摘要
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 con- firmed by PCR and sequencing. After linearization, this recombinant vector was transformed into yeast GSl15 by electric shock. Methanol was used to induce the expression of TM-D2, and the TM-D2 was identified by SDS-polyacrylamide gel eleetrophoresis 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.
出处
《诊断学理论与实践》
2017年第1期55-59,共5页
Journal of Diagnostics Concepts & Practice
基金
国家卫生和计划生育委员会"2013-2014年度国家临床重点专科建设项目"
上海市卫生计生系统重要薄弱学科建设项目(2016zb0203)
关键词
血栓调节蛋白
重组质粒
毕赤酵母
Thrombomodulin
Recombinant vector
Pichia pastoris
