乙型脑炎病毒E蛋白在巴斯德毕赤酵母中的表达

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JEV E蛋白基因亚克隆人真核表达载体pPIC9K，以电穿孔法转化酵母SMD1168。用MD平板筛选重组子、G418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113000和78000，表达量约为50mg/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。

Expression of Japanese encephalitis virus E protein in methylotrophic yeast Pichia pastoris

AIM: To express Japanese encephalitis virus (JEV) E protein in methylotrophic yeast Pichia pastoris . METHODS: The gene coding for JEV E protein was sub cloned into vector pPIC9k. The constructed plasmid was transformed into yeast SMD1168 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of E protein in yeast was induced by the addition of methanol and analyzed by SDS PAGE and Western blot. RESULTS: The protein was produced at a yield of 50 mg per litre of culture. Owing to heterogeneous glycosylation, relative molecular mass ( M r) of the expressed E protein was sized about 113 000 and 78 000. CONCLUSION: JEV E protein was expressed successfully in yeast Pichia pastoris , which should be useful for the production of diagnostic reagents and genetically engineered vaccine of JEV.