目的 构建血栓调节蛋白表皮生长因子样结构功能域（thrombomodulin-domain 2,TM-D2）的毕赤酵母表达体系,以获得重组TM-D2蛋白。方法 以人cDNA 序列为模板,用PCR 扩增出TM-D2片段,并将其插入pPICZaB质粒,构建重组表达质粒TM-D2-pPICZaB,重组质粒电击转化入毕赤酵母菌株GS115,用甲醇诱导酵母进行蛋白表达,行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl-sulfate polyacrylamide gel electrophoresis,SDS-PAGE）和蛋白印迹法鉴定蛋白表达及纯化情况。结果 PCR产物经琼脂糖凝胶电泳和质粒测序证实,TM-D2片段已正确克隆到表达载体中;SDS-PAGE和蛋白印迹法检测结果证实,上清液中含His-tag的重组TM-D2的表达,且其相对分子质量约为55 000。结论 TM-D2-pPICZaB初步判断TM-D2蛋白可在毕赤酵母中表达。
Objective: To establish a Pichia yeast expression system expressing EGF-like domains of thrombomo-dulin domain 2(TM-D2) to obtain recombinant TM-D2 protein. Methods: The nucleotide sequence encoding TM-D2 was amplified by PCR before being ligated into pPICZaB vector. Then TM-D2-pPICZaB vector was constructed and confirmed by PCR and sequencing. After linearization, this recombinant vector was transformed into yeast GS115 by electric shock. Methanol was used to induce the expression of TM-D2, and the TM-D2 was identified by SDS-polyacrylamide gel electrophoresis and Western blotting. Results: Sequencing and PCR revealed that recombinant fragment of TM-D2 with his-tag was cloned into pPICZaB vector, and SDS-PAGE and Western blotting confirmed that TM-D2 protein was present in the supernatant. Conclusions: The recombinant vector TM-D2-pPICZaB is successfully constructed, and TM-D2 is well expressed in Pichia pastoris.
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