摘要
目的 从人 T细胞中克隆 Akt的 PH结构域及其调节区基因并进行 6 - his和 GST融合表达 ,研究二者的体外结合情况 ,为进一步研究 Akt的调节区在其分子激活中的作用打下基础 .方法 以 RT- PCR的方法从人 T细胞中扩增目的基因片段 ,测序证明序列正确 .再将正确的目的基因片段定向克隆到表达载体 p RSET- A和 p GEX- 4 T- 1中 .以 IPTG诱导 ,获得 Akt的 PH结构域及其调节区与 6 - his和 GST的融合表达 .表达的 PH结构域融合蛋白经谷胱甘肽耦联的琼脂糖珠纯化 ,以 pull- down法检测该 PH结构域与 6 - his调节区的结合 .结果 Akt的 PH结构域和调节区基因序列完全正确 ,得到与 GST和 6 - his的融合表达 ,表达产物有较高可溶性 .结论 SDS- PAGE检测到 PH结构域可在体外与调节区特异地结合 .
AIM To probe the role of Akt regulatory domain (RD) in the activation of Akt. METHODS The gene fragments encoding Akt PH domain and RD were cloned from T cells and expressed in fusion proteins with GST and 6 his, respectively. RT PCR was employed to clone the gene frag ments. After confirmation of the sequences, the target gene fragments were cloned into expression vector pGEX 4T 1 and pRSET A. The recombinant vectors were transformed into E.coli which were induced by IPTG and the fusion proteins GST Akt PH domain. RESULTS GST Akt RD domain and 6his RD were expressed in soluble forms. The fusion protein GST Akt PH domain and GST were purified by glutathione agarose beads. In the pull down assay and analysis of SDS PAGE, the 6 his RD domain was detected in GST Akt PH domain lane. CONCLUSION Akt PH domain can be associated specifically with Akt RD in vitro .
出处
《第四军医大学学报》
北大核心
2002年第22期2027-2031,共5页
Journal of the Fourth Military Medical University
基金
国家杰出人才科学基金资助项目 (3 982 5 113 )
国家自然科学基金资助项目 (3 9870 718)
