摘要
目的：从大鼠脾脏克隆信号分子ｇｕａｎｉｎｅ－ｎｕｃｌｅｏｔｉｄｅ－ｒｅｌｅａｓｉｎｇｆａｃｔｏｒ（ＧＲＦ）的ｐｌｅｃｋｓｔｒｉｎｈｏｍｏｌｏｇｙ（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ｃＤＮＡ，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，以双脱氧终止法测序．然后再克隆到表达载体ｐＧＥＸ－４Ｔ－１中作ＧＳＴ融合表达．结果：信号分子ＧＲＦ的ＰＨ结构域基因完全正确，内部不含终止码．并在表达载体ｐＧＥＸ－４Ｔ－１中获得融合表达．结论：从组织细胞中克隆ＰＨ结构域基因是可行的方法，可在大肠杆菌中以ＧＳＴ融合蛋白的形式表达．
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.
出处
《第四军医大学学报》
1999年第6期461-463,共3页
Journal of the Fourth Military Medical University
