牛副流感病毒3型抗体间接ELISA检测方法的建立与初步应用

旨在建立牛副流感病毒3型(BPIV3)抗体间接ELISA检测方法。克隆NP-HN截短串联基因并构建原核表达载体pET28a(+)-NP-HN,诱导纯化NP-HN重组蛋白作为包被抗原,优化ELISA反应条件,建立BPIV3抗体间接ELISA检测方法,进一步与病毒中和试验和进口ELISA试剂盒进行比较,应用本方法对270份临床血清样本进行了检测。结果表明,表达的NP-HN重组蛋白大小为44ku,具有良好的反应原性,建立的ELISA检测方法特异性强,与牛的主要呼吸道病原如牛病毒性腹泻病毒、牛传染性鼻气管炎病毒等均无交叉反应,批内和批间重复性试验的变异系数小于8%,与病毒中和试验和进口商品化ELISA试剂盒的总符合率分别为96.67%和98.89%。对采自山东、辽宁和天津的270份临床血清样本检测后总的阳性率为82.59%(223/270)。建立的BPIV3抗体间接ELISA方法具有较好的特异性和敏感性,可应用于BPIV3的流行病学调查和抗体检测研究。

Establishment and Preliminary Application of an Indirect ELISA for Detecting Antibodies against Bovine Parainfluenza Virus Type 3

In order to establish an indirect ELISA (iELISA)for detecting antibodies against bovine parain-fluenza virus type 3 (BPIV3),the truncated NP-HN gene was cloned and pET28a(+)-NP-HN vector of prokaryotic expression was constructed.We optimized the ELISA reaction conditions using the NP-HN re-combinant protein as coating antigen,and established an iELISA method to specifically detect the positive serum of BPIV3.Further the iELISA method was compared with virus neutralization test and imported ELISA kits,respectively.Finally,270 clinical serum samples were tested.The results showed that the re-combinant 44 ku protein could highly expressed in the form of soluble and inclusion bodies in E.coli,which had good antigenic specificity and reactionogenicity,and had no cross reaction with positive serum of bovine viral diarrhea virus,infectious bovine rhinotracheitis virus and other respiratory pathogens.The coefficients of variation for intra and inter-assay were lower than 8%.The total coincidence rate with virus neutraliza-tion test and imported ELISA kit were 96.67% and 98.89%.The total positive rate of 270 clinical serum samples from Shandong,Liaoning,and Tianjin was 82.59% (223/270).These results demonstrated that BPIV3 indirect ELISA method was specific and sensitive,and can be used for epidemiological investigation and antibody detection for BPIV3.