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

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.