信号分子GRF的PH结构域基因片段的克隆与表达

目的：从大鼠脾脏克隆信号分子ｇｕａｎｉｎｅ－ｎｕｃｌｅｏｔｉｄｅ－ｒｅｌｅａｓｉｎｇ ｆａｃｔｏｒ（ＧＦＲ）的ｐｌｅｃｋｓｔｒｉｎ ｂｏｍｏｌｏｇｙ（ＰＨ）结构域基因并进行谷胱甘肽转移酶（ＧＳＴ）融合表达，为进一步寻找其新配基、研究其功能打下基础。方法：采用一步法从大鼠新鲜脾组织中提取总ＲＮＡ，反转录合成ｃＮＤＡ，ＰＣＲ方法扩增目的基因片段，并克隆到载体ｐＵＣ１９中。引物末端标记后，以双脱氧终止法

Cloning and expression of gene encoding PH domains of signaling molecules GRF

AIM: To screen for novel ligands of the PH domains and to investigate the functions of the PH domains in signal transduction. METHODS: Rat spleens were employed to clone the gene encoding PH domain of GRF. Total RNA molecules were isolated from fresh spleens and mRNAs were reversely transcriped into cDNAs. After amplification by PCR, the fragments of DNA were cloned into vector pUC19. The method of dideoxytermination was employed to sequence with labeled primmers, and then the gene was cloned into vector pGEX4T1 to express in fusion with GST. RESULTS: The sequence of the gene was correct and the gene was successfully expressed in pGEX4T1. CONCLUSION: The methods of cloning PH domains of signaling molecules are feasible and useful in study of signal transduction. The fragments of the gene can correctly be expressed in E. coli as fusion with GST.