利用酵母Two-hybrid系统筛选编码产物与p53相作用的人类基因

Using the Yeast Two hybrid System to Identify Gene Encoding p53 associated Proteins

用酵母ｔｗｏ－ｈｙｂｒｉｄ系统在人胚胎脑组织ｃＤＮＡ表达文库中寻找编码产物与ｐ５３相互作用的基因。在１．５×１０６个转化子中筛选到６８个初级阳性克隆。通过测定转化子第二轮β－半乳糖苷酶活力发现，人胚胎脑组织ｃＤＮＡ表达文库中有一个阳性克隆中的ｃＤＮＡ编码产物与ｐ５３反应结果为明显阳性，说明两个蛋白之间存在较强的相互作用。用热测序法测得这段ｃＤＮＡ的序列通过查询基因数据库发现，这段ｃＤＮＡ编码人的泛肽交联酶，可认为是泛肽交联酶参与ｐ５３在细胞内浓度的调节。

p53 interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function involving cell cycle control, gene regulation, cell differentiation, apoptosis and tumor suppression. To identify these proteins, the yeast two hybrid system was used to screen a human fatal brain cDNA library. Of the transformants, 1.5×10 6 were screened for HIS3 and Lacz expression. One pGAD fusion that was dependent on the presence of the GBD p53 fusion plasmid for β galactosidase activity assay was isolated. The cDNA sequence of the positive colony was determined. The gene p53 1 was cloned. p53 1 has the 97% similarity of human ubiquitin conjugating enzyme, indicating ubiquitin conjugating enzyme invloves in the modulation of p53 in cells.