马疱疹病毒糖蛋白G基因C-末端的原核表达

为获得马疱疹病毒1型(EHV1)和4型(EHV4)糖蛋白G(gG),对克隆载体pMD-1gG和pMD-4gG分别进行BamHⅠ+SalⅠ和EcoRⅠ+HindⅢ双酶切,将所得片段分别插入表达载体pGEX-6P-1和pET28a(+)中,构建了重组质粒pGEX-1gG和pET-4gG。经双酶切和序列鉴定为阳性后,将重组质粒pGEX-1gG转化入大肠杆菌BL21(DE3)、重组质粒pET-4gG转化入大肠杆菌Rosetta(DE3)中,经IPTG诱导表达后对表达产物进行SDS-PAGE分析。结果显示,分别在35和17ku处出现与预期大小相符的目的条带。Western-blot结果证实,目的蛋白均具有良好的反应原性。表明,成功实现了EHV1gG和EHV4gG蛋白的正确表达,初步鉴定其具有良好的反应原性。

Prokaryotic expression of glycoprotein G genes of EHV1 and EHV4

To obtain the gG proteins of EHV1 and EHV4 by prokaryotic expression,the recombinant cloning plasmids pMD-1gG and pMD-4gG were digested with BamHⅠ+SalⅠ and EcoRⅠ+HindⅢ respectively,and the gG fragments were cloned into the BamHⅠ and SalⅠ sites of plasmid pGEX-6P-1 and the EcoRⅠ and HindⅢ sites of pET-28a(+)to construct recombinant expression plasmids pGEX-1gG and pET-4gG.The recombinant plasmids were verified by digestion and sequencing analysis and then transformed into Escherichia coli BL21(DE3) and Rosetta(DE3) respectively,which were then induced with IPTG at 37 ℃.Expressed proteins of 35 ku and 17 ku as expected were showed by SDS-PAGE.In the Western-blot test,the expressed products can react with the antibodies to EHV1 or EHV4,which indicated that fusion proteins were expressed in the prokaryote system and showed good reactionogenicity.