Construction and identification of eukaryotic expression vector of mouse SGK1 gene
10.13481/j.1671-587X.20250107
- VernacularTitle:小鼠SGK1基因真核表达载体的构建及鉴定
- Author:
Lina ZHANG
1
;
Long BA
;
Jun MENG
Author Information
1. 内蒙古医科大学附属医院临床检验诊断教研室,内蒙古呼和浩特 010059
- Keywords:
Serum and glucocorticoid-induced kinase 1;
Eukaryotic expression vector;
HEK293 cells;
Vector construction;
Plasmids
- From:
Journal of Jilin University(Medicine Edition)
2025;51(1):51-57
- CountryChina
- Language:Chinese
-
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.
