风疹病毒E1—374糖蛋白生物活性检测及其初步应用

Expression and bioassay of rubella virus E1-374 glycoprotein in yeast cells

目的分泌表达风疹病毒E1-374糖蛋白并检测其免疫原性。方法将E1—374蛋白的cDNA插入表达载体pGAPZotA中构建出表达质粒pGAPZctA—E1—374，经BlnI酶线性化后通过电转的方法导入毕赤酵母菌，博来霉素筛选阳性菌落。间接免疫荧光和WesternBlot检测E1—374蛋白的表达及免疫反应性。免疫小鼠后应用间接ELISA检测风疹病毒IgG抗体。结果SDS-PAGE和WesternBlot显示E1-374蛋白的相对分子质量为46．89×10^3。ELISA法检测免疫小鼠的抗血清为阳性。建立的间接ELISA方法的最佳工作条件是抗原包被浓度为5．5μg／ml，血清最佳稀释度为1：100，板内变异系数值为0．36％-12．45％。结论E1-374蛋白能够引起小鼠体液免疫应答，是风疹亚单位疫苗的重要候选成分，并可应用于风疹病毒IgG抗体的检测。

Objective To express the rubella virus E1-374 glycoprotein in Pichia pastoris and study the immunogenecity of the recombinant protein. Methods The cDNA of protein E1-374 was cloned into the expression vector pGAPZctA and transformed into Pichia pastoris GS115 cells by electrotransfection. The expressed protein was confirmed by indirect immunofluorescenee and demonstrated immunoreactivity by Western Blot. Rubella virus IgG antibody was assayed with ELISA after mice were inmmunized by E1-374 glyeoprotein. Results SDS-PAGE analysis and Western Blot analysis of E1-374 protein revealed this protein to be 46. 89 x 103. Antiserum （1： 100） and E1-374 （5.5 μg/ml） was chosen for ELISA optimization. The intra-assay coefficient of variation for the ELISA was 0. 36% -12, 45%. Conclusion Protein E1-374 was highly expressed in Pichia pastoris cells, and it was a good choice to prepare rubella virus recombinant protein vaccines.