牛病毒性腹泻病毒E0蛋白的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2022.08.029

摘要/Abstract

摘要： 【目的】应用大肠杆菌表达系统表达牛病毒性腹泻病毒(BVDV) E0蛋白,纯化后免疫小鼠制备E0蛋白多克隆抗体用于BVDV的检测技术研究。【方法】采用PCR扩增BVDV的E0基因,将其连接至pET-28a (+)构建重组表达载体pET28a-E0。将重组质粒转化大肠杆菌BL21感受态细胞,经IPTG诱导、亲和层析纯化后,通过SDS-PAGE和Western blotting鉴定E0蛋白的表达情况。将纯化的E0蛋白免疫小鼠制备BVDV E0多克隆抗体,并通过Western blotting、细胞免疫荧光试验、ELISA等试验检测该多克隆抗体的特异性和效价,以及在BVDV检测中的应用。【结果】成功构建原核表达载体pET28a-E0,表达并纯化E0蛋白,制备的BVDV E0多克隆抗体可特异性识别纯化的E0蛋白及pET28a-E0在BL21中表达的总蛋白。进一步Western blotting、细胞免疫荧光试验、双抗夹心法ELISA证明制备的E0多克隆抗体可用于BVDV的检测。间接ELISA结果表明,E0多克隆抗体效价高于1∶64 000。【结论】制备的BVDV E0多克隆抗体效价高、抗原结合特异性强,为BVDV E0蛋白的生物学功能研究及BVDV的检测提供了材料支持。

关键词: 牛病毒性腹泻病毒(BVDV); E0基因; 原核表达; 多克隆抗体

Abstract: 【Objective】 The E0 protein of Bovine viral diarrhea virus (BVDV) was expressed by E.coli,after purification,the E0 protein polyclonal antibody was prepared by immunizing mice,which was then used for BVDV detection.【Method】 The BVDV E0 gene was amplified by PCR and ligated into pET-28a(+) to construct recombinant expression vector pET28a-E0.The recombinant plasmid was transformed into E.coli BL21 competent cells.After IPTG induction and affinity chromatography purification,the expression of E0 protein was identified by SDS-PAGE and Western blotting.The purified E0 protein was immunized into mice to prepare BVDV E0 polyclonal antibodies,and the specificity and titer of the polyclonal antibodies were detected by Western blotting,cell immunofluorescence,ELISA and other tests,as well as the application in BVDV virus detection.【Result】 The prokaryotic expression vector pET28a-E0 was successfully constructed,and the E0 protein was expressed and purified.The prepared BVDV E0 polyclonal antibody could specifically recognize the purified E0 protein and the total protein expressed by pET28a-E0 in BL21.Further,Western blotting,cellular immunofluorescence and double antibody sandwich ELISA proved that the prepared E0 polyclonal antibody could be used for the detection of BVDV.The results of indirect ELISA showed that the titer of E0 polyclonal antibody was higher than 1:64 000.【Conclusion】 The prepared BVDV E0 polyclonal antibody had a high titer and strong antigen-binding specificity,which provided material support for the biological functional study of BVDV E0 protein and the detection of BVDV.

Key words: Bovine viral diarrhea virus (BVDV); E0 gene; prokaryotic expression; polyclonal antibodies

中图分类号:

S852.65⁺3

王炜, 韩慧慧, 丁乃峥, 何成强. 牛病毒性腹泻病毒E0蛋白的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(8): 3131-3139.

WANG Wei, HAN Huihui, DING Naizheng, HE Chengqiang. Prokaryotic Expression of Bovine Viral Diarrhea Virus E0 Protein and Preparation of Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3131-3139.

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