弓形虫GRA3基因的原核表达及单克隆抗体的制备与初步应用研究

发布时间：2018-11-22 13:48

【摘要】：弓形虫病(toxoplasraosis),又称弓形体病,它是由一种球虫,即弓形体(Toxoplasma gandii)所引起的一种寄生原虫病。弓形虫体内存在一种重要的分泌抗原致密性颗粒蛋白,经临床人体和动物试验结果显示,致密性颗粒蛋白的反应原性和免疫原性均很高,而目前对GRA3抗原的研究显示,该抗原可以诱发机体产生体液免疫,所以该抗原对于将来用于该病的临床诊断及其疫苗的研究均具有很大价值。本研究旨在成功构建致密性颗粒蛋白GRA3(Dense granle protein GRA3)原核表达载体,进一步利用表达的重组GRA3蛋白抗原制备抗重组GRA3的单克隆抗体,并进行单克隆抗体的鉴定及应用,进一步的研究该抗原的应用价值,为弓形虫病的诊断及亚单位疫苗的研制奠定基础。本研究根据GenBank AF414079已发表的弓形虫GRA3基因序列设计了一对特异性的引物,利用PCR技术成功扩增出了一段687bp的基因片段,对该基因进行了序列分析,结果显示该基因片段为弓形虫GRA3基因；并成功构建了原核表达载体pGEX-GRA3;将构建好的表达载体,经IPTG诱导,在大肠杆菌BL21中进行了表达,经SDS-聚丙烯酰胺凝胶电泳显示,获得了分子量为50ku的融合蛋白GST-GRA3,经分析大部分以可溶性蛋白的形式存在。Western-Blotting免疫印迹分析,该重组蛋白与弓形虫阳性血清发生特异性反应,说明该蛋白具有一定的反应原性。为进一步探讨重组蛋白作为诊断抗原的可能性。本研究利用高效表达的重组GRA3蛋白免疫BALB/c小鼠,采用细胞融合技术,经间接ELISA方法筛选和有限稀释法克隆后获得一株抗重组GRA3蛋白的杂交瘤细胞株(5E4)。经鉴定,其McAb的亚型为IgGl型。ELISA和western blotting试验结果表明,获得的5E4McAb可特异性的识别弓形虫GRA3蛋白,表明5E4McAb识别的表位可能位于GRA3蛋白的保守区域。并以兔抗弓形虫IgG为捕获抗体,利用抗重组GRA3蛋白单克隆抗体为检测抗体,成功建立了用于弓形虫病检测的单抗双夹心ELISA方法,该方法对弓形虫检病检测灵敏度达0.154ug/mL。用该法对延边某猪场152头猪进行检测,结果阳性率为13.2%。并与常规ELISA相比较,该法更加敏感、特异,更有利于准确诊断。为临床研制弓形虫诊断试剂、治疗试剂奠定了基础,为制备疫苗的候选基因的筛选提供依据,同时为弓形虫的检测和区域流行病学调查提供了一种简便快速的诊断方法。
[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.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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