猪流行性腹泻病毒病S蛋白单抗制备及夹心ELISA检测PEDV方法的建立与应用
发布时间：2018-08-30 20:21
【摘要】：应用PCR扩增出PEDV毒株的纤突糖蛋白S基因序列,并将其克隆到原核表达载体pGEX-4T-1中,构建出pGEX-4T-1-S表达载体。应用IPTG诱导表达重组S蛋白,并将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,并以此建立了检测PEDV抗原的双抗体夹心ELISA方法,确定、优化了所建立的ELISA反应条件。以建立的双抗体夹心ELISA和RT-PCR方法对PEDV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%。该研究为PED诊断及流行病学调查提供了一种特异、敏感、快速和易于在基层应用的技术方法及本病的防治提供了流行病学理论依据。
[Abstract]:The protein S gene of PEDV 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. Mice were immunized with Freund's complete / incomplete adjuvant to prepare monoclonal antibody against PEDV S protein. A sandwich ELISA method for detecting PEDV antigen was established. The ELISA reaction conditions were optimized. The double antibody sandwich ELISA and RT-PCR method were used to compare and detect the PEDV samples. The coincidence rate of the two methods was 100. ELISA was used to detect fecal samples of suspected PED epidemic pig farm in Jilin province, and the positive rate of PEDV was 68%. This study provides an epidemiological theoretical basis for the diagnosis and epidemiological investigation of PED, which is specific, sensitive, rapid and easy to be applied at the grass-roots level.
【作者单位】： 吉林大学动物医学学院;吉林大学人兽共患病研究所;吉林大学动物科学学院;
【基金】：国家重点研发计划资助项目(2017YFD0500104,2016YFD0500904)
【分类号】：S852.651
[Abstract]:The protein S gene of PEDV 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. Mice were immunized with Freund's complete / incomplete adjuvant to prepare monoclonal antibody against PEDV S protein. A sandwich ELISA method for detecting PEDV antigen was established. The ELISA reaction conditions were optimized. The double antibody sandwich ELISA and RT-PCR method were used to compare and detect the PEDV samples. The coincidence rate of the two methods was 100. ELISA was used to detect fecal samples of suspected PED epidemic pig farm in Jilin province, and the positive rate of PEDV was 68%. This study provides an epidemiological theoretical basis for the diagnosis and epidemiological investigation of PED, which is specific, sensitive, rapid and easy to be applied at the grass-roots level.
【作者单位】： 吉林大学动物医学学院;吉林大学人兽共患病研究所;吉林大学动物科学学院;
【基金】：国家重点研发计划资助项目(2017YFD0500104,2016YFD0500904)
【分类号】：S852.651
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