發(fā)布時間：2019-04-10 06:59

【摘要】：斑點熱(spotted fever)是由斑點熱群立克次體(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)是斑點熱立克次體最主要的表面蛋白抗原,被認為是發(fā)展斑點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斑點熱立克次體的ompA和ompB部分基因片段,并對所表達的重組蛋白的抗原性進行研究。 采用PCR方法,從斑點熱立克次體的基因組中擴增到ompA和ompB基因的基因片段,將兩個基因片段分別與原核表達載體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誘導轉(zhuǎn)化菌表達目的蛋白。SDS-PAGE鑒定pQE30/ompA和pQE30/ompB轉(zhuǎn)化菌分別表達了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(IFA)該重組蛋白能有效地誘導家兔產(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立氏立克次體的特異性抗體。 血清學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、斑點熱群立克次體精河株等斑點熱立克次體免疫血清發(fā)生交叉反應(yīng),提示該OmpA重組蛋白具有斑點熱群特異性。用OmpA重組蛋白免疫小鼠和豚鼠,采用IFA檢測免疫動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動物產(chǎn)生較高水平的特異性IgG。為了了解OmpA重組蛋白誘導細胞免疫應(yīng)答的能力,我們采用MTT法分析OmpA免疫小鼠脾細胞在該抗原刺激下的增殖水平。結(jié)果顯示免疫小鼠脾細胞在OmpA的刺激下的細胞增殖水平顯著高于對照,提示OmpA具有T細胞表位,能夠有效刺激機體產(chǎn)生特異性細胞免疫應(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)免疫動物受立克次體攻擊后的發(fā)熱和臟器的病理學改變
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392.1

【相似文獻】

相關(guān)期刊論文 前10條

1 孟艷芬;段長松;王錫樂;熊小路;溫博海;;黑龍江立克次體感染血管內(nèi)皮細胞的初步研究[J];中國人獸共患病學報;2011年06期

2 ;[J];;年期

3 ;[J];;年期

4 ;[J];;年期

5 ;[J];;年期

6 ;[J];;年期

7 ;[J];;年期

8 ;[J];;年期

9 ;[J];;年期

10 ;[J];;年期

相關(guān)會議論文 前2條

1 王怡丹;劉建國;姜晶;王偉;亓鵬;白朋元;管曉燕;;防齲基因疫苗pVAX1-SPG免疫BALB/c小鼠的實驗研究[A];全國第三次牙體牙髓病學臨床技術(shù)研討會論文匯編[C];2009年

2 岳朝暉;劉建國;王晶;趙靖;王偉;姜晶;管曉燕;張劍;;防齲基因疫苗pcDNA3-PAc不同途徑免疫BALB/c小鼠的實驗研究[A];全國第三次牙體牙髓病學臨床技術(shù)研討會論文匯編[C];2009年

相關(guān)重要報紙文章 前1條

1 鄭靈巧　朱玉;暑期預防腸道及蟲媒傳染病[N];北京日報;2003年

相關(guān)博士學位論文 前1條

1 牛東升;立氏立克次體外膜蛋白B基因的應(yīng)用研究[D];中國人民解放軍軍事醫(yī)學科學院;2007年

相關(guān)碩士學位論文 前4條

1 焦艷梅;斑點熱立克次體ompA和ompB基因片段的克隆與表達及重組蛋白抗原性的研究[D];中國人民解放軍軍事醫(yī)學科學院;2005年

2 釗守鳳;鉤端螺旋體屬特異性表面蛋白抗原融合基因lipL32/1-ompL1/1原核表達系統(tǒng)的構(gòu)建、表達和鑒定及酵母表達系統(tǒng)的構(gòu)建[D];浙江大學;2004年

3 楊曉;檢測斑疹傷寒病原體實時熒光定量PCR方法的建立黑龍江立克次體和立克次體精河株毒力和抗原性初步研究[D];中國人民解放軍軍事醫(yī)學科學院;2007年

4 林英姿;斑點熱群立克次體海南株的分離和鑒定[D];江西醫(yī)學院;2001年

，

本文編號：2455583