Construction,Protein Expression,and Subcellular Localization of Various Kinase Mutant Plasmids of GATA1
10.12007/j.issn.02584646.2018.01.006
- VernacularTitle: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
- Author:
Danni LI
1
;
Yunpeng LIU
Author Information
1. 中国医科大学附属第一医院肿瘤内科
- Keywords:
GATA1;
PAK5;
phosphorylation;
gene cloning;
fusion protein;
transfect
- From:Journal of China Medical University
2018;47(1):22-26
- CountryChina
- Language:Chinese
-
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.
