人SNARE复合物相关蛋白的表达与纯化

Expression and purification of human SNARE complex associated proteins

[目的]构建人SNARE复合物中的SNAP25、Syntaxin 1a和VAMP1蛋白真核表达载体,并于体外进行表达纯化。[方法]将重组质粒转化至大肠杆菌DH10Bac感受态细胞,经抗性和蓝白斑筛选,获得重组杆粒;用脂质体转化法,将重组杆粒转染至Sf9昆虫细胞中使其产生重组病毒,最后对病毒进行扩增后再感染Sf9昆虫细胞表达目的蛋白;所得蛋白进行Western Blot鉴定及纯化。[结果]测序结果表明SNAP25、Syntaxin 1a和VAMP1真核表达载体构建成功,Western Blot检测出三种蛋白的特异性条带,利用His标签蛋白纯化出Syntaxin 1a蛋白。[结论]利用杆状病毒表达系统成功表达出了SNARE复合物相关蛋白。

[Objective]The eukaryotic expression vectors of SNAP25,VAMP1 and Syntaxin 1a protein in human SNARE complex were constructed, and then expressed and purified in vitro.[Method] The recombinant plasmid was transformed into competent E.coli DH10Bac cells to construct the recombined bacmid and then transfected into Sf9 insect cells by liposome transformation to produce recombinant virus, after that, the virus was amplified and infected Sf9 insect cells to express the target protein.The obtained proteins were identified and purified by Western Blot.[Result]The sequencing results showed that the eukaryotic expression vectors SNAP25,Syntaxin 1a and VAMP1 were successfully constructed.The specific bands of the three proteins were detected by Western Blot, and Syntaxin 1a was purified by His tag protein.[Conclusion] SNARE complex related proteins were successfully expressed by baculovirus expression system.