类泛素家族SUMO-1和UBC9的克隆、融合表达及纯化

目的：克隆类泛素化家族SUMO-1和UBC9基因,表达并纯化二者与谷胱甘肽S-转移酶（GST）的融合蛋白。方法：用PCR方法从乳腺文库中扩增SUMO-1和UBC9的编码序列,分别将其以正确相位与pGEX-KG载体中的GST编码序列融合,得到重组质粒pGST-SUMO-1和pGST-UBC9,分别转化大肠杆菌DH5α,表达融合蛋白GST-SUMO-1和GST-UBC9;用谷胱甘肽-Sepharose4B亲和纯化融合蛋白;用Western印迹检测融合蛋白的表达及纯化。结果：分别构建了SUMO-1和UBC9的融合表达载体;Western印迹检测表明,GST-SUMO-1和GST-UBC9融合蛋白获得表达;纯化得到了融合蛋白。结论：克隆、表达并纯化了SUMO-1和UBC9与GST的融合蛋白。

Cloning, Fusion Expression and Purification of SUMO-1 and UBC9WEI Wei1,2, DING Li-Hua1, ZHANG Hao1, YANG Zhi-Hong1,

Objective：To clone,express and purify the fusion proteins of sumoylation family members SUMO-1 and UBC9.Methods：The coding sequences of SUMO-1 and UBC9 were amplified by PCR and fused in frame with the coding region of GST in the pGEX-KG vector to generate the pGST-SUMO-1 and pGST-UBC9 recombinant plasmids.The GST-SUMO-1 and GST-UBC9 fusion proteins were expressed in E.coli DH5α and purified by Glutathione-Sepharose 4B affinity chromatography.The fusion proteins were characterized by Western blot.Results：The pGST-SUMO-1 and pGST-UBC9 recombinant plasmids were successfully constructed.Western blot analysis showed that the fusion proteins were expressed.Conclusion：The genes of SUMO-1 and UBC9 were successfully cloned and their fusion proteins were purified.