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牛病毒性腹泻病毒E2蛋白的原核表达及抗体间接ELISA检测方法的建立
孙丹,黄小洁,秦义娴,高月异,孙淼,刘丹
0
(北京市兽药饲料监测中心;中国兽医药品监察所)
摘要:
为建立检测牛病毒性腹泻病毒抗体的间接ELISA检测方法,利用大肠杆菌表达系统成功表达E2蛋白并纯化,利用纯化后的蛋白作为包被抗原,用方阵法对影响ELISA试验的各个因素进行优化,并进行特异性、敏感性和重复性试验。确定E2抗原最佳包被浓度为1 μg/mL,最佳血清稀释度为1∶40,最佳封闭液为1%明胶,最佳血清作用方式为37 ℃作用30 min,酶标抗体的最佳作用方式为1∶2000稀释、37 ℃作用30 min;最佳底物作用时间为室温20 min,阳性临界值为OD450nm≥0.255。与血清中和试验和商品化试剂盒进行比较,总符合率分别为98%和96%,批内和批间重复性试验的变异系数均小于10%。该方法与其它常见牛病毒阳性血清均无交叉反应。本研究成功建立了间接ELISA方法,为进一步用于大批量牛血清样本的抗体检测和流行病学研究奠定了基础。
关键词:  牛病毒性腹泻病毒  E2蛋白  原核表达  间接ELISA方法
DOI:
投稿时间:2023-05-19修订日期:2023-10-20
基金项目:中国兽医药品监察所兽药行业公益性重点专项“牛羊主要病原检测新技术与制品效力检验替代方法研究”(GY202103)
E.coli Expression of Bovine Viral Diarrhea Virus E2 Protein and Establishment of Indirect ELISA Method for Antibody Detection
(Beijing veterinary drug and feed monitoring center)
Abstract:
In order to establish an indirect ELISA method for the detection of bovine viral diarrhea virus antibodies, E2 protein was successfully expressed in E.coli expression system. The purified protein was used as the coating antigen, and the factors of ELISA test were optimized by square matrix method. Specificity, sensitivity and repeatability tests were performed. The optimal conditions of ELISA were as follows: 1 μg/mL E2 antigen coated at 4 ℃ for overnight, 1% gelatin sealed at 37 ℃for 1 h, 1∶40 dilution serum incubation at 37 ℃for 30 min, 1∶2000 diluted second antibody incubated at 37 ℃ for 30 min and colored for 20 min, and the positive critical value was ODOD450nm≥0.255. Compared with the serum neutralization test and IDEXX ELISA, the total coincidence rate was 98% and 96%, respectively.The intra-assay and inter-assay coefficient of variation was less than 10%. This method had no cross reaction with common bovine virus positive serum. Therefore, the established indirect ELISA method can be used for antibody detection of large quantities of samples and epidemiological studies.
Key words:  bovine viral diarrhea virus  E2 protein  E.coli expression  indirect ELISA

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