摘要
目的 :克隆akt1的调节结构域 (regulationdomainRD)的编码基因 ,表达并纯化AktRD蛋白 ,为研究RD的功能奠定基础 .方法 :用RT PCR方法从胚胎肝细胞中获得Akt1调节结构域RD的编码基因 ,克隆至pMD18 T载体并测序 ,结果正确后亚克隆到含有 6个His的融合表达载体pRSET A中 ,转化大肠杆菌BL2 1(DE3 ) ,经IPTG诱导表达RD/6×His融合蛋白 ,超声裂菌 ,将上清通过金属螯合层析进行纯化 .结果 :从人胚胎肝细胞中扩增出编码RD的cDNA ,序列测定证实与文献报道的序列一致 ;成功构建了 6×His融合的表达载体pRSET A RD ;表达了RD/6×His融合蛋白 ,并纯化得到了RD/6×His蛋白 .结论 :成功克隆了人akt1的调节结构域RD基因 ,构建了6×His融合的表达载体 。
AIM: To clone the coding sequences of human RD (regulation domain of PKB/Akt), to express the corresponding protein and to get the purified protein. METHODS: The RD cDNA (regulation domain of PKB/Akt) fragment was amplified using PCR in fetal hepatocytes. The PCR product was then ligated into pMD18 T vector. After sequence analysis, it was subcloned into pRSET A which can express the target protein as a (His)6 tagged fusion. The bacterial strain BL21 (DE3) was used as the host cells to induce the expression of the fusion protein by IPTG. Cell pellets were lysed by sonication to get the supernatant. RESULTS: RD coding region was cloned into pRSET A and the sequence was confirmed by comparison with the published one. Recombinant pRSET A RD was successfully constructed. RD/6×His fusion protein was correctly expressed and the purification was easily achieved on a Ni2 NTA affinity column by virtue of the (His)6 tag on the protein. CONCLUSION: Human RD of akt1 gene has been successfully cloned and RD/6×His recombinant plasmid constructed. RD/6×His fusion protein has been correctly expressed in E.coli and the soluble fusion protein purified by Ni2 NTA agarose beads.
出处
《第四军医大学学报》
北大核心
2003年第6期481-484,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金 (3982 51 1 3
30 1 70 4 65)
军队十五重点课题(0 1Z0 80 )
