A型禽流感病毒M2蛋白胞外功能区(M2e)基因的原核表达和免疫原性分析  

Prokaryotic expression of the ectodomain of the conserved influenza matrix protein M2(M2e) gene of avian influenza A virus and analysis of immunogenicity of the expressed protein

作　　者：张新涛[1] 刘明[1] 杜金玲[1] 刘春国[1] 李洪涛[1,2] 孙恩成[3] 石薇琳[1,2] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室农业部动物流感开放实验室及国家禽流感参考实验室OIE禽流感参考实验室,黑龙江哈尔滨150001 [2]东北农业大学动物医学院,黑龙江哈尔滨150030 [3]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国兽医科学》2010年第4期357-362,共6页Chinese Veterinary Science

基　　金：国家“十一五”科技支撑计划项目(2006BAD06A05);黑龙江省自然科学基金项目(ZJN0702-02)

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。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.

关 键 词：A型禽流感病毒 M2e基因 原核表达 免疫原性 

分 类 号：S852.659.5[农业科学—基础兽医学]