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目的 :用巴斯德毕赤酵母系统表达乙型脑炎病毒 (JEV)E蛋白。方法 :将JEVE蛋白基因亚克隆入真核表达载体 pPIC9K ,以电穿孔法转化酵母SMD116 8。用MD平板筛选重组子、G4 18筛选高拷贝转化子 ,经甲醇诱导表达后 ,SDS PAGE和免疫印迹分析表达产物。结果 :由于糖基化不同 ,所表达产物的相对分子质量 (Mr)约为 1130 0 0和 780 0 0 ,表达量约为5 0mg/L。结论 :在巴斯德比赤酵母中成功地表达了JEVE蛋白 ,为制备JEV的诊断抗原和基因工程重组疫苗打下基础
Objective: To express Japanese encephalitis virus (JEV) E protein using Pichia pastoris system. Methods: The JEVE protein was subcloned into eukaryotic expression vector pPIC9K and transformed into yeast SMD116 by electroporation. Recombinant plasmids were screened by MD plates and high copy transformants were screened by G418. After induced by methanol, the expressed products were analyzed by SDS PAGE and immunoblotting. Results: Due to the different glycosylation, the relative molecular mass (Mr) of the expressed product was about 1130 0 0 and 780 0 0, and the expression level was about 50 mg / L. Conclusions: The JEVE protein was successfully expressed in Pasteurella multocida, laying a foundation for the production of diagnostic and genetically engineered recombinant vaccines for JEV