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

Abstract

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

CLC Number:

S852.65⁺3

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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Prokaryotic Expression of Bovine Viral Diarrhea Virus E0 Protein and Preparation of Polyclonal Antibody

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