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

摘要:

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。

Abstract:

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 GAl1 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 yGⅡ) 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. Com-bining precursors’ achievements, we will draw a conclusion that trans-membrane domain (TD) and cyto-plasmic domain have a negative regulation on RV-G antigen immunogenicity in S. cerevisiae.