狂犬病病毒糖蛋白在酿酒酵母中的表达

Expression of Rabies Virus Glycoprotein Gene in Saccharomyces cerevisiae

利用酿酒酵母表达系统表达狂犬病病毒糖蛋白G,可获得大量无致病的抗原,为研究新型狂犬病疫苗提供条件。构建Tat-G融合基因,通过EcoR I和Xba I酶切位点克隆至pYes2表达载体中,醋酸锂法转化酿酒酵母,URA3筛选鉴定阳性克隆,阳性重组子经半乳糖诱导20 h后,提取蛋白,SDS-PAGE和Western blot分析鉴定融合蛋白。SDS-PAGE结果显示糖蛋白基因在酿酒酵母中可能表达为2种形式的蛋白,yGI和yGII,分子大小分别为66 kD和56 kD,Western blot显示在56 kD处有特异性条带。结合前人的研究成果,初步判断狂犬病病毒糖蛋白基因的跨膜TD区和膜内编码区对RV-G蛋白分子的正确折叠和免疫活性等有至关重要的影响,从而为进一步提高yGII蛋白的表达奠定基础。

To obtain non-pathogenic rabies virus glycoprotein （RV-G）, we expressed RV-G in Saccaromyces Cerevisiae （S. cerevisiae）. In our study, tat-G fusion gene was cloned into the expression vector pYes2.0, which allows expression of a foreign gene in the yeast cells under the control of GAll promoter. Transformation was performed by using lithium-treated yeast cells and several Ura＋ -tranformants were isolated. According to the relative mobility in SDS-PAGE, we know probably two forms （designated as yGI and yG II ） of RV-G analogues produced in S. cerevisiae, their molecular weights were estimated as 66 kD and 56 kD, respectively. On the other hand, there was a specific band about 56 kD shown in western blot result. Combining precursors＇ achievements, we will draw a conclusion that trans-membrane domain （TD） and cytoplasmic domain have a negative regulation on RV-G antigen immunogenicity in S. cerevisiae.