基于重组E2蛋白的牛病毒性腹泻病毒间接ELISA抗体检测方法的建立与评价

doi:10.16431/j.cnki.1671-7236.2024.12.032

摘要/Abstract

摘要： 【目的】通过真核系统表达牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)E2蛋白，以此为包被蛋白建立一种BVDV间接ELISA抗体检测方法，以解决中国BVDV抗体检测技术不足的问题。【方法】将BVDV E2基因克隆至真核表达载体pTT5，转染293F细胞，待50%细胞死亡后进行超声破碎，并使用Ni离子亲和层析的方法纯化重组E2(rE2)蛋白；以rE2蛋白作为包被抗原，通过对各反应条件进行优化后建立检测BVDV抗体的间接ELISA方法，进行敏感性、特异性、重复性评价，并与中和试验进行比对。用所建方法对不同省份的牛血清样本进行检测。【结果】采用293F细胞表达了rE2蛋白，经亲和层析方法纯化，rE2蛋白的纯度为99%，浓度为1.74 mg/mL；通过对各个反应条件优化，成功建立了BVDV间接ELISA抗体检测方法。该方法与商品化试剂盒的灵敏度一致，均可检出抗体效价临界血清(VN＝1)；具有良好的特异性，与传染性牛鼻气管炎病毒(IBRV)、牛副流感病毒-3(BPIV-3)、牛呼吸道合胞病毒(BRSV)和牛疱疹病毒-1(BHV-1)抗体阳性血清均无交叉反应；板内和板间重复性试验的变异系数均在5%以内，证明该方法重复性较好；与血清中和试验的符合率为97.5%。临床样本检测结果显示，共检测出364份阳性血清和64份阴性血清。【结论】基于真核表达的rE2蛋白所建立的BVDV间接ELISA抗体检测方法具有良好的特异性和敏感性，可用于BVDV抗体检测，本研究结果为中国牛病毒性腹泻防控工作提供一种有效的技术手段。

关键词: 牛病毒性腹泻病毒(BVDV); E2蛋白; 间接ELISA

Abstract: 【Objective】 To provide an effective technique for serological investigation,prevention and control of bovine viral diarrhea (BVD) in China，the E2 protein of Bovine viral diarrhea virus (BVDV) was expressed by eukaryotic system,and used as the coating material to establish a BVDV indirect ELISA antibody detection method for clinic detection.【Method】 The BVDV E2 gene was cloned into the eukaryotic expression vector pTT5,and 293F cells were transfected.After 50% of 293F cells died,the cells were collected,ultrasonicated and the recombinant E2 (rE2) protein was purified by using Ni ion affinity chromatography.rE2 protein was used as coating antigen,and the conditions of each reaction were optimized,an indirect ELISA method for detection of BVDV antibody was developed,and its specificity,sensitivity,repeatability and conformity with the classical neutralisation test were evaluated.The established method was used for detection of bovine serum samples from different provinces of China.【Result】 The rE2 protein was expressed by 293F cells and purified by affinity chromatography,and the purity was 99% with a concentration of 1.74 mg/mL.Through the optimization of each reaction condition,an indirect ELISA antibody detection method for BVDV was successfully established.The method was sensitive enough to detect the weak antibody potency serum (VN＝1),which was consistent with that of the commercial kit.It had good specificity and no cross-reaction with the Infections bovine rhinotracheitis virus (IBRV),Bovine parainfluenza virus 3,(BPIV-3),Bovine respiratory syncytial virus (BRSV) and Bovine herpesvirus 1 (BHV-1) antibody-positive sera.The coefficients of variation of the intra- and inter-batch reproducibility tests were within 5%,which showed good reproducibility.And the conformity with the classical neutralisation test was 97.5%.Bovine serum samples from different provinces were detected and total 364 sera were positive and 64 sera were negative sera.【Conclusion】 The indirect ELISA detection method for antibody against BVDV based on eukaryotic expression of rE2 protein had good specificity and sensitivity,and provided an effective means for the prevention and control of BVD in China.

Key words: Bovine viral diarrhea virus (BVDV); protein E2; indirect ELISA

中图分类号:

S852.65⁺3

张宜宸, 孙明军, 邓昌顺, 丁家波, 徐宝梁, 陈磊, 全春兰, 赵康, 范学政, 秦彤. 基于重组E2蛋白的牛病毒性腹泻病毒间接ELISA抗体检测方法的建立与评价[J]. 中国畜牧兽医, 2024, 51(12): 5459-5469.

ZHANG Yichen, SUN Mingjun, DENG Changshun, DING Jiabo, XU Baoliang, CHEN Lei, QUAN Chunlan, ZHAO Kang, FAN Xuezheng, QIN Tong. Development and Evaluation of an Indirect ELISA Based on Recombinant E2 Protein for Detection of Bovine Viral Diarrhea Virus Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5459-5469.

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