為98%的炭疽桿菌黏附,表明BslA蛋白可能是一種潛在的保護性抗原。 第二部分工作中,本實驗通過蛋白結構預測軟件,確定核心功能結構域可能存在的位置,從而進行分段研究。為了更好、更方便的檢測,我們將蛋白片段與EGFP蛋白融合表達,使EGFP成為標簽。再通過免疫熒光等方法檢測(?)BslA蛋白片段功能。結果表明,成功構建了pET28a-EGFP/Rosetta(DE3)和實現(xiàn)了可溶性表達,并對其進行了純化。表達分析和流式細胞檢測結果表明,構建的EGFP蛋白有很好的熒光性。另外,根據(jù)蛋白結構軟件的預測我們對BslA蛋白進行了首次分段,成功構建了pET28a-EGFP-BslA(260-500)/Rosetta(DE3)、pET28a-EGFP-BslA(411-560)/Rosetta(DE3)、pET28a-EGFP-BslA(511-652)/Rosetta(DE3),進行了截短BslA的表達與純化。經熒光電鏡檢測結果表明,BslA(260-500)、BslA(411-560)具有黏附功能,以此推斷核心功能結構域位于BslA蛋白的411-500位氨基酸。根據(jù)此結果,進行了第二次分段,成功構建pET28a-EGFP-BslA(411-455)/Rosetta(DE3)、pET28a-EGFP-BslA(431-485)/Rosetta(DE3)、pET28a-EGFP-BslA(456-500/Rosetta(DE3)重組菌,對表達的蛋白進行了純化。免疫熒光檢測的結果表明BslA(411-455)、BslA(431-485)片段具有粘附功能,進一步確定了BslA蛋白的核心功能結構域位于431455位點。 綜上,BslA是一種重要的保護性抗原,可能在新一代炭疽疫苗的研究和開發(fā)中扮演重要的角色。BslA蛋白是具有細胞粘附功能,其核心功能結構域的確定為進一步研究炭疽芽胞桿菌的致病機制與免疫預防打下了良好的基礎。
[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.
【學位授予單位】：安徽大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378;S855.12

【相似文獻】

相關期刊論文 前10條

1 馬曉冬,馬文麗,孫朝暉,呂梁,鄭文嶺,王洪敏,石嶸;制備炭疽芽胞桿菌檢測基因芯片的初步研究[J];微生物學報;2004年03期

2 展德文,王們,王令春,張兆山;炭疽芽胞桿菌疫苗研究進展[J];微生物學報;2005年01期

3 田國忠;俞東征;海榮;魏建春;馬鳳琴;蔡虹;張建華;鄭玉紅;付秀萍;張志凱;張恩民;徐冬蕾;;炭疽芽胞桿菌基因組中串聯(lián)重復序列拷貝數(shù)的分析[J];中華微生物學和免疫學雜志;2006年09期

4 魏建春;張恩民;馬鳳琴;張建中;;中國炭疽芽胞桿菌致死因子基因中單核苷酸多態(tài)性位點分析[J];中華流行病學雜志;2006年07期

5 田國忠;海榮;俞東征;魏建春;馬鳳琴;蔡虹;張建華;鄭玉紅;付秀萍;張志凱;張恩民;徐冬蕾;;利用串聯(lián)重復序列研究炭疽芽胞桿菌的基因分型[J];中華流行病學雜志;2006年08期

6 榮光華;夏懿;朱詩應;童一民;戚中田;;炭疽芽胞桿菌染色體特異序列的篩選及實時定量檢測[J];微生物學報;2006年06期

7 呂和平;閔勇;楊東明;鄭應華;李旭;武軼;;炭疽芽胞桿菌(Bacillus anthracis)檢測質粒的構建及其應用[J];微生物學雜志;2007年03期

8 李冰;李建民;徐俊杰;劉樹玲;張羽;付玲;陳薇;;抗炭疽保護性抗原單克隆抗體可變區(qū)基因的克隆及序列分析[J];生物技術通訊;2007年02期

9 王艷春;姜娜;展德文;邱炎;袁盛凌;陶好霞;王令春;張兆山;劉純杰;;Cre-LoxP系統(tǒng)在炭疽芽胞桿菌基因敲除中的應用及eag基因的敲除[J];生物化學與生物物理進展;2009年07期

10 董樹林;蠟樣芽孢桿菌的分類地位和鑒定標準[J];微生物學免疫學進展;1985年03期

相關會議論文 前5條

1 張利軍;胡小華;;類炭疽芽胞桿菌噬菌體生物學特性研究[A];中華醫(yī)院管理學會第十二屆全國醫(yī)院感染管理學術年會論文匯編[C];2005年

2 徐秀華;;全球感染性疾病發(fā)展趨勢與醫(yī)院感染控制[A];中華醫(yī)院管理學會第十屆全國醫(yī)院感染管理學術年會論文匯編[C];2003年

3 陳世平;;炭疽及生物武器防護[A];中華醫(yī)院管理學會醫(yī)院感染管理專業(yè)委員會第九屆醫(yī)院感染管理學術年會論文匯編[C];2002年

4 孫明;;蘇云金芽胞桿菌YBT-1520的功能組分析[A];2008年中國微生物學會學術年會論文摘要集[C];2008年

5 孫長貴;;2011年CLSI M100-S21文件主要更新內容介紹[A];2011年浙江省檢驗醫(yī)學學術年會論文匯編[C];2011年

相關博士學位論文 前2條

1 邱寧;蘇云金芽胞桿菌YBT-1520基因組中插入序列組的研究[D];華中農業(yè)大學;2010年

2 周，

本文編號：1991687