摘要
SH2D4A是SH2蛋白家族成员之一,可能参与酪氨酸蛋白激酶相关受体介导的信号转导,调节细胞的生长、增殖和分化,进而影响人类疾病的发生。文章运用酵母双杂交技术筛选SH2D4A相互作用蛋白,并利用酵母交配(mating)实验和GST pull-down实验进行了初步鉴定。首先成功构建了酵母诱饵蛋白重组表达载体pGBKT7-SH2D4A;利用该重组体对人肾脏cDNA文库进行逐级筛选,共得到46个阳性克隆;经目的基因的分离、DNA测序及BLAST软件对序列进行比对分析,发现5种可能的SH2D4A互作蛋白(AZGP1、DAD1、HSD17B10、KAT5和PKM2);NetPhos 2.0 Server软件预测结果显示除HSD17B10外其他4种蛋白均含有磷酸化酪氨酸;进一步以KAT5和HSD17B10作为代表进行了酵母交配和GST pull-down,证实两者均可直接结合SH2D4A。
SH2D4A is a member of SH2 signaling protein family, which is involved in the signal transduction mediated by protein tyrosine kinase-related receptor, cell growth, proliferation, differentiation, and thereby affects the development of human disorders. To determine the role of SH2D4A in the cell signal transduction pathway, SH2D4A interactive proteins were screened using yeast two-hybrid system, and yeast mating and GST pull-down assays were carried out to further confirm the interaction. We successfully generated a bait protein expression construct-pGBKT7-SH2D4A, screened the human kidney cDNA library, and obtained 46 positive yeast clones. After isolation of positive colonies, DNA sequencing, and sequence alignment analysis with BLAST software, we obtained 5 potential SH2D4A interactive proteins, AZGP1, DAD1, HSD 17B 10, KAT5, and PKM2, which were predicted by NetPhos 2.0 Server software and were all shown to be phosphorylated tyrosine （pY）-containing proteins except for HSD17B10. KAT5 and HSD17B10 were selected to perform yeast mating and GST pull-down experiments, indicating their direct binding to SH2D4A.
出处
《遗传》
CAS
CSCD
北大核心
2010年第7期712-718,共7页
Hereditas（Beijing)
基金
国家自然科学基金项目(编号:30370785)资助
