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为建立基于重组外膜蛋白A的检测禽源多杀性巴氏杆菌抗体的间接ELISA方法,扩增去除信号肽的外膜蛋白A(outer membrane protein A,OmpA)基因,定向克隆到载体pET32a,构建重组表达质粒pET32a-PmOmpA,转化至Escherichia coli BL21(DE3)以IPTG进行诱导,通过镍离子亲和层析纯化重组蛋白,进行SDS-PAGE和Westernblotting分析。以重组蛋白为包被抗原建立间接ELISA方法,方阵滴定法确定其最佳包被浓度和血清的最佳稀释度。结果,重组蛋白能与阳性血清发生良好的免疫反应。重组蛋白的包被浓度为4mg/L,血清的最佳稀释度为1∶80。SPF鸡的新城疫、鸡白痢和大肠杆菌病阳性血清,隔离饲养的番鸭鸭瘟、鸭病毒性肝炎和鸭疫里默氏菌病阳性血清,用该方法检测均为阴性。板内变异系数和板间变异系数均小于10%。ELISA方法的敏感性比直接凝集试验高出80倍以上。以重组外膜蛋白A建立的ELISA方法具有良好的特异性、重复性和敏感性,可以用于禽霍乱感染性抗体的检测,也可用于禽霍乱灭活苗和荚膜疫苗免疫效果的检测。
In order to establish an indirect ELISA method based on recombinant outer membrane protein A for detection of Pasteurella multocida antibodies, the outer membrane protein A (OmpA) gene was amplified and cloned into the vector pET32a, The recombinant plasmid pET32a-PmOmpA was constructed and transformed into Escherichia coli BL21 (DE3) to induce by IPTG. The recombinant protein was purified by nickel ion affinity chromatography and analyzed by SDS-PAGE and Western blotting. The recombinant protein was used as an antigen to establish an indirect ELISA method. The titration method was used to determine the optimal concentration and serum dilution. As a result, the recombinant protein can react well with positive sera. Recombinant protein coating concentration of 4mg / L, the best serum dilution 1:80. SPF chickens Newcastle disease, Shigella and E. coli disease-positive sera, isolated reared duck feces duck plague, duck viral hepatitis and duck immunodeficiency virus-positive sera, using this method were negative. The intraplate variation coefficient and interplate variation coefficient were both less than 10%. ELISA method is more than 80 times more sensitive than direct agglutination test. The ELISA method established by recombinant outer membrane protein A has good specificity, repeatability and sensitivity and can be used for the detection of avian cholera infectious antibodies, and also for the detection of immune effects of inactivated vaccine and capsule vaccine of avian cholera.