H3N2亚型猪流感病毒HA1蛋白的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.04.026

Abstract

Abstract: 【Objective】 The purpose of this study was to obtain the HA1 protein,a structural domain of the haemagglutinin (HA) head of Swine influenza virus (SIV) subtype H3N2 with good immunogenicity,and prepare specific polyclonal antibodies for the identification and functional study of the HA1 protein.【Method】 In this study,the HA1 fragment of SIV type A (A/swine/Guangdong/04/2005(H3N2)) strain was amplified,the prokaryotic expression vector pET-28a(＋)-H3N2-HA1 was constructed by homologous recombination,and the recombinant positive plasmid were transformed into Escherichia coli BL21(DE3) competent cells after identification by PCR and sequencing.The conditions of HA1 protein expression (including induction temperature,induction time and IPTG concentration) were optimised,and protein purification was carried out by nickel column affinity chromatography.The recombinant protein obtained was mixed with QuickAntibody-Mouse3W adjuvant and then immunised with BALB/c mice by intramuscular injection to prepare mouse polyclonal antibody against HA1 protein of H3N2 SIV,and the titer,reactivity and specificity of the polyclonal antibody were determined by ELISA,Western blotting and immunofluorescence assay (IFA).【Result】 In this study,the prokaryotic expression vector pET-28a(＋)-H3N2-HA1 was successfully constructed.The highest expression of HA1 recombinant protein of H3N2 SIV was induced by 1 mmol/L IPTG at 26 ℃ for 10 h,and it was mainly expressed in the form of inclusion body,with the protein molecular mass size of about 39.5 ku.Five polyclonal antibodies against HA1 protein of H3N2 SIV were successfully prepared,with a titer of 1∶1 638 400.Western blotting and IFA identification results showed that the prepared polyclonal antibodies could specifically recognize HA1 protein of H3N2 SIV eukaryotic expressed,and did not react with the HA1 protein of other subtypes of influenza viruses,such as H1N1 SIV and H7N9 Avian influenza virus (AIV),with a good reactivity and specificity.【Conclusion】 In this study, HA1 protein of H3N2 SIV with good immunogenicity and was successfully expressed and purified,and mouse derived polyclonal antibodies with good reactivity and specificity were prepared,which provided an important tool for further study of the biological function of HA1 protein of H3N2 SIV.

Key words: Swine influenza virus (SIV); H3N2 subtype; HA1 protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺1

SI Wei, TAN Xiyu, XU Lingyun, LUO Tingrong, LI Xiaoning, GU Jinyan. Prokaryotic Expression of HA1 Protein of Swine Influenza Virus Subtype H3N2 and Preparation of Polyclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(4): 1739-1749.

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Prokaryotic Expression of HA1 Protein of Swine Influenza Virus Subtype H3N2 and Preparation of Polyclonal Antibodies

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