H3N2犬流感病毒M1蛋白的原核表达及鉴定

为制备犬流感病毒(H3N2) M1蛋白纯品,针对M1基因序列设计引物,用聚合酶链式反应(PCR)扩增目的基因片段,扩增产物克隆至表达载体pET-SUMO中并转化至宿主菌BL21(DE3),诱导表达目的蛋白,探索纯化工艺,制备目的蛋白,并用Western blot检测纯化的M1目的蛋白。通过PCR成功扩增出大小为771 bp的M1基因,成功构建p ET-SUMO-M1表达载体,表达的融合蛋白相对分子量为41 kD,主要以可溶形式表达,纯化后获得蛋白纯品,Western blot检测显示用M1蛋白(28 k D)免疫小鼠制备的多抗能与制备的蛋白纯品发生特异性反应,从而证明蛋白纯品为M1目的蛋白。试验制备出的M1蛋白纯品可为进一步制备通用型抗犬流感病毒抗体提供纯品抗原。

Prokaryotic expression and preparation of H3N2 canine influenza virus M1 protein

To prepare the high purity recombinant M1 protein, the canine influenza virus (H3N2) M1 gene was cloned and prokaryotically expressed. Primers targeting the canine influenza virus (H3N2) M1 gene were designed, then the H3N2 M1 gene was amplified by PCR and cloned into the expression vector pET-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The expressed product was purified by a series procedure, including chromatography and some subsequent treatment, then identified by Western blot. This study successfully constructed pET-SUMO-M1 and expressed the high purity recombinant M1 protein. The acquired high purity M1 protein provides a pure antigen for further preparation of a universal anti-canine influenza virus antibody.