非洲猪瘟病毒B602L蛋白的原核表达、纯化及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.09.026

Abstract

Abstract: 【Objective】 African swine fever virus (ASFV) is a pathogen that is very harmful to the swine industry, and the B602L protein, as an important non-structural protein, is one of the chaperone molecules for the folding of the coat protein p72 into the correct tertiary structure, which plays a key role in the assembly of the virus into an icosahedral structure.Specific antibodies are of great importance for the study of the mechanism of the anti-African swine fever immune response and serological detection methods.This study was aimed to obtain ASFV B602L protein and prepare polyclonal antibodies against ASFV B602L protein, and provide materials for the functional research of ASFV B602L protein and the development of related diagnostic reagents. 【Method】 The present study was conducted on the B602L gene of ASFV China/2018/AnhuiXCGQ strain.After the prediction of the physicochemical properties and the analysis of the antigenicity of B602L protein, the B602L gene was cloned into the prokaryotic expression vector pET-28a(+) by homologous recombination and expressed in Escherichia coli BL21(DE3) competent cells.The protein was purified by nickel column affinity chromatography, the purification effect and specificity were verified by SDS-PAGE and Western blotting.And the purified B602L protein was immunized against BALB/c mice to prepare polyclonal antibodies.The titer of polyclonal antibodies was determined by indirect ELISA method, and the specificity of polyclonal antibodies was detected by Western blotting and Dot blotting. 【Result】 Bioinformatics analysis showed that B602L protein was an unstable hydrophilic protein without transmembrane region and signal peptide.The secondary structure mainly consisted of alpha helix (54.91%), extended chain (9.81%) and random coil (32.08%).ASFV B602L recombinant plasmid was successfully constructed.SDS-PAGE and Western blotting results showed that the recombinant strain pET-28a-B602L-BL21 was highly expressed under the condition of 16 ℃ and 0.5 mmol/L, and the obtained recombinant protein was mainly expressed in the supernatant of strain BL21.It was about 70 ku in size and binded specifically to ASFV-positive serum.Indirect ELISA showed that the prepared murine polyclonal antibody against B602L had a titer of 1∶409 600.Western blotting and Dot blotting results showed that the murine polyclonal antibody against B602L could specifically recognize B602L protein. 【Conclusion】 The prokaryoticly expressed B602L recombinant protein had good immunogenicity, which could specifically bind to ASFV-positive serum, and the prepared polyclonal antibody against mouse anti-ASFV B602L protein had good biological activity.The results could provide experimental materials and theoretical support for further research on the biological function of ASFV B602L protein and the establishment of diagnostic methods for African swine fever.

Key words: African swine fever virus (ASFV); B602L protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺1

GUO Chenyun, SONG Jinxing, WANG Mengxiang, SUN Junru, YAN Ruoqian, XIE Caihua, WU Yanan, ZHANG Gaiping. Prokaryotic Expression,Purification and Polyclonal Antibodies Preparation of African Swine Fever Virus B602L Protein[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 3980-3989.

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Prokaryotic Expression,Purification and Polyclonal Antibodies Preparation of African Swine Fever Virus B602L Protein

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