PKBAkt 调节结构域克隆，表达及初步纯化.docVIP

PKB/Akt 调节结构域的克隆，表达及初步纯化 作者：王立峰，张健，李莹，苏金，药立波，刘新平 【关键词】 基因表达 　　【Abstract】 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 pMD18T vector. After sequence analysis, it was subcloned into pRSETA which can express the target protein as a (His)6tagged 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 pRSETA and the sequence was confirmed by comparison with the published one. Recombinant pRSETARD was successfully constructed. RD/6×His fusion protein was correctly expressed and the purification was easily achieved on a Ni2NTA 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 Ni2NTA agarose beads. 　　【Keywords】PKB/AKT; cloning, molecular; gene expression; purification 　　【摘要】 目的： 克隆akt1的调节结构域 (regulation domain RD )的编码基因，表达并纯化 Akt RD蛋白，为研究 RD的功能奠定基础.方法： 用RTPCR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pMD18T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pRSETA中，转化大肠杆菌BL21(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pRSETA 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,表达了RD/6×His融合蛋白并得到了纯化的RD/6×His蛋白. 　　【关键词】 PKB /Akt ；克隆,分子； 基因表达 ；纯化 　　0引言 　　PKB/Akt分子由三部分组成，第一部分为N端的pH结构域，第二部分为中间的催化结构域，第三部分为C端的调节结构域（regulation domain, RD）［１－３］ .PKB/Akt参与某些生长因子或淋巴因子诱导的靶细胞应答，影响胞内糖类的转运、糖原的合成及蛋白质的合成，抑制细胞凋亡等，是机体维持正常生命活动的一个重要调节分子［４］.PKB/Akt催化结构域中的Thr308和调节结构域中的Ser473都发生磷酸化后，使其活性被激活，进而调节下游分子，执行