弓形虫PAPS抗体诊断液及SAG1表达研究

发布时间：2018-04-16 08:15

本文选题：弓形虫 + SAG1基因　；参考：《西南大学》2006年硕士论文

【摘要】：弓形虫是一种机会性致病原虫，分布十分广泛，由其导致的弓形虫病也是危害严重的人畜共患病之一。本文针对诊断方法和诊断所用抗原展开研究，以期建立快速简便的诊断方法和获得特异性强的诊断抗原。一、PAPS(Polyaledehyde Polystyrene,PAPS)弓形虫抗体诊断液的制备研究。为了满足基层现场疾病诊断和动物检疫的需要，对用聚苯乙烯做载体的乳胶凝集试验(Latex agglutination test,LAT)进行了优化。通过对聚苯乙烯乳胶载体的改造，使其在较温和的化学反应条件下表面被聚醛化，聚苯乙烯载体携带功能团后，载体变得更加疏松且易混匀，颗粒直径变大，改变了以往聚苯乙烯乳胶以物理吸附的方式结合蛋白质，使聚醛化后的聚苯乙烯可以化学吸附的方式结合蛋白质；对PAPS微球载体进行保存期试验，结果发现，于4℃保存九个月其功能团和结合蛋白的稳定性没有发生变化；对PAPS弓形虫抗体诊断液进行保存期试验，结果表明，于4℃保存九个月其敏感性、稳定性没有发生变化；以市售标准间接血凝试剂盒作对照，利用所购买试剂盒内的阳性血清和阴性血清对PAPS弓形虫抗体诊断液进行敏感性检测，结果显示，PAPS弓形虫抗体诊断液敏感性比间接血凝试剂高出两个稀释度以上；利用血吸虫、锥虫、旋毛虫、肺吸虫、肝片吸虫、囊虫阳性血清与PAPS弓形虫抗体诊断液进行凝集试验，结果均呈阴性，未见发生交叉反应；运用镜检见虫的弓形虫阳性猪血清和经小鼠传代接种方法证实无弓形虫的健康猪血清进行PAPS凝集试验，结果发现阴阳性符合率均为100％；对上海市某区的九个地方2800只犬进行弓形虫抗体调查，总的阳性犬数485只，平均阳性率为17.3％。本方法可以使聚苯乙烯乳胶载体更稳定地结合蛋白质，可延长诊断液的保存时间，实验结果也更容易通过肉眼观察，且具有特异性强、灵敏度高、重复性好、快速、简便、无需特殊仪器设备等优点。二、截短弓形虫SAGI(surface antigen 1)表面抗原的原核表达研究。 SAG1作为弓形虫速殖子的表面抗原，已被证明能有效的刺激机体产生抗体，也是目前公认最有潜力的免疫诊断抗原，试验将速殖子SAG1的基因片段截短使其在大肠杆菌中表达，，以求获得具有良好抗原活性的重组抗原。本研究利用小白鼠进行腹腔传代收获弓形虫速殖子，然后直接利用它提取基因组DNA进行PCR，将PCR产物回收后通过T-A克隆的方式直接与pGEM-T-easy载体相连，将连接物转化到DH5α感受态细胞中，经过蓝白筛选和抗性筛选，选挑取阳性克隆，构建pGEM-T-easy+SAGI重组质粒；将pGEM-T-easy+SAG1重组质粒经EcoRI和HindⅢ双酶切，回收目的片断，连接到pGEX-2T上，将连接物转化到DH5α感受态细胞中，经过抗性筛选，选挑取阳性克隆，构建重组质粒pGEX-2T+SAG1；再将pGEX-2T+SAG1转入E.coli(BL21)中用IPTG诱导进行表达，经过确定最佳诱导时间和最适IPTG浓度来优化表达条件，经过一系列缓冲液的处理，离心纯化包涵体，将纯化产物进行SDS-PAGE电泳，然后将其转移到NC膜上进行Western-Blotting杂交来分析抗原活性。结果显示，在DH5α感受态细胞中成功构建pGEM-T-easy+SAG1和pGEX-2T+SAG1重组质粒，通过IPTG诱导，此片断在大肠杆菌中成功高效表达，相对分子量约61.5kD，据Western-Blotting显示，表达产物能被
[Abstract]:Toxoplasma gondii is an opportunistic protozoan, is widely distributed, which causes toxoplasmosis is serious zoonotic disease. According to the diagnostic methods and diagnostic antigen used research, in order to establish a rapid diagnosis method is simple and highly specific diagnostic antigen.
A study on the preparation of PAPS (Polyaledehyde Polystyrene, PAPS) antibody diagnostic solution for Toxoplasma gondii.
In order to meet the needs of primary site disease diagnosis and animal quarantine, the latex agglutination test using polystyrene as carrier (Latex agglutination test, LAT) were optimized. Through modification of polystyrene latex carrier, the chemical reactions in mild conditions by surface aldehyde, polystyrene carrier group. The carrier has become more loose and easy to mix, larger particle diameter, changed polystyrene latex by physical adsorption method combined with protein, poly formaldehyde after the polystyrene chemical adsorption way binding protein; PAPS microspheres for preservation test, results showed that at 4 DEG C for nine months the functional group and combined with the protein stability did not change; the PAPS of Toxoplasma gondii antibody in the diagnosis of liquid storage test, results showed that at 4 DEG C for nine months the sensitivity, no stability A change to a commercially available standard; indirect hemagglutination test kit were purchased by the kit in positive and negative sera sensitivity detection, Toxoplasma antibody diagnostic reagents on PAPS results showed that PAPS of Toxoplasma gondii antibody diagnostic sensitivity than indirect hemagglutination test was higher than two dilution degrees above; the use of Schistosoma japonicum, Trypanosoma, Trichinella, paragonimiasis, Fasciola hepatica, cysticercosis and Toxoplasma PAPS positive serum antibody in the diagnosis of liquid agglutination test results were negative, no cross reaction; using microscopic insect Toxoplasma gondii positive pig and PAPS agglutination test method isolated mice confirmed that Toxoplasma free healthy pigs results serum, positive and negative coincidence rate was 100%; for Toxoplasma antibody survey of a district of Shanghai city in nine places in 2800 dogs, the total number of positive dogs was 485, the average positive rate was 17.3%. The method can make the polystyrene latex carrier bind to the protein more stable, prolong the storage time of the diagnostic solution, and the experimental result is more easily observed through the naked eye. It has the advantages of high specificity, high sensitivity, good reproducibility, fast and simple, and no special instrument and equipment are needed.
Two, study on the prokaryotic expression of the surface antigen of SAGI (surface antigen 1) of truncated Toxoplasma gondii.
SAG1 as a surface antigen of Toxoplasma gondii tachyzoites, has been shown to be effective in stimulating the body to produce antibodies, immune antigen is currently recognized as the most promising test, the gene fragment truncated tachyzoites. The expression of SAG1 in Escherichia coli, in order to obtain good immunological activity. In this study, the recombinant antigen intraperitoneal passage harvest of Toxoplasma gondii in mice, and then directly use it to extract genomic DNA by PCR, the product of PCR was cloned by T-A after the way is directly connected with the pGEM-T-easy carrier, will be connected into DH5 alpha feeling state in the cell, through the blue white screening and resistance screening, selection of positive clones pGEM-T-easy+SAGI, construction of recombinant plasmid; recombinant plasmid pGEM-T-easy+SAG1 by EcoRI and Hind III restriction enzyme digestion, amplified fragments, connected to the pGEX-2T, the connection is transformed into DH5 competent cells by alpha. After resistance screening, selected positive clones, recombinant plasmid pGEX-2T+SAG1; then pGEX-2T+SAG1 into E.coli (BL21) induced by IPTG for expression. After determining the optimal induction time and the optimal concentration of IPTG to optimize the expression conditions, through a series of processing buffer, centrifugation and purification of inclusion bodies, the purified product was SDS-PAGE. And then transferred to NC membrane by Western-Blotting hybridization analysis of antigenic activity. The results showed that the DH5 alpha feeling pGEM-T-easy+SAG1 was successfully constructed and recombinant plasmid pGEX-2T+SAG1 cells, induced by IPTG, the fragment in E.coli successfully expressed the relative molecular weight of about 61.5kD, according to Western-Blotting, the expression product can be

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392.1

【参考文献】