小鼠SGK1基因真核表达载体的构建及鉴定  

Construction and identification of eukaryotic expression vector of mouse SGK1 gene

作　　者：张丽娜 巴隆 孟峻[2] ZHANG Lina;BA Long;MENG Jun(Department of Clinical Laboratory Diagnosis,Affiliated Hospital,Inner Mongolia Medical University,Hohhot 010059,China;Department of Clinical Laboratory,Affiliated Hospital,Inner Mongolia Medical University,Hohhot 010050,China)

机构地区：[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 [2]内蒙古医科大学附属医院检验科,内蒙古呼和浩特010050

出　　处：《吉林大学学报(医学版)》2025年第1期51-57,共7页Journal of Jilin University:Medicine Edition

基　　金：国家自然科学基金项目(81360109,81660267);内蒙古自治区科技厅自然科学基金项目(2021MS08158)。

摘　　要：目的:构建含有小鼠血清和糖皮质激素诱导激酶(SGK)1基因的真核表达载体pcDNA3.1-MYC-SGK1-mcherry,并观察载体在转染HEK293细胞后的表达情况。方法:PCR法扩增SGK1目的基因片段,并与经过HindⅢ和SbfⅠ双酶切的pcDNA3.1-MYC-C-mcherry载体连接。酶切和测序验证成功后,采用脂质体转染法将pcDNA3.1-MYC-SGK1-mcherry转染至HEK293细胞中,Western blotting法测定HEK293细胞中真核表达载体的表达情况。结果:酶切载体条带位于5200 bp,目的基因条带位于3100 bp,与预期结果相符。Snap Gene软件比对,pcDNA3.1-MYC-SGK1-mcherry的测序结果与预期序列一致,表明真核表达载体pcDNA3.1-MYC-SGK1-mcherry构建成功。Western blotting法观察到转染后的HEK293细胞在相对分子质量49000附近显现出明显的条带,真核表达载体pcDNA3.1-MYC-SGK1-mcherry成功表达。结论:成功构建真核表达载体pcDNA3.1-MYC-SGK1-mcherry,为后续研究SGK1基因对小鼠受精卵细胞早期发育的作用机制奠定了基础。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 HindⅢand SbfⅠ.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 5200 bp and the target gene band was located at 3100 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 49000.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.

关 键 词：血清和糖皮质激素诱导激酶1 真核表达载体 HEK293细胞 载体构建 质粒 

分 类 号：Q132[生物学—普通生物学]