牛传染性鼻气管炎病毒gE基因的克隆与表达

Coloning and Expresseion of Infectious Bovine Rihinotracheitis Virus gE Gene

为了克隆表达牛传染性鼻气管炎病毒gE因,根据牛传染性鼻气管炎病毒(infectious Bovine rhinotraeheitis virus,IBRV)全基因组序列设计合成gE因的特异性引物,采用PCR方法扩增gE基因,并将其克隆至pGEM-T3载体。然后对携带gE因的质粒进行双酶切处理,连接到经相同酶切处理的原核表达载体pET-32a,进而转化宿主菌BL21(DE3),通过获得阳性菌落进行IPTG诱导表达,SDS-PAGE对蛋白表达及纯化情况进行分析。SDSPAGE电泳结果显示,表达产物融合蛋白大小约为87kD,与预测值相符。利用Ni层析柱纯化重组蛋白,Western-blot和ELISA鉴定纯化的重组蛋白浓度为0.4mg/mL(0.123A)。在大肠杆菌中成功表达了pET-32a-gE融合蛋白,纯化后通过免疫印迹分析具有良好的免疫原性。

This study aimed to clone and express of recombinant infectious Bovine rhinotraeheitis virus(IBRV)gEgene.Specific primers were designed,and gE gene were ampilified by PCR.The PCR product were purifiedand ligated into pGEM-T3 vector,The target DNA fragments were separated by EcoRI and BamHI and purifiedThen the gE were ligated into the prokaryotic expression vector pET-32 a,which was double-digested with the EcoRIand BamHI.The gene of gE were expressed with N-terminal His-tag fusion proteins by IPTG inducton in E.coli BL21(DE3).S The results of protein expression were analyzed by SDS-PAGE.The results showed that IBRV gE protein were 87 kD.The purified gE protein =0.4mg/mL(0.123A)which were examined by ELISA and western blot.The recombinant protein could react with the antibody against IBRV by western bolt,and with good antigenicity.