【摘要】 为研究A型禽流感病毒M2蛋白胞外功能区(M2e)蛋白亚单位疫苗在鸡体的免疫保护力,利用笔者所在实验室已构建的含M2e基因的质粒pGEM-T-1459,获得顺次串联的基因片段3M2e,将其克隆到原核表达载体pMAL-C2x中,经酶切和DNA测序鉴定正确后转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达,SDS-PAGE和Western-blot分析,结果显示,表达出一分子质量为60 ku的可溶性融合蛋白(MBP-3M2e),与预期值相符,该蛋白表达量约占菌体表达量的59.8%。间接免疫荧光和间接ELISA检测结果显示,用纯化后的融合蛋白免疫的SPF鸡血清可与流感病毒的M2蛋白发生反应,且其抗体效价较高,表明纯化后的重组融合蛋白具有良好的免疫原性。更多还原

【Abstract】 In order to study the immunoprotective efficacy of a M2e-based recombinant subunit vaccine against avian influenza A virus in chickens,three tandem copies of M2e were obtained from a plasmid pGEM-T-1459 containing M2e gene,then inserted into the prokaryotic expression vector pMAL-C2x.After being identified by restrictive enzyme digestion and sequencing analysis,the recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells.The transformed bacteria were induced by IPTG and produced a soluble fusion protein(MBP-3M2e).The expressed recombinant protein of 60ku in mass was identified by SDS-PAGE and Western-blot.The SPF chickens were immunized with the purified MBP-3M2e protein.Indirect immunofluorescence assay and indirect enzyme-linked immunosorbent assay showed that the purified fusion protein possessed good immunogenicity.更多还原