人Era结合蛋白基因的克隆

Since 1887 we have been studying on the function of the novel gene era. In 1999, we had cloned the human era cDNA gene from the human fetal brain. In order to discover the function of human era gene, we fished the human Era-binding protein gene from the human fetal liver cDNA library by utilizing the yeast two hybrid system. And we got 16 candidate gene that possibly binding with human Era. On the basis of bioinformatic analysis, we highly expressed the complement component C3 (NTR domain) in E. coli, and proved that complement C3 (NTR domain) could bind with human Era in vitro. By adopting the RNAi technique in HEK293 cell, we got the " loss of function" phenotype of the human Era protein. The flow cytometric results indicated that there was G2/M cell cycle arrest in HEK293 cell with this phenotype. The acquired evidence that human Era worked in the cell cycle will give us some good hints on the human era gene research, and it lays a good foundation for elucidating the mechanism that human era gene functions in the cell.

采用酵母双杂交技术，从人胎脑MATCHMAKER LexAcDNA文库中钓取全长人Era结合蛋白cDNA基因，分析其特征。用基因工程手段高表达人Era结合蛋白，经纯化后测定其与人Era结合的理化特性。构建人Era结合蛋白基因的正义和反义真核表达载体，转染人细胞，获取人Era结合蛋白对细胞周期作用的证据，为人细胞周期调控的研究提供新的资料。