非洲猪瘟病毒p72蛋白原核表达及单克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.11.032

Abstract

Abstract: 【Objective】 The aim of this experiment was to express and purify African swine fever virus (ASFV) p72 protein and prepare the monoclonal antibodies.【Method】 The prokaryotic expression vector pET-28b-p72 was constructed and transformed into Escherichia coli BL21(DE3) competent cells for inducing expression and protein purification.The purified p72 protein was used to immunize BALB/c mice, and the spleen of the immunized mice was fused with myeloma cells to prepare monoclonal antibodies.The positive hybridoma cells secreting antibodies were screened by indirect ELISA and subcloning.Antibody types were detected by mouse antibody type detection kit.Ascites of monoclonal antibody against ASFV p72 protein was prepared by in vivo induction method, purified by saturated ammonium sulfate precipitation method, and the titer and specificity of monoclonal antibody were detected by indirect ELISA, indirect immunofluorescence assay (IFA) and Western blotting.【Result】 The recombinant protein of ASFV p72 was expressed in the form of inclusion bodies in Escherichia coli.Four monoclonal cell lines secreting IgG1 antibody were obtained, named 6F8, 7C3, 8H7 and 9G2, respectively.Indirect ELISA results showed that the titer of the antibody secreted by 6F8 and 8H7 cell lines was 1:6 400, and the titer of the antibody by 7C3 and 9G2 cell lines was 1:28 000.In addition, the antibody secreted by four cell lines was positive in reaction with p72 protein, but negative for Pseudorabies virus (PRV), Classical swine fever virus (CSFV), Porcine parvovirus (PPV), Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine epidemic diarrhea virus (PEDV). The antibodies secreted by four monoclonal cell lines could react specifically with p72 eukaryotic protein.【Conclusion】 Four cell lines that could secrete monoclonal antibodies specifically bound to ASFV p72 protein were successfully prepared, which laid a foundation for further investigation of the function of ASFV p72 protein and the prevention and diagnosis of African swine fever.

Key words: African swine fever virus; p72 protein; prokaryotic expression; monoclonal antibody

CLC Number:

S852.65⁺1

CHEN Guie, RONG Fang, FENG Xianing, SUN Ronghang, MA Shaozhao, HAO Wenqian, YE Zian, LIU Yufu, LI Zuosheng, CHEN Ruiai. Prokaryotic Expression of p72 Protein of African Swine Fever Virus and Preparation of Its Monoclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4646-4654.

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Prokaryotic Expression of p72 Protein of African Swine Fever Virus and Preparation of Its Monoclonal Antibodies

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