基于N蛋白的牛冠状病毒抗体间接ELISA检测方法的建立与应用

Establishment and Application of Indirect ELISA Detection Method for Bovine Coronavirus Antibody Based on N Protein

为建立牛冠状病毒(Bovine coronavirus,BCoV)抗体间接ELISA检测方法,对BCoV的N基因进行克隆,利用原核表达制备重组N蛋白,以纯化的重组N蛋白作为包被抗原,建立ELISA检测方法,并对随机收集的奶牛血清样本进行检测。结果显示,重组N蛋白大小为50 ku,经Western blot鉴定重组N蛋白具有良好的反应原性;建立的ELISA检测方法抗原包被量为1.5μg/mL,血清以1∶50稀释,酶标二抗以1∶10000稀释,临界值为0.256。用该方法检测牛病毒性腹泻病毒、牛轮状病毒、牛传染性鼻气管炎病毒、口蹄疫病毒阳性血清均无交叉反应;当BCoV标准阳性血清1∶2560稀释时检测结果仍为阳性,灵敏性较好;批内和批间变异系数分别为1.83%~5.80%和2.34%~7.45%。对陕西省部分牛场收集的296份牛血清进行检测,BCoV抗体阳性率为18.24%。建立了一种快速检测BCoV抗体的间接ELISA方法,为BCoV的实验室诊断和流行病学调查提供了技术支持。

In order to establish an indirect ELISA detection method for bovine coronavirus(BCoV)antibodies,the BCoV N gene was cloned,and the recombinant N protein was prepared by prokaryotic expression.The purified recombinant N protein was used as the coating antigen to establish an ELISA detection method,and the bovine serum samples collected randomly were detected.The results showed that the size of the recombinant N protein was 50 ku,and the recombinant N protein showed good reactivity by Western blot.The established ELISA method for detection of antigen was 1.5μg/mL,the serum was diluted by 1∶50 times,and HRB-conjugated secondary antibody was diluted by 1∶10000 times.The critical value was 0.256.The positive sera of bovine viral diarrhea virus,bovine rotavirus,bovine infectious rhinotracheitis virus and foot-and-mouth disease virus were all detected negative by this method.When the BCoV standard positive serum was diluted at 1:2560,the detection result was still positive.The intra-batch and inter-batch variation coefficients were 1.83%-5.80%and 2.34%-7.45%,respectively.The positive rate of BCoV antibody was 18.24%in 296 samples of bovine sera collected from different cattle farms in Shaanxi province.In this study,an indirect ELISA method for rapid diagnosis of BCoV antibodies was established,which provided technical support for the laboratory diagnosis and epidemiological investigation of BCoV.