Abstract:

Objective To construct an eukaryotic expression vector pcDNA3.1-MYC-SGK1-mcherry which containing mouse serum and glucocorticoid-induced kinase （SGK）1 gene， and to observe its expression in the transfected HEK293 cells. Methods The SGK1 target gene segments were amplified by PCR method， and the segments were ligated to the pcDNA3.1-MYC-C-mcherry vector which was doubly-digested with Hind Ⅲ and Sbf Ⅰ. After successful verification by enzyme digestion and sequencing， the pcDNA3.1-MYC-SGK1-mcherry expression vector was transfected into the HEK293 cells by liposome transfection. Western blotting method was used to determine the expression level of eukaryotic expression vector in the cells. Results The vector band was located at 5 200 bp and the target gene band was located at 3 100 bp， which was consistent with the expected results. The sequencing results were also consistent when compared with the expected sequence by Snap Gene software， which indicated that the eukaryotic expression vector pcDNA3.1-MYC-SGK1-mcherry was successfully constructed. Successful expression of the eukaryotic expression vector pcDNA3.1-MYC-SGK1-mcherry was observed by Western blotting method， in which the transfected cells showed well-defined bands near the relative molecular mass of 49 000. Conclusion The eukaryotic expression vector pcDNA3.1-MYC-SGK1-mcherry is successfully constructed， laying a solid foundation for the subsequent study on the transition mechanism of SGK1 gene in the early development of mouse fertilized egg cells.

Key words: Serum and glucocorticoid-induced kinase 1, Eukaryotic expression vector, HEK293 cells, Vector construction, Plasmids