分化抑制蛋白N端保守域结构预测及其基因突变体的构建  

N-Terminal Conserved Structure Prediction and Mutant Construction of Inhibitor of Differentiation

作　　者：姜凡[1] 伍志强[1] 赵亚力[1] 杨洁[1] 尹肖云[1] 韩为东[1] 

机构地区：[1]解放军总医院基础医学研究所,北京100853

出　　处：《生物技术通讯》2008年第6期795-798,共4页Letters in Biotechnology

基　　金：解放军总医院苗圃基金(07MP69)

摘　　要：目的:对分化抑制蛋白(Id)家族的N端序列进行保守性结构分析,并构建其基因突变体。方法:用ClustalX(1.81)软件对Id蛋白家族中的3个成员(Id1～Id3)的N端序列进行同源性分析,用Swiss-PdbViewer3.7(SP5)软件模拟高同源区域中关键性氨基酸突变前后的三维结构模型;用PCR方法将突变点引入Id序列,再通过重叠PCR方法扩增出全长编码序列,酶切与测序确证突变序列的准确性。结果:Id1～Id3蛋白的N端存在一个由11个氨基酸残基形成的高度保守的环-螺旋(Loop-Helix)结构,将其中最保守的丝氨酸与亮氨酸分别突变为甘氨酸与缬氨酸,将突变后的Id基因序列重组到pGEX原核表达载体中。结论:在Id1～Id3蛋白N端识别了一个保守的Loop-Helix结构,为深入研究其协同的功能特征提供了结构依据;突变其中的丝氨酸为研究Id蛋白潜在的磷酸化修饰及相应功能特征的改变奠定了基础。Objective： To analyze N-terminal domain in inhibitor of differentiation（Id） 1-Id3, looking for functional sites, and to build up prokaryotic expression vectors for mutant Id genes. Methods： ClustalX（1.81） software was used to analyze the homology of N-terminal domain in Id1～Id3 proteins. Three-dimensional protein structures were obtained by Swiss-Pdb- Viewer3.7 （SP5） to compare the changes of conserved domain in Id proteins before and after mutation of the key amino acids. Amplifications were carried out with mutagenic primers, and then DNA products were inserted into prokaryotic ex- pression vectors. Id1～Id3 mutant gene sequences were determined by double digestion and sequencing. Results： N-terminal structure in Id1～Id3 proteins involves a highly conserved Loop-Helix domain. In addition, prokaryotic expression vectors of mutant Id1～Id3 genes were established successfully. Conclusion： Conserved Loop-Helix domain was initially recognized in our study, approving the synergetic function of Id proteins. Serine mutants of Id1～Id3 proteins might be convenient tools for in-depth studies on the phosphorylation of Id protein family.

关 键 词：分化抑制因子 突变 三维结构建模 

分 类 号：Q78[生物学—分子生物学]