作　　者： ; ; ; ; (任向荣）; (唐永红）; (粟宽源）; (余宙耀）;

机构地区： 暨南大学

出　　处： 《上海免疫学杂志》 2003年第3期168-172,共5页

摘　　要： 为了从巴氏毕赤酵母中获得分泌表达的非融合人源抗HBsAg单链抗体 (HBscFv ) ,设计引物从pGEM HBscFv上扩增HBscFv,亚克隆至P pastoris表达载体pPICZαA中 ,然后转化P pastorisGS115、KM71,菌落PCR、高浓度Zeocin抗性筛选鉴定转化子 ,甲醇诱导目的蛋白表达并对其性质进行鉴定。结果发现 ,3%～ 5 %的转化子具有 2 0 0 0mg/LZeocin抗性 ;转化子诱导后 ,可以合成并分泌相对分子质量为 32 0 0 0的HBscFv ,表达量占酵母培养上清中总蛋白的 2 2 % ;酵母表达产物可被针对大肠杆菌来源HBscFv的单克隆抗体特异性识别 ,并具有结合HBsAg活性。 To express the anti HBsAg single chain Fv (HBscFv),the HBscFv,being amplified from plasmid pGEM HBscFv was subcloned into expression vector pPICZαA and then transformed into Pichia pastoris GS115 and KM71 The recombinant Pichia strains,identified by direct PCR and Zeocin resistant screening of Pichia transformants,were cultivated and induced with methanol The target protein was determined by Western blotting and indirect ELISA It was found that high Zeocin resistant transformants were 3%～5% of the total transformants screened Those transformants induced could secrete HBscFv with an apparent mass weight of 32 kD into the cultural supernatants up to a level of 22% of the total proteins.The HBscFv produced in Pichia pastoris could be recognized specifically by a McAb against the HBscFv of the E coli origin and possessed ability of HBsAg binding

关 键 词： 乙肝表面抗原 单链抗体 巴氏毕赤酵母 表达

领　　域： [生物学]