分泌型Survivin真核表达载体的构建及其在HeLa细胞中的表达

目的:构建含6个组氨酸标签(His6)的小鼠存活素(Survivin)融合蛋白的真核表达载体,及其在真核细胞中的表达。方法:根据小鼠Survivin序列设计引物,经过RT-PCR克隆Survivin的cDNA并进一步构建分泌型Sur-vivin的真核表达载体,经测序鉴定后在大肠杆菌中扩增在HeLa细胞中表达;以金属离子螯合层析富集,再用免疫印迹法鉴定。结果:克隆到Survivin编码区全长序列,经DNA测序后证明与已报道序列相同。构建氨基端融合小鼠IL-2信号肽,羧基端带有His6标签的Survivin融合蛋白的真核表达载体,在HeLa细胞中转染成功并且表达;细胞上清经HisTrap HP柱层析富集后,进行免疫印迹分析,表明该融合蛋白以分泌型方式在HeLa细胞中表达。结论:成功克隆Survivin的cDNA,并构建其分泌型真核表达载体。

Construction of eukaryotic expression plasmid survivin for fusion protein and its expression in HeLa cells

Aim:To construct the eukaryotic expression vector for mouse survivin fusion protein with a His6 tag and to express it in eukaryotic cells. Methods: The primers were designed according to the mouse survivin gene,and the cDNA of survivin was cloned by RT-PCR.The eukaryotic expression vector for secreting type of survivin was constructed.After sequencing analysis,the plasmid was amplified in E.coli and expressed in HeLa cells.Then the recombinant protein was enriched by metal ion chelating chromatography and identified by Western blotting.Results: DNA sequence analysis showed that the cloned cDNA encoding survivin was identical to the published sequence.The eukaryotic expression vector encoding survivin which fused with an IL-2 signal sequence at amino terminus and a His6 tag at carboxyl terminus was successfully expressed in HeLa cells.The fusion protein enriched by HisTrap HP column was assessed by Western blotting,indicating that the protein was expressed in secreting form in HeLa cells.Conclusion:The cDNA of survivin was cloned,and the eukaryotic expression vector for the secreting survivin fusion protein was constructed.This study would be useful for research of vaccine which targeted to survivin in future.