【摘要】 目的:探讨毕赤酵母能否高效表达具有生物学活性的弓形虫主要表面抗原P30与免疫佐剂CTA2/B重组蛋白(P30-CTA2/B)。方法:将目的基因定向亚克隆入pGAPZαA表达载体,构建酵母表达载体pGAPZαA-P30-CTX;线性化重组酵母表达载体,电穿孔法导入毕赤酵母GS115,抗生素Zeocin筛选、PCR法鉴定阳性转化子;酵母工程菌摇瓶表达,取上清液行SDS-PAGE和Westernblotting分析。结果:在毕赤酵母菌中分泌表达P30-CTA2/B重组蛋白,产量高达0.57g/L;SDS-PAGE显示其在49000处有一条明显表达蛋白条带,Westernblotting显示表达蛋白具有P30抗原特异性。结论:大量具有免疫活性的P30-CTA2/B重组蛋白在毕赤酵母中表达,为弓形虫亚单位复合疫苗的研制和大规模动物实验创造了条件。更多还原

【Abstract】 Objective: To explore high-level secretive expressed fusion protein of toxoplasma major surface antigen P30 with cholera toxin A2/B in Pichia pastoris. Methods: A compound gene fragment including P30 and cholera toxin A2/B was inserted into expression vector pGAPZαA by subclone technique , then the recombinants were transferred into yeast GS115 by electroporation and Pichia transformants were identified by PCR. The expression product was analyzed by means of SDS-PAGE and Western bolting. Results: The positive recombinant plasmids pMD18T-P30-CTA2/B and pGAPZαA-P30-CTA2/B were constructed successfully. A specific 49kDa fusion protein was expressed in Pichia pastoris, the product amounted to 570 mg/ L. The fusion protein P30-CTA2/B had good antigenicity. Conclusion: High-level expression of secreted P30-CTA2/B fusion protein is achieved in Pichia pastoris expression system.更多还原