發(fā)布時(shí)間：2019-04-10 06:59

【摘要】：斑點(diǎn)熱(spotted fever)是由斑點(diǎn)熱群立克次體(spotted fever group rickettsia,SFGR)所引起的一組疾病的總稱。190kDa(外膜蛋白A,OmpA)和120kDa(外膜蛋白B,OmpB)是斑點(diǎn)熱立克次體最主要的表面蛋白抗原,被認(rèn)為是發(fā)展斑點(diǎn)熱的診斷試劑和亞單位疫苗的靶分子。本研究旨在克隆與表達(dá)斑點(diǎn)熱立克次體的ompA和ompB部分基因片段,并對(duì)所表達(dá)的重組蛋白的抗原性進(jìn)行研究。 采用PCR方法,從斑點(diǎn)熱立克次體的基因組中擴(kuò)增到ompA和ompB基因的基因片段,將兩個(gè)基因片段分別與原核表達(dá)載體pQE30連接,構(gòu)建重組質(zhì)粒pQE30/ompA和pQE30/ompB。將該重組質(zhì)粒分別轉(zhuǎn)化大腸桿菌,并用IPTG誘導(dǎo)轉(zhuǎn)化菌表達(dá)目的蛋白。SDS-PAGE鑒定pQE30/ompA和pQE30/ompB轉(zhuǎn)化菌分別表達(dá)了56kDa OmpA和44kDa OmpB重組蛋白。 免疫印跡分析證明立氏立克次體的OmpB重組蛋白僅與立氏立克次體全菌免疫血清發(fā)生特異反應(yīng)。用純化的OmpB重組蛋白免疫家兔,間接免疫熒光試驗(yàn)(IFA)該重組蛋白能有效地誘導(dǎo)家兔產(chǎn)生高水平的特異性IgG。IFA分析發(fā)現(xiàn)該重組蛋白免疫血清與立氏立克次體全菌抗原發(fā)生特異性反應(yīng),而與其它立克次體的全菌抗原無明顯交叉反應(yīng)。這些結(jié)果說明本次研究制備的OmpB重組蛋白為立氏立克次體種特異性抗原,該重組蛋白免疫血清可以用作檢測(cè)立氏立克次體的特異性抗體。 血清學(xué)分析顯示黑龍江立克次體56kDa OmpA重組蛋白能與黑龍江立克次體、立氏立克次體、斑點(diǎn)熱群立克次體精河株等斑點(diǎn)熱立克次體免疫血清發(fā)生交叉反應(yīng),提示該OmpA重組蛋白具有斑點(diǎn)熱群特異性。用OmpA重組蛋白免疫小鼠和豚鼠,采用IFA檢測(cè)免疫動(dòng)物的血清特異性抗體水平,結(jié)果顯示該OmpA重組蛋白能有效地誘導(dǎo)動(dòng)物產(chǎn)生較高水平的特異性IgG。為了了解OmpA重組蛋白誘導(dǎo)細(xì)胞免疫應(yīng)答的能力,我們采用MTT法分析OmpA免疫小鼠脾細(xì)胞在該抗原刺激下的增殖水平。結(jié)果顯示免疫小鼠脾細(xì)胞在OmpA的刺激下的細(xì)胞增殖水平顯著高于對(duì)照,提示OmpA具有T細(xì)胞表位,能夠有效刺激機(jī)體產(chǎn)生特異性細(xì)胞免疫應(yīng)答。 用黑龍江立克次體和立氏立克次體分別攻擊黑龍江立克次體OmpA重組蛋白免疫豚鼠,結(jié)果發(fā)現(xiàn)免疫動(dòng)物受立克次體攻擊后的發(fā)熱和臟器的病理學(xué)改變
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392.1

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