牛IRF7蛋白的原核表达及多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2021.10.034

Abstract

Abstract: The purpose of this study was to express bovine interferon regulatory factor 7 (IRF7) protein by prokaryotic expression system, and purify the protein, so as to prepare high purity bovine IRF7 rabbit polyclonal antibody. Bioinformatics software was used to predict and analyze the potential biological functions of bovine IRF7, with reference to the bovine IRF7 gene sequence (GenBank accession No. :NM_001105040.1), primers for bovine IRF7 were designed and PCR was used to amplify bovine IRF7 gene from Bovine viral diarrhea virus (BVDV) infected MDBK cells. The bovine IRF7 gene was attached to the pMD19-T clone vector, and then attached to the prokaryotic expression vector pET-28a(+) to transform into Escherichia coli DH5α competent cells, recombination plasmid pET-28a-IRF7 was constructed. The recombinant plasmid pET-28a-IRF7 was transformed into Escherichia coli BL21 (DE3) competent cells. After induction by IPTG, the expression products were analyzed and identified by SDS-PAGE. The specificity of the polyclonal antibody was identified by Western blotting, and the titer of rabbit anti-bovine IRF7 polyclonal antibody was determined by indirect ELISA. After BVDV infected cells, the expression of the antiviral factor IRF7 was detected by Real-time quantitative PCR. Bioinformatics analysis showed that IRF7 protein had no transmembrane domain, no signal peptide, and many phosphorylation sites. The secondary structure was mainly random coil, which was consistent with the three-dimensional model of the tertiary structure, indicating that this protein might have a good antigenic potential. A 1 497 bp fragment of IRF7 gene was successfully amplified by PCR, and the bovine IRF7 protein was successfully expressed with a molecular weight of about 60 ku. The titer of rabbit anti-bovine IRF7 polyclonal antibody was 1:128 000 by indirect ELISA. The expression of IRF7 was decreased in MDBK cells infected with BVDV strain, and the expression of IFN-β was significantly promoted by BVDV infection of cells overexpressing IRF7 (P<0.05). In conclusion, bovine IRF7 protein was successfully expressed and purified in this study, and rabbit polyclonal antibody against bovine IRF7 protein was prepared, which provided materials for elucidating the molecular mechanism of bovine IRF7 in innate immune antiviral response.

Key words: bovine; IRF7; protein purification; polyclonal antibody

CLC Number:

S852.4⁺3

ZHANG Jiangwei, ZHANG Chao, HE Jinke, XI Jing, SUN Tianhao, HE Yanhua, CHEN Chuangfu. Prokaryotic Expression of Bovine IRF7 Protein and Preparation of Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3812-3822.

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Prokaryotic Expression of Bovine IRF7 Protein and Preparation of Polyclonal Antibody

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