猪传染性胃肠炎病毒纤突糖蛋白在毕赤酵母中的分泌表达

目的:利用巴斯德毕赤酵母表达系统表达猪传染性胃肠炎病毒(TGEV)纤突糖蛋白S。方法:根据GenBank中猪TGEV纤突糖蛋白S全基因设计一对引物,并在5'引物和3'引物中引入EcoRⅠ、NotⅠ酶切位点,2.2kb的目的基因S经PCR扩增后克隆于pBS-T载体,再将S基因经双酶切从T载体切下并与穿梭质粒pPIC9k连接,SalⅠ线性化重组穿梭质粒pPIC9k-S,电转化于毕赤酵母GS115感受态细胞,G418筛选鉴定阳性重组子,经甲醇诱导,SDS-PAGE检测诱导后上清。结果:对pPIC9k-S重组酵母表达载体的测序证实已成功克隆了猪TGEVS基因;重组酵母菌诱导表达后,SDS-PAGE检测结果显示表达产物的相对分子质量约为82×103,且S蛋白以可溶性形式分泌表达于胞外。结论:利用巴斯德毕赤酵母真核表达系统成功表达了猪传染性胃肠炎病毒(河北分离株)纤突糖蛋白S。

The Secreted Expression of the Spike Glycoprotein of Transmissible Gastroenteritis Virus of Swine in Pichia pastoris

Objective：To express the recombinant spike glycoprotein of transmissible gastroenteritis virus（TGEV） of swine in Pichia pastoris expression system.Methods：The 5＇ and 3＇ primer were designed according to the complete genome sequence of TGEV of swine in GenBank with EcoRⅠ and NotⅠ endonuclease sites.After amplied by PCR,partial spike gene S（2.2 kb long） was sub-cloned into vector pBS-T.Then S gene was inserted into shuttle plasmid pPIC9K by cleaved from T-vector.After sequencing,recombinant shuttle plasmid DNA,linearized with SalⅠ,was integrated into the genome of the methylotrophic yeast P.pastoris GS115 by electroporation and positive clones were screened with G418.At last,the supernatant of medium was analyzed by SDS-PAGE after induced with methanol.Results：The TGEV S gene was confirmed by the sequencing result of the recombinant yeast expression vector pPIC9k-S.SDS-PAGE results indicated that spike protein was secreted into the culture supernatant in a form of soluble protein and the molecular weight of spike protein is approximately 82 kD.Conclusion：The result reveals that the spike protein has been expressed successfully in the P.pastoris expression system.