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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 obtain secreted, expressed, non-human anti-HBsAg single-chain antibody (HBscFv) secreted from P. pastoris, primers were designed to amplify HBscFv from pGEM HBscFv, subcloned into the P pastoris expression vector pPICZαA and then transformed into P pastoris GS115, KM71, colony PCR, high concentration of Zeocin resistance screening identified transformants, methanol induced expression of the target protein and its properties were identified. The results showed that 3% ~ 5% of the transformants had a resistance of 200 mg / L of zococin. After induction with the transformants, the HBscFv with a relative molecular mass of 3200 was synthesized and secreted, accounting for the total amount of HBcFv in the culture supernatant 2 2% of the protein; the yeast expression product can be specifically recognized by the monoclonal antibody against E. coli origin HBscFv and has the activity of binding to HBsAg.