非洲猪瘟病毒p37蛋白生物信息学分析及其多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.01.030

Abstract

Abstract: 【Objective】 This study was aimed to obtain ASFV p37 protein and prepare polyclonal antibody against ASFV p37 protein to provide materials for structural and functional studies of ASFV p37 protein.【Method】 In this study, bioinformatics tools were used to analyze the p37 protein of ASFV HLJ/2018 (GenBank accession No.:MK333180.1), and the p37 gene was designed and synthesized, and the pET32a-p37 recombinant expression vector was constructed.The recombinant plasmid was transformed into of Escherichia coli BL21 (DE3) competent cells.After induced by IPTG, the soluble expression of the recombinant protein was analyzed by SDS-PAGE.The bacterial precipitate was denatured by 8 mol/L urea, centrifuged, filtered through a 0.45 μm membrane, then purified by nickel affinity chromatography, and their purification effect and specificity were verified by SDS-PAGE and Western blotting.The polyclonal antibody against ASFV p37 protein was prepared by immunizing mice with 50 μg of purified p37 protein per mouse.The titer of polyclonal antibody was determined by indirect ELISA. The specificity of the polyclonal antibody was detected by Western blotting and indirect immunofluorescence assay.【Result】 Bioinformatics analysis showed that the p37 protein was a stable hydrophilic protein with no transmembrane region or signal peptide.The secondary structure mainly contained alpha helix (45.28%), extended stran (15.09%) and random coil (31.54%).SDS-PAGE and Western blotting results showed that the prokaryotic expressed recombinant protein p37 was successfully expressed after induction by IPTG, with a size around 62 ku, mainly in the form of inclusion bodies, and could react with His monoclonal antibody.Indirect ELISA results showed that the titer of the prepared anti-mouse polyclonal antibody reached 1:256 000.Western blotting results showed a specific band at around 42 ku.Indirect immunofluorescence showed a specific red fluorescence, indicating that the polyclonal antibody reacted with the recombinant baculovirus-expressed p37 protein with good specificity.【Conclusion】 ASFV p37 protein was successfully expressed and purified, and a polyclonal antibody against ASFV p37 protein was prepared, indicating that the prepared recombinant protein had good immunogenicity and could generate strong immune response, which laid the foundation for the subsequent structural and functional study of ASFV p37 protein structure function, antibody and related vaccine development.

Key words: African swine fever virus (ASFV); p37 protein; protein expression; polyclonal antibody

CLC Number:

S852.65^＋1

ZHANG Mengyang, HE Jian, LIU Yangkun, YAO Lunguang. Bioinformatics Analysis and Polyclonal Antibody Preparation of African Swine Fever Virus p37 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 300-309.

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Bioinformatics Analysis and Polyclonal Antibody Preparation of African Swine Fever Virus p37 Protein

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