从SARS冠状病毒(GD322株)组织培养上清中提取 RNA,进行 RT PCR扩增其 M基因并克隆到巴氏毕赤酵母表达载体 pPICZαB,电穿孔法将重组体整合入酵母菌 P. pastoris,经抗生素 Zeocin筛选产生的转化子进行表型鉴定,取Mut+菌用甲醇诱导表达,SDS PAGE检测其表达产物。Mut+ 酵母转化菌经甲醇诱导可分泌表达约 65kDa和42 kDa的蛋白质,与SARS恢复期病人血清的免疫印迹证实它们为特异的重组M蛋白质,且获得的重组M蛋白质具有免疫反应性。 RNA was extracted from the tissue culture supernatant of SARS coronavirus (GD322 strain), and the M gene was amplified by RT PCR and cloned into the expression vector pPICZαB of Pichia pastoris. The recombinant was integrated into the yeast P. pastoris by electroporation, The phenotypes of the transformants were identified by Zeocin screening. Mut + bacteria were induced by methanol and detected by SDS PAGE. Mut + yeast transformed cells induced methanol-secretable proteins secreting about 65 kDa and 42 kDa, and immunostained with SARS convalescent patient sera to confirm that they are specific recombinant M proteins, and the obtained recombinant M proteins are immunoreactive.