斑点热立克次体ompA和ompB基因片段的克隆与表达及重组蛋白抗原性的研究

发布时间：2019-04-10 06:59

【摘要】：斑点热(spotted fever)是由斑点热群立克次体(spotted fever group rickettsia,SFGR)所引起的一组疾病的总称。190kDa(外膜蛋白A,OmpA)和120kDa(外膜蛋白B,OmpB)是斑点热立克次体最主要的表面蛋白抗原,被认为是发展斑点热的诊断试剂和亚单位疫苗的靶分子。本研究旨在克隆与表达斑点热立克次体的ompA和ompB部分基因片段,并对所表达的重组蛋白的抗原性进行研究。 采用PCR方法,从斑点热立克次体的基因组中扩增到ompA和ompB基因的基因片段,将两个基因片段分别与原核表达载体pQE30连接,构建重组质粒pQE30/ompA和pQE30/ompB。将该重组质粒分别转化大肠杆菌,并用IPTG诱导转化菌表达目的蛋白。SDS-PAGE鉴定pQE30/ompA和pQE30/ompB转化菌分别表达了56kDa OmpA和44kDa OmpB重组蛋白。 免疫印迹分析证明立氏立克次体的OmpB重组蛋白仅与立氏立克次体全菌免疫血清发生特异反应。用纯化的OmpB重组蛋白免疫家兔,间接免疫荧光试验(IFA)该重组蛋白能有效地诱导家兔产生高水平的特异性IgG。IFA分析发现该重组蛋白免疫血清与立氏立克次体全菌抗原发生特异性反应,而与其它立克次体的全菌抗原无明显交叉反应。这些结果说明本次研究制备的OmpB重组蛋白为立氏立克次体种特异性抗原,该重组蛋白免疫血清可以用作检测立氏立克次体的特异性抗体。 血清学分析显示黑龙江立克次体56kDa OmpA重组蛋白能与黑龙江立克次体、立氏立克次体、斑点热群立克次体精河株等斑点热立克次体免疫血清发生交叉反应,提示该OmpA重组蛋白具有斑点热群特异性。用OmpA重组蛋白免疫小鼠和豚鼠,采用IFA检测免疫动物的血清特异性抗体水平,结果显示该OmpA重组蛋白能有效地诱导动物产生较高水平的特异性IgG。为了了解OmpA重组蛋白诱导细胞免疫应答的能力,我们采用MTT法分析OmpA免疫小鼠脾细胞在该抗原刺激下的增殖水平。结果显示免疫小鼠脾细胞在OmpA的刺激下的细胞增殖水平显著高于对照,提示OmpA具有T细胞表位,能够有效刺激机体产生特异性细胞免疫应答。 用黑龙江立克次体和立氏立克次体分别攻击黑龙江立克次体OmpA重组蛋白免疫豚鼠,结果发现免疫动物受立克次体攻击后的发热和脏器的病理学改变
[Abstract]:Spotted fever (spotted fever) is a group of diseases caused by spotted fever group Rickettsia (spotted fever group rickettsia,SFGR. 190 kDa (outer membrane protein A, OmpA) and 120kDa (outer membrane protein B, OmpB) are the main surface protein antigens of spotted fever rickettsia. It is believed to be a target molecule for the development of Dot Fever diagnostic reagents and subunit vaccines. The aim of this study was to clone and express part of ompA and ompB gene fragments of Dot Rickettsia, and to study the antigenicity of the recombinant protein. The gene fragments of ompA and ompB genes were amplified from the genome of Dot Rickettsia by PCR method. The two gene fragments were ligated with prokaryotic expression vector pQE30 to construct recombinant plasmids pQE30/ompA and pQE30/ompB.. The recombinant plasmid was transformed into E. coli, and the recombinant protein was induced by IPTG. The recombinant proteins of 56kDa OmpA and 44kDa OmpB were identified by SDS-PAGE in the transformed strain pQE30/ompA and pQE30/ompB, respectively. Western blot analysis showed that the recombinant OmpB protein of Rickettsia rickettsiae only reacted specifically with the immune serum of Rickettsia rickettsiae. Rabbits were immunized with purified OmpB recombinant protein, Indirect immunofluorescence assay (IFA) showed that the recombinant protein could effectively induce the rabbit to produce a high level of specific IgG.IFA. It was found that the recombinant protein reacted specifically with the whole bacterial antigen of Rickettsia rickettsiae. However, there was no obvious cross-reaction with other Rickettsia antigens. These results suggest that the recombinant OmpB protein prepared in this study is a rickettsia specific antigen, and the immune serum of the recombinant protein can be used as a specific antibody to detect Rickettsia rickettsiae. Serological analysis showed that the recombinant 56kDa OmpA protein of Heilongjiang Rickettsia could cross-react with the immune sera of Heilongjiang Rickettsia, Rickettsia rickettsiae, spotted fever group Rickettsia Jinghe strain, etc. The results indicated that the recombinant protein of OmpA had the specificity of dot fever group. OmpA recombinant protein was used to immunize mice and guinea pigs, and the serum specific antibody level of immunized animals was detected by IFA. The results showed that the recombinant OmpA protein could effectively induce the production of high level of specific IgG. in mice and guinea pigs. In order to understand the ability of OmpA recombinant protein to induce cellular immune response, we used MTT method to analyze the proliferation level of spleen cells in OmpA immunized mice under the stimulation of this antigen. The results showed that the proliferation level of spleen cells in immunized mice was significantly higher than that in control mice stimulated by OmpA, suggesting that OmpA had T cell epitopes and could effectively stimulate the organism to produce specific cellular immune responses. Guinea pigs were immunized with Rickettsia Heilongjiang and Rickettsia rickettsiae OmpA recombinant protein, respectively. The results showed that the immune animals had fever and pathological changes of organs after Rickettsia challenge.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1

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