人源性抗HBsAg单链Fab基因在Pichia pastoris中的表达

目的：研究重组Fab的结构与亲和活性的关系：方法：通过重叠PCR，将人源性抗HBsAg Fab的H和L链基因融合构建单链Fab基因，并将其转入毕赤酵母表达载体pPICZαA中。以单链Fab基因表达载体通过氯化锂转化法转化毕赤酵母GS115。将获得的重组酵母在摇瓶中培养进行重组单链Fab的可溶性表达。表达上清经硫酸铵沉淀及亲和层析纯化后，用直接ELISA检测表达产物和纯化Fab的活性：结果：SDS—PAGE和Western blot分析显示，单链Fab在毕赤酵母中获得分泌型表达。薄层扫描显示，在摇瓶中培养毕赤酵母表达的单链Fab约为5～10mg／L。经亲和层析纯化获得纯度达97．8％的重组单链Fab。经直接ELISA测定的结果显示，重组单链Fab具有较好的结合HBsAg的活性。结论：通过重叠PCR构建的融合单链Fab基因，可成功地在毕赤酵母中获得分泌型表达，表达产物具有较好的结合HBsAg的活性。

Expression of human anti-HBsAg single chain Fab gene in Pichia pastoris

AIM: To investigate the relation of the affinity activity to the structure of recombinant Fab. METHODS: The human anti-HBsAg single chain Fab gene was obtained by overlaping PCR from the H and L chains of Fab, and was introduced into the expression system of Pichia pastoris. The expression vector of single chain Fab was transformed into GS115 cells by the method of LiCl transformation. The transformants were obtained and cultured in shake flask. The culture supernatant of the recombinant yeast was deposited in (NH_4)_2SO_4 and purified by affinity chromatography. The affinity of culture supernatant of recombinant yeast and purified single chain Fab was analyzed by ELISA. RESULTS: The analysis of SDS-PAGE and Western blot showed that the single chain Fab gene was expressed successfully in Pichia pastoris, and the expression quantity was about 5-10 mg/L in shake flask. The recombinant Fab was purified by affinity chromatography and the purity reached 97.8%. The ELISA analysis showed that the recombinant single chain Fab had good HBsAg binding properties. CONCLUSION: The single chain Fab gene could be expressed successfully in Pichia pastoris and the recombinant single chain Fab can efficiently bind with HBsAg.