目的 采用大引物法构建GATA1不同激酶活性突变体GATA1 S161A S187A(死型)和GATA1 S161D S187D(激活型)真核表达载体,并证实其融合蛋白在细胞内的表达及定位,旨在进一步探讨其生物学功能和潜在肿瘤治疗靶点.方法 以GFP-GATA1WT为模板,采用大引物法扩增S161A S187A、S161D S187D突变体片段,双酶切克隆至pEGFP-C1表达载体中,将重组质粒转染至HEK293中,经免疫印迹鉴定融合蛋白的表达.结果 用大引物PCR法成功构建GATA1不同激酶活性突变体的真核表达载体pEGFP-GATA1 S161A S187A和pEGFP-GATA1 S161D S187D,验证了其融合蛋白表达.共聚焦激光显微镜技术显示,融合蛋白主要定位于细胞核内.结论 利用大引物法成功构建GATA1不同激酶活性突变体真核表达载体,并为进一步进行该突变体的结构和功能研究奠定了基础.%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.
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