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目的 在毕赤酵母表达系统中表达风疹病毒 (rubellavirus ,RV)JR2 3株包膜糖蛋白E1 ,为研究E1蛋白的结构功能、开发基因工程疫苗和重组蛋白诊断试剂盒奠定基础。方法 E1基因的表达质粒 pGAPZαA E1经AvrⅡ线性化后用LiCl法转化酵母菌 ,在YPD(含 1 0 0 μg/mlZeocinTM)平板上两次筛选 ,挑出单菌落 ,于液体YPD中培养 ,并在不同时间收集培养物 ,用SDS PAGE和Westernblot进行分析。结果 SDS PAGE显示E1重组蛋白在毕赤酵母中高效稳定表达 ,在 4 8h时表达量达到最高 ,之后趋于稳定 ,上清和细胞中均有蛋白表达。Westernblot结果表明 ,上清中的E1重组蛋白能够分别与抗RV的阳性血清和单克隆抗体反应 ,而细胞中的E1重组蛋白只能与抗RV的阳性血清反应 ,不能与单克隆抗体反应。这说明所表达的蛋白一部分在信号肽的引导下分泌出胞 ,并经过折叠形成了正确的构像 ,能够与单克隆抗体结合 ;而另一部分由于蛋白本身的跨膜区连在了细胞膜上没有分泌出去 ,没有形成能够被单抗识别的构像 ,不能与单抗结合。结论 E1包膜糖蛋白在酵母菌中成功表达 ,且免疫反应性良好。
Objective To express the envelope glycoprotein E1 of rubella virus (JR2) JR3 in Pichia pastoris expression system, and lay the foundation for the study of the structural function of E1 protein and the development of genetic engineering vaccine and recombinant protein diagnostic kit. Methods The E1 gene expression plasmid pGAPZαA E1 was linearized with AvrⅡ and transformed into Saccharomyces cerevisiae by LiCl method. The plasmid was screened twice on YPD (containing 100 μg / ml ZeocinTM) plate, single colonies were picked out and cultured in liquid YPD Cultures were harvested at different times and analyzed by SDS PAGE and Western blotting. Results SDS PAGE showed that E1 recombinant protein was efficiently and stably expressed in Pichia pastoris and reached the highest level at 48 h, then stabilized. The protein expression was observed in both supernatant and cell. The result of Western blot showed that the E1 recombinant protein in the supernatant could react with the anti-RV positive serum and the monoclonal antibody respectively. However, the E1 recombinant protein in the cell only reacted with the anti-RV positive serum and could not react with the monoclonal antibody. This shows that part of the expressed protein secreted by the signal peptide under the guidance of the cell and folded to form the correct conformation, with the monoclonal antibody; the other part of the protein itself due to the transmembrane region connected to the cell membrane is not Secreted, there is no conformation that can be recognized by Mab, but not Mab. Conclusion E1 envelope glycoprotein was successfully expressed in yeast and immunoreactivity was good.