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

摘要/Abstract

摘要： 【目的】获得免疫原性良好的H3N2亚型猪流感病毒(Swine influenza virus,SIV)血凝素(haemagglutinin,HA)头部结构域HA1蛋白，并制备特异性多克隆抗体，用于HA1蛋白的鉴定及功能研究。【方法】本研究通过扩增A型SIV (A/swine/Guangdong/04/2005(H3N2))毒株的HA1片段，采用同源重组方法构建原核表达载体pET-28a (＋)-H3N2-HA1，经PCR和测序鉴定正确后将重组阳性质粒转化大肠杆菌BL21(DE3)感受态细胞。对HA1蛋白表达条件(诱导温度、诱导时间、IPTG浓度)进行优化，通过镍柱亲和层析法纯化蛋白。将获得的重组蛋白与QuickAntibody-Mouse3W佐剂混合后以肌内注射方式免疫BALB/c小鼠，制备H3N2亚型SIV HA1蛋白多克隆抗体，并通过ELISA、Western blotting和免疫荧光试验(IFA)对多克隆抗体的效价、反应性及特异性进行鉴定。【结果】本研究成功构建原核表达载体pET-28a (＋)-H3N2-HA1；H3N2亚型SIV HA1重组蛋白在1 mmol/L IPTG 26 ℃诱导10 h时表达量最高，且主要以包涵体形式表达，蛋白分子质量大小约为39.5 ku。成功制备5株H3N2亚型SIV HA1蛋白多克隆抗体，效价均为1∶1 638 400。Western blotting和IFA鉴定结果显示，制备的多克隆抗体可特异性识别真核表达的H3N2亚型SIV HA1蛋白，与H1N1亚型SIV、H7N9亚型禽流感病毒(AIV)等其他亚型流感病毒的HA1蛋白均不发生反应，具有良好的反应性及特异性。【结论】本研究成功表达并纯化了免疫原性良好的H3N2亚型SIV HA1蛋白，制备了反应性及特异性良好的鼠源多克隆抗体，为深入研究H3N2亚型SIV HA1蛋白的生物学功能提供了重要工具。

关键词: 猪流感病毒(SIV); H3N2亚型; HA1蛋白; 原核表达; 多克隆抗体

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

中图分类号:

S852.65⁺1

司维, 谭茜宇, 徐玲芸, 罗廷荣, 李晓宁, 顾金燕. H3N2亚型猪流感病毒HA1蛋白的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2025, 52(4): 1739-1749.

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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