禽流感病毒HA1蛋白的原核表达及其免疫原性的初步研究

Prokaryotic Expression and Immunogenicity of HA1 Protein of Avian influenza virus

从H9N2亚型禽流感病毒感染的鸡胚尿囊液中提取病毒RNA,RT-PCR克隆全长血凝素(He-magglutinin,HA)基因,并进一步克隆HA的主要抗原区HA1基因。将HA1克隆到原核表达载体pGEX-KG,与谷胱苷肽(GST)融合表达,表达的融合蛋白GST-HA1以包涵体形式存在。包涵体经变性、复性处理,Western blot分析表明,表达蛋白具有良好的免疫学活性。ELISA检测发现,GST-HA1只能与H9亚型禽流感病毒抗体发生反应,而与H5和H7亚型禽流感病毒抗体无交叉反应。进一步将纯化的融合蛋白与佐剂混合后免疫Balb/c小鼠,免疫小鼠体内产生了较高滴度的特异性抗体。制备的HA1蛋白特异性强,具有良好的免疫原性,为禽流感病毒的鉴别诊断和禽流感疫苗开发奠定了基础。

The HA gene was cloned by RT-PCR from allantoic fluid infected with H9N2 AIV.The major antigen domain HA1 was cloned from the full-length HA and further subcloned to the prokaryotic expression vector pGEX-KG resulting in the recombinant plasmid pKG-HA1 which was used to express the fusion protein GST-HA1 in E.coli.The immunocompetence of the purified GST-HA1 was demonstrated by Western blot and ELISA.The results suggested that the fusion GST-HA1 proteins could only react with the positive antibodies against H9 subtypes AIV but not with H5 and H7 antibodies.The high titer antibodies from Balb/c mice vaccinated with the commixture of the purified expression products and adjuvant could specifically react with H9N2 AIV.The HA1 protein in this study layed a foundation for the distinguishable diagnosis and the vaccine＇s development.