A型禽流感病毒不同拷贝数基质蛋白胞外区基因的原核表达及免疫保护力研究  

Prokaryotic expression and protective efficacy of the multicopy ectodomain of the matrix protein gene of avian influenza A virus

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

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：《中国预防兽医学报》2010年第10期795-799,共5页Chinese Journal of Preventive Veterinary Medicine

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

摘　　要：为研究含禽流感病毒(AIV)不同拷贝数基质蛋白胞外功能区(M2e)亚单位疫苗在SPF鸡体中的免疫保护效力差异,根据已发表的高致病性AIV M2基因序列设计合成两对引物,利用PCR技术扩增单个M2e基因片段,以此为基础顺次串联得到不同拷贝数的M2e基因片段,并将其克隆到pMAL-C2x载体中,构建不同拷贝数的A型AIV M2e基因的重组表达质粒,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达,SDS-PAGE鉴定。表达产物经纯化后免疫家兔、SPF鸡,制备抗M2e融合蛋白的多克隆抗体。Western blot和间接免疫荧光试验检测纯化的融合蛋白免疫原性,间接ELISA测定鸡血清抗体水平,表明MBP-3M2e融合蛋白的抗体水平较高,且MBP-3M2e亚单位疫苗产生最好的免疫保护力。To evaluate the protective efficacy of the matrix protein ectodomain of avian influenza A virus（AIV）,the single ectodomain copy of the matrix protein 2 gene（M2e） was amplified by PCR using two pairs of primers derived from the M2 gene of AIV.Multiple tandem copies of M2e were constructed by linking the single ectodomain copys and then inserted into the pMAL-C2x vector.The resultant plasmids M2en was transformed into E.coli BL21（DE3） competent cells,and the fusion proteins were induced with IPTG and identified by SDS-PAGE.Anti-M2e polyclonal sera were obtained by inoculating rabbits and SPF chickens with the purified fusion proteins.The results showed the expressed proteins retained excellent immunogenicity as detected by western blot and indirect immunofluorescence assay.The 3M2e fusion protein was shown to induce higher antibody levels and immune protection in chickens.

关 键 词：A型禽流感病毒 M2e基因 原核表达 多克隆抗体 免疫保护力 

分 类 号：S852.65[农业科学—基础兽医学]