重组人CLK1的真核表达及鉴定

目的:合成真核细胞CLK1(Cdc2-like kinase 1)编码基因,构建CLK1/pEGFP-N2真核表达载体并在真核细胞HEK293A中过表达,为CLK1的生物学功能研究奠定基础。方法:从人脐静脉血管内皮细胞中提取总RNA,采用RT-PCR技术用已知引物合成cDNA,将CLK1基因扩增后插入真核细胞表达载体pEGFP-N2,将重组质粒热转化至大肠杆菌感受态Trans 10细胞中获得重组菌株,提取质粒进行酶切鉴定及插入基因测序;将构建的重组质粒转染HEK293A细胞,用Western印迹及免疫荧光检测CLK1的表达水平,同时对其下游的磷酸化SF2/ASF蛋白进行检测。结果:构建了CLK1/pEGFP-N2真核表达载体,将其转染HEK293A细胞后24 h,CLK1蛋白表达水平最高;同时,CLK1过表达后使得下游的SF2/ASF蛋白磷酸化水平升高。结论:构建了人CLK1基因的真核细胞表达载体CLK1/pEGFP-N2,并在HEK293A细胞中过表达,其生物活性也得到了验证。本研究为外源性CLK1基因在真核细胞中过表达提供了一种途径,为CLK1的生物学功能研究奠定了基础,也可为真核细胞其他蛋白表达体系的构建提供借鉴。

Eukaryotic Expression and Identification of Recombinant Homo CLK1

Objective： To investigate synthesis of the gene sequence encoding eukaryotic Cdc2-like kinase 1 （CLK1）, construction of the eukaryotic vectors CLK1/pEGFP-N2 and its over-expression in HEK293A cells. This work provides guideline for research in CLK1 biological function. Methods： Total RNA was extracted from human umbilical vein endothelial cells. The CLK1 gene was amplified by RT-PCR, and then was inserted reversely into the eukaryotic expression vector pEGFP-N2. The recombined plasmid was sequenced and transformed into E.coli Trans 10. Subsequently, the plasmid was transfected into HEK293A cells. The expression level of CLK1 was mea-sured by Western blot and immunofluorescence. Then the phosphorylation level of SF2/ASF protein was detected. Results： The CLK1/pEGFP-N2 eukaryotic over-expression vector was successfully constructed. The optimum time of highest expression level of CLK1 in HEK293A cells was 24 h. And the phosphorylation level of SF2/ASF pro-tein was increased. Conclusion： A recombinant eukaryotic expression plasmid containing CLK1 gene was success-fully constructed and expressed in HEK293A. This research provides a means for the exogenous CLK1 gene ex-pression in eukaryotic cells, and laid the foundation for the further study of CLK1 biological function. At the same time, it makes a reference for the construction of other protein expression system in eukaryotic cells.