猪流行性腹泻病毒S蛋白的单抗制备及夹心ELISA检测PEDV方法建立与应用

发布时间：2018-07-23 08:20

【摘要】：猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行腹泻病毒(PEDV)引起的一种临床上以新生仔猪急性腹泻、呕吐、脱水及高死亡率为特征的高度接触性传染病,给全球养猪国家造成了巨大经济损失。目前有关本病的诊断方法多偏重于分子生物学方法,如RT-PCR。虽然RT-PCR方法具有特异、敏感、准确等特点,但该方法成本昂贵、难以应用于大规模临床样品的诊断和流行病学调查。因此,迫切需要建立一种特异、敏感、快速简便诊断PED的方法。本实验应用PCR扩增出PEDV-BS-14毒株的纤突糖蛋白S基因的部分序列,并将其克隆到原核表达载体p GEX-4T-1中,构建出p GEX-4T-1-S表达载体。应用IPTG诱导表达重组S蛋白,并将纯化的重组蛋白以弗氏完全/不完全佐剂乳化后免疫小鼠,制备抗PEDV S蛋白的单克隆抗体。以制备的抗体建立了检测PEDV抗原的双抗体夹心ELISA方法,并确定、优化了所建立的ELISA反应条件。捕获抗体的最佳包被量为0.6μg,酶标抗体的最佳稀释倍数为1:1000,最佳封闭液为2%BSA,最佳封闭时间为1 h,酶标抗体最佳作用时间为1 h,最佳显色时间为10 min。以建立的双抗体夹心ELISA和RT-PCR方法对PEDV粪便样品进行了比较检测,两者的符合率为100%。应用ELISA检测了吉林省疑似PED流行猪场的粪便样品,发现PEDV阳性检出率为68%。综上所述,本研究成功表达出重组PEDV S蛋白,制备抗PEDV的单克隆抗体,建立了特异、敏感、快速检测PEDV病毒抗原的双抗体夹心ELISA方法,并对吉林省猪群感染PEDV进行了病原流行病学的研究,发现猪群的PEDV阳性感染率为68%,该研究结果将为PEDV诊断及流行病学研究提供技术手段和流行病学理论依据。
[Abstract]:Porcine epidemic diarrhea (PEDV) is a highly contagious disease caused by porcine epidemic diarrhea virus (PEDV), which is characterized by acute diarrhea, vomiting, dehydration and high mortality in newborn piglets. At present, the diagnosis of this disease is mainly focused on molecular biological methods, such as RT-PCR. Although RT-PCR has the characteristics of specificity, sensitivity and accuracy, it is difficult to be used in the diagnosis and epidemiological investigation of large-scale clinical samples because of its high cost. Therefore, it is urgent to establish a specific, sensitive, rapid and simple method for the diagnosis of PED. In this experiment, the partial sequence of fibrin S gene of PEDV-BS-14 strain was amplified by PCR, and cloned into prokaryotic expression vector pGEX-4T-1 to construct pGEX-4T-1-S expression vector. IPTG was used to induce the expression of recombinant S protein. The purified recombinant protein was emulsified with Freund's complete / incomplete adjuvant to prepare monoclonal antibody against PEDV S protein. A sandwich Elisa method for the detection of PEDV antigen was established by using the prepared antibodies, and the conditions of Elisa reaction were optimized. The best encapsulation amount of the capture antibody is 0.6 渭 g, the best dilution multiple of the enzyme labeled antibody is 1: 1000, the best blocking liquid is 2BSA, the best blocking time is 1 hour, the best time of the enzyme labeled antibody is 1 hour, the best color reaction time is 10 min. Double antibody sandwich Elisa and RT-PCR were used to detect PEDV fecal samples and the coincidence rate was 100. The fecal samples of suspected PED epidemic pig farm in Jilin province were detected by Elisa, and the positive rate of PEDV was 68%. In conclusion, the recombinant PEDV S protein was successfully expressed in this study, and the monoclonal antibody against PEDV was prepared. A sandwich Elisa was established for the specific, sensitive and rapid detection of PEDV virus antigen. The pathogenic epidemiology of PEDV in swine herd in Jilin Province was studied. It was found that the positive infection rate of PEDV in swine herd was 68. The results of the study would provide technical means and epidemiological theoretical basis for PEDV diagnosis and epidemiological study.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28

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