【摘要】：弓形蟲病(toxoplasraosis),又稱弓形體病,它是由一種球蟲,即弓形體(Toxoplasma gandii)所引起的一種寄生原蟲病。弓形蟲體內(nèi)存在一種重要的分泌抗原致密性顆粒蛋白,經(jīng)臨床人體和動(dòng)物試驗(yàn)結(jié)果顯示,致密性顆粒蛋白的反應(yīng)原性和免疫原性均很高,而目前對(duì)GRA3抗原的研究顯示,該抗原可以誘發(fā)機(jī)體產(chǎn)生體液免疫,所以該抗原對(duì)于將來用于該病的臨床診斷及其疫苗的研究均具有很大價(jià)值。本研究旨在成功構(gòu)建致密性顆粒蛋白GRA3(Dense granle protein GRA3)原核表達(dá)載體,進(jìn)一步利用表達(dá)的重組GRA3蛋白抗原制備抗重組GRA3的單克隆抗體,并進(jìn)行單克隆抗體的鑒定及應(yīng)用,進(jìn)一步的研究該抗原的應(yīng)用價(jià)值,為弓形蟲病的診斷及亞單位疫苗的研制奠定基礎(chǔ)。 本研究根據(jù)GenBank AF414079已發(fā)表的弓形蟲GRA3基因序列設(shè)計(jì)了一對(duì)特異性的引物,利用PCR技術(shù)成功擴(kuò)增出了一段687bp的基因片段,對(duì)該基因進(jìn)行了序列分析,結(jié)果顯示該基因片段為弓形蟲GRA3基因；并成功構(gòu)建了原核表達(dá)載體pGEX-GRA3;將構(gòu)建好的表達(dá)載體,經(jīng)IPTG誘導(dǎo),在大腸桿菌BL21中進(jìn)行了表達(dá),經(jīng)SDS-聚丙烯酰胺凝膠電泳顯示,獲得了分子量為50ku的融合蛋白GST-GRA3,經(jīng)分析大部分以可溶性蛋白的形式存在。Western-Blotting免疫印跡分析,該重組蛋白與弓形蟲陽性血清發(fā)生特異性反應(yīng),說明該蛋白具有一定的反應(yīng)原性。 為進(jìn)一步探討重組蛋白作為診斷抗原的可能性。本研究利用高效表達(dá)的重組GRA3蛋白免疫BALB/c小鼠,采用細(xì)胞融合技術(shù),經(jīng)間接ELISA方法篩選和有限稀釋法克隆后獲得一株抗重組GRA3蛋白的雜交瘤細(xì)胞株(5E4)。經(jīng)鑒定,其McAb的亞型為IgGl型。ELISA和western blotting試驗(yàn)結(jié)果表明,獲得的5E4McAb可特異性的識(shí)別弓形蟲GRA3蛋白,表明5E4McAb識(shí)別的表位可能位于GRA3蛋白的保守區(qū)域。 并以兔抗弓形蟲IgG為捕獲抗體,利用抗重組GRA3蛋白單克隆抗體為檢測(cè)抗體,成功建立了用于弓形蟲病檢測(cè)的單抗雙夾心ELISA方法,該方法對(duì)弓形蟲檢病檢測(cè)靈敏度達(dá)0.154ug/mL。用該法對(duì)延邊某豬場(chǎng)152頭豬進(jìn)行檢測(cè),結(jié)果陽性率為13.2%。并與常規(guī)ELISA相比較,該法更加敏感、特異,更有利于準(zhǔn)確診斷。為臨床研制弓形蟲診斷試劑、治療試劑奠定了基礎(chǔ),為制備疫苗的候選基因的篩選提供依據(jù),同時(shí)為弓形蟲的檢測(cè)和區(qū)域流行病學(xué)調(diào)查提供了一種簡(jiǎn)便快速的診斷方法。
[Abstract]:Toxoplasmosis (toxoplasraosis),) is a parasitic protozoa caused by a coccidiosis, I. e., Toxoplasma gondii (Toxoplasma gandii). In Toxoplasma gondii, there is an important antigen-secreting dense granule protein, which has been shown to be highly reactive and immunogenicity by clinical human and animal trials. This antigen can induce humoral immunity, so it has great value for clinical diagnosis and vaccine research in the future. The purpose of this study was to construct the prokaryotic expression vector of dense granule protein (GRA3 (Dense granle protein GRA3) successfully, and to further use the expressed recombinant GRA3 protein antigen to prepare monoclonal antibody against recombinant GRA3, and to identify and apply the monoclonal antibody. Further study on the application value of this antigen will lay a foundation for the diagnosis of toxoplasmosis and the development of subunit vaccine. In this study, a pair of specific primers were designed according to the GRA3 gene sequence of Toxoplasma gondii published by GenBank AF414079. A segment of 687bp gene was successfully amplified by PCR technique, and the gene was sequenced. The results showed that the gene fragment was GRA3 gene of Toxoplasma gondii. The prokaryotic expression vector pGEX-GRA3; was successfully constructed. The constructed expression vector was induced by IPTG and expressed in Escherichia coli BL21. The fusion protein GST-GRA3, with molecular weight of 50ku was obtained by SDS- polyacrylamide gel electrophoresis. Western-Blotting Western blot analysis showed that the recombinant protein reacted specifically with Toxoplasma gondii positive serum, which indicated that the recombinant protein had a certain reactivity. In order to further explore the possibility of recombinant protein as diagnostic antigen. In this study, BALB/c mice were immunized with highly expressed recombinant GRA3 protein. A hybridoma cell line (5E4) against recombinant GRA3 protein was obtained by indirect ELISA screening and limited dilution method. The McAb subtype of Toxoplasma gondii was identified as IgGl. The results of ELISA and western blotting tests showed that the obtained 5E4McAb could specifically recognize GRA3 protein of Toxoplasma gondii, indicating that the epitope recognized by 5E4McAb might be located in the conserved region of GRA3 protein. Using rabbit anti-Toxoplasma IgG as capture antibody and monoclonal antibody against recombinant GRA3 protein as detection antibody, a single-antibody double sandwich ELISA method for detection of Toxoplasma gondii was successfully established. The sensitivity of this method for detection of Toxoplasma gondii was 0.154 ugmL. mL. This method was used to detect 152 pigs in a pig farm in Yanbian, and the positive rate was 13.22. Compared with conventional ELISA, this method is more sensitive, specific and more helpful for accurate diagnosis. It lays a foundation for the clinical development of Toxoplasma gondii diagnostic reagents and therapeutic reagents and provides the basis for screening candidate genes for the preparation of vaccines. It also provides a simple and rapid diagnostic method for the detection of Toxoplasma gondii and the investigation of regional epidemiology.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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