布鲁氏菌分泌蛋白BspI生物信息学分析及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2022.08.027

Abstract

Abstract: 【Objective】 The purpose of this study was to analyze the Brucella secreted protein BspI by bioinformatics,construct prokaryotic expression vector,and obtain BspI protein and prepare its polyclonal antibody,so as to provide materials for the follow-up study of the biological function of BspI protein.【Method】 Bioinformatics analysis of the amino acid sequence of BspI protein was performed using online software,referring to the BspI gene sequence (GenBank accession No.:DK63_1233) of Brucella 16M strain,after PCR amplification,BspI gene was ligated into pMD19-T cloning vector and screened for positive clones.The target gene was ligated into pET-28a expression vector,transformed into Escherichia coli BL21(DE3) competent cells,induced protein expression by IPTG,and analyzed by SDS-PAGE.The protein was purified by nickel column affinity chromatography, the purified protein was mixed with Freund's adjuvant to immunize rabbits,and blood was collected to isolate serum for analysis of polyclonal antibody specificity and antibody titer by Western blotting and indirect ELISA,respectively.【Result】 BspI protein was unstable hydrophilic protein,presented a transmembrane structure with no signal peptide region,had 13 phosphorylation sites and 7 antigenic determinants,and the secondary structure of mainly contained alpha helix,extended chain,and random coil.The 675 bp of BspI target gene was successfully amplified by PCR,and the pET-28a-BspI expression vector was successfully constructed.SDS-PAGE and Western blotting analysis results showed that the 25.3 ku protein was successfully expressed with no obvious heterobands after purification,and the prepared polyclonal antibody was able to specifically bind to BspI protein.The indirect ELISA results represented the polyclonal antibody titer of BspI protein was 1:409 600.【Conclusion】 The prepared polyclonal antibody against BspI protein of rabbit origin could specifically recognize BspI protein,and the protein was relatively reactogenic,which provided a reference for further research on the role played by BspI protein in the endoparasitism of Brucella.

Key words: Brucella; bioinformatic analysis; secreted protein BspI; polyclonal antibody

CLC Number:

S852.61⁺4

XIAO Yangyang, MA Zhongchen, LI Ruirui, CHEN Chuangfu, ZHENG Wei, WANG Yong, WANG Pengyan. Bioinformatic Analysis and Polyclonal Antibody Preparation of Brucella Secreted Protein BspI[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3112-3121.

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Bioinformatic Analysis and Polyclonal Antibody Preparation of Brucella Secreted Protein BspI

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