中国医科大学学报2018，Vol.47Issue(1)：22-26,5.DOI:10.12007/j.issn.02584646.2018.01.006
GATA1不同激酶活性突变体质粒的构建及蛋白表达和亚细胞定位
Construction,Protein Expression,and Subcellular Localization of Various Kinase Mutant Plasmids of GATA1
摘要
Abstract
Objective The GATA1 mutant GATA1 S161A S187A (death type) and GATA1 S161D S187D (activated) eukaryotic expression vectors were constructed using the large primer method,and,to explore their biological function and potential tumor treatment targets,the expression and localization of the fusion protein in cells were confirmed. Methods S161A,S187A,S161D,and S187D mutants were amplified by GFP-GATA1 WT,which served as the template. The recombinant plasmid was cloned into a pEGFP-C1 expression vector and transfected into HEK293 cells by immunoblotting expression of the fusion protein. Results The eukaryotic expression vectors pEGFP-GATA1 S161A S187A and pEGFP-GATA1 S161D S187D were successfully constructed using the high primer PCR method,and expression of the fusion protein was verified. Confocal laser microscopy showed that the fusion protein was mainly located in the nuclei of HEK293 cells. Conclusion A eukaryotic expression vector of a GATA1 mutant was successfully constructed using the large primer method. This work lays the foundation for further studies on the structure and function of the mutant.关键词
GATA1/PAK5/磷酸化/基因克隆/融合蛋白/转染Key words
GATA1/PAK5/phosphorylation/gene cloning/fusion protein/transfect分类
医药卫生引用本文复制引用
李丹妮,刘云鹏..GATA1不同激酶活性突变体质粒的构建及蛋白表达和亚细胞定位[J].中国医科大学学报,2018,47(1):22-26,5.基金项目
国家自然科学基金(81372546) （81372546）
