狂犬病病毒糖蛋白在酿酒酵母中的分泌表达

目的使狂犬病病毒糖蛋白在酿酒酵母中获得分泌表达,为制备口服狂犬病疫苗奠定基础。方法克隆狂犬病病毒糖蛋白基因,与菊粉酶信号肽序列连接为融合基因InG,该融合基因克隆至pYes2载体Gal1启动子下游,构建成诱导分泌表达载体pYes2-InU-G,该重组载体转化酿酒酵母筛选阳性克隆,阳性重组子经半乳糖诱导12h后,收集上清进行SDS-PAGE电泳和Western-blot鉴定。结果 RT-PCR和Western-blot鉴定显示狂犬病病毒糖蛋白已获得分泌表达,并且具有良好的抗原性。结论 CVS24病毒株的糖蛋白在酿酒酵母中获得分泌表达且具有良好的抗原性,为进一步研究重组酵母能否在消化道中存活并继续进行分泌表达提供了研究条件。

Expression of Recombinant Rabies Virus Glycoprotein in Saccharomyces cerevisiae

Objective To construct a secretory expression vector pYes-InG for the future development of an oral rabies virus vaccine.Methods Coding sequence of the rabies virus glycoprotein（G） was removed and the signal sequence was inserted in-frame with the inulinase signal sequences（INU）.The INU-G fusion gene was then subcloned into pYes2 with GAl1 promoter.Transformation was performed by using lithium-treated yeast cells and several Ura ＋ tranformants were isolated.12 hours after the induction of positive recombinants,supernatant was collected and analyzed by SDS-PAGE and Western-blot.mRNA was extracted and analyzed by RT-PCR.Results Rabies virus glycoproteins were successfully expressed and secreted into culture medium.Antigenicity of the expressed protein was confirmed.Conclusion This research result can be used in the future research on livability and secretory expression of recombinant yeast cells in gastrointestinal tract.