检测猪丹毒杆菌抗体重组SpaA ELISA的建立

发布时间：2018-03-08 19:42

本文选题：猪丹毒杆菌　切入点：表面保护性蛋白SpaA　出处：《微生物学通报》2017年03期 　论文类型：期刊论文

【摘要】：【目的】利用表达纯化的猪丹毒杆菌表面保护性蛋白SpaA,建立检测猪丹毒杆菌抗体的间接ELISA方法。【方法】克隆扩增猪丹毒杆菌SpaA基因,并将SpaA基因与原核表达载体p GEX-6P-1连接,通过PCR、双酶切及测序鉴定后,将阳性重组质粒转化入受体菌E.coli Rosetta(DE3),并利用IPTG进行诱导表达,SDS-PAGE和Western blot鉴定表达产物。将SpaA重组蛋白按不同浓度包被酶标板,通过方阵滴定法确定最佳抗原包被浓度及血清稀释度,并对其他条件进行优化,最终建立检测猪丹毒杆菌抗体的间接ELISA方法。【结果】利用克隆表达的猪丹毒杆菌SpaA蛋白作抗原,通过方阵滴定法确定蛋白最佳包被浓度为1.0 mg/L,血清的最佳稀释度为1:100,建立了检测猪丹毒杆菌抗体的间接ELISA方法,批内及批间变异系数均小于10%,具有较好的重复性及特异性。用建立的间接ELISA方法检测猪丹毒疫苗免疫后的健康猪血清样品,检测结果与美国TSZ公司猪丹毒杆菌抗体检测试剂盒和Western blot鉴定结果进行对比,两者总符合率分别为92.20%、92.59%。【结论】试验利用原核表达的SpaA重组蛋白作抗原建立的检测猪丹毒杆菌抗体的间接ELISA方法,特异性强、重复性好、敏感性高,可用于猪丹毒杆菌的抗体检测及流行病学调查。
[Abstract]:[objective] to establish an indirect ELISA method for the detection of porcine erysipelas antibody using purified SpaA protein. [methods] SpaA gene was cloned and amplified, and the SpaA gene was ligated with prokaryotic expression vector p GEX-6P-1. The positive recombinant plasmid was transformed into E. coli Rosetta-DE3 by PCR, double enzyme digestion and sequencing. The expressed product was identified by IPTG induced expression of SDS-PAGE and Western blot. The recombinant protein of SpaA was coated with different concentration of enzyme labeling plate. The best antigen coating concentration and serum dilution were determined by square array titration, and other conditions were optimized. An indirect ELISA method was established to detect the antibody of erysipelas. [results] the cloned SpaA protein of erysipelas was used as antigen. The best coating concentration of protein was 1.0 mg / L and the optimal dilution of serum was 1: 100. An indirect ELISA method was established for the detection of erysipelas antibodies. The coefficient of variation within and between batches was less than 10, with good reproducibility and specificity. The established indirect ELISA method was used to detect the serum samples of healthy pigs immunized with porcine erysipelas vaccine. The results were compared with those of TSZ's antibody detection kit and Western blot. The total coincidence rates of the two methods were 92.20 and 92.59.Conclusion the indirect ELISA method using prokaryotic expressed SpaA recombinant protein as antigen to detect the antibody of erysipelas is highly specific, reproducible and sensitive. It can be used for antibody detection and epidemiological investigation of erysipelas.
【作者单位】：安徽农业大学动物科技学院;安徽省畜禽产业共性技术研究院;
【基金】：国家星火计划重点项目(No.2014GA710002) 安徽省质量工程项目(No.2013sxzx008) 安徽农业大学大学生创新基金项目(No.XJ2015142) 安徽省生猪产业体系基金项目~~
【分类号】：S852.6

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