小鼠SGK3真核表达载体的构建及鉴定  

Construction and identification of mouse SGK3 eukaryotic expression vector

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

机构地区：[1]内蒙古医科大学附属医院临床检验诊断学教研室,呼和浩特010059 [2]内蒙古医科大学附属医院检验科

出　　处：《山西医科大学学报》2024年第7期849-854,共6页Journal of Shanxi Medical University

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

摘　　要：目的构建含有小鼠血清和糖皮质激素诱导蛋白激酶3(serum and glucocorticoid-induced protein kinase 3,SGK3)基因的真核表达载体pcDNA3.1-MYC-SGK3-mCherry,并观察和验证其在转染细胞HEK293中的表达。方法通过聚合酶链式反应将实验室保存的真核表达质粒pcDNA3.1-MYC-SGK3中目的基因SGK3与mCherry融合并扩增出来,然后定向克隆至pcDNA3.1-MYC质粒中,经限制性内切酶消化和测序证实后,通过脂质体法转染HEK293细胞,Western blotting法检测目的基因的蛋白表达情况。结果测序结果与之前预期结果相符,证实pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。Western blotting结果显示,转染pcDNA3.1-MYC-SGK3-mCherry的HEK293细胞出现清晰的阳性反应条带,说明目的片段成功表达。结论pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。Objective To construct the eukaryotic expression vector pcDNA3.1-MYC-SGK3-mCherry containing mouse serum and glucocorticoid-induced protein kinase 3(SGK3)gene,and verify its expression in HEK293 cells after transfection.Methods The target gene SGK3 in the eukaryotic expression plasmid pcDNA3.1-MYC-SGK3 preserved in the laboratory was fused with mCherry and amplified by polymerase chain reaction,and then cloned into pcDNA3.1-MYC plasmid.After restriction enzyme digestion and sequenc-ing,it was transfected into HEK293 cells by liposome method,and the protein expression of the target gene was detected by Western blotting.Results The sequencing results were consistent with the previous expected results,confirming that the pcDNA3.1-MYC-SGK3-mCherry eukaryotic expression vector was successfully constructed.Western blotting results showed that there was clear positive reaction band in HEK293 cells transfected with pcDNA3.1-MYC-SGK3-mCherry,indicating that the target fragment was successfully expressed.Conclusion The eukaryotic expression vector pcDNA3.1-MYC-SGK3-mCherry is successfully constructed.

关 键 词：血清和糖皮质激素诱导蛋白激酶3 真核表达载体 聚合酶链式反应 限制性内切酶 HEK293细胞 载体构建 

分 类 号：Q78[生物学—分子生物学]