非洲猪瘟病毒p30蛋白单克隆抗体制备和鉴定

doi:10.16431/j.cnki.1671-7236.2023.03.038

Abstract

Abstract: 【Objective】 The aim of this experiment was to prepare monoclonal antibody to African swine fever virus (ASFV) p30 protein,and lay the foundation for the establishment of a rapid diagnostic test for ASFV and the research of a vaccine.【Method】 The p30 prokaryotic expression plasmid pET-30a(+)-p30 was constructed,transformed into E.coli BL21 (DE3) competent cells for induced expression,and then the target protein was obtained by nickel column affinity chromatography.p30 was compounded and immunized to BALB/c mice for three times every two weeks,and the spleen of immunized mice was fused with myeloma cells to prepare monoclonal antibody by ELISA method and subclonal screening of antibody-secreting hybridoma cells.The screened monoclonal cells were injected into the peritoneal cavity of mice to prepare ascites.The monoclonal antibodies were identified by Western blotting detection,immunofluorescence assay (IFA) and antibody isotype analysis.【Result】 The p30 protein prokaryotic expression plasmid was successfully constructed,and the recombinant p30 protein expressed in the prokaryotic system was purified using IPTG to induce.Four monoclonal antibody hybridoma cell lines,named 1H3B4,3F2F5,6E3A1 and 9D6B9,which could stably secrete the ASFV p30 protein,were successfully prepared.The potencies of the antibodies were determined by ELISA in the range of 1∶100 000 to 1∶1 000 000.The screened monoclonal antibodies were identified by Western blotting and IFA and those antibodies were specifically reacted with p30 protein.The monoclonal antibodies were identified as IgG2b for the heavy chain and Kappa for the light chain of 3F2F5 and 9D6B9.6E3A1 had IgM for the heavy chain and Kappa for the light chain.1H3B4 had IgG2b for the heavy chain and Lambda for the light chain.【Conclusion】 Four monoclonal antibodies against ASFV p30 protein were successfully prepared in this experiment,and the immunological characteristics of the monoclonal antibodies were analyzed,the results provided basis for the biological function research of p30 protein and the serological detection of ASFV.

Key words: African swine fever virus (ASFV); p30 protein; monoclonal antibody; CP204L gene

CLC Number:

S852.65⁺1

HOU Haoyu, ZHENG Nannan, WU Hongju, CHEN Yinlong, LI Chao, ZHANG Angke, JI Pengchao, WAN Bo, DU Yongkun, ZHANG Gaiping. Preparation and Characterization of Monoclonal Antibody Against p30 Protein of African Swine Fever Virus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1241-1249.

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Preparation and Characterization of Monoclonal Antibody Against p30 Protein of African Swine Fever Virus

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