小鼠SGK1基因真核表达载体的构建及鉴定

doi:10.13481/j.1671-587X.20250107

Abstract

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

CLC Number:

Q132

Lina ZHANG,Long BA,Jun MENG. Construction and identification of eukaryotic expression vector of mouse SGK1 gene[J].Journal of Jilin University(Medicine Edition), 2025, 51(1): 51-57.

Figures/Tables 3

References 25

1	LI X D， WANG Z Y， JIAO C Y， et al. Hepatocyte SGK1 activated by hepatic ischemia-reperfusion promotes the recurrence of liver metastasis via IL-6/STAT3［J］. J Transl Med， 2023， 21（1）： 121.
2	SANG Y W， KONG P P， ZHANG S Z， et al. SGK1 in human cancer： emerging roles and mechanisms［J］. Front Oncol， 2020， 10： 608722.
3	CICENAS J， MESKINYTE-KAUSILIENE E， JUKNA V， et al. SGK1 in cancer： biomarker and drug target［J］. Cancers， 2022， 14（10）： 2385.
4	LANG F， STOURNARAS C， ZACHAROPOULOU N， et al. Serum- and glucocorticoid-inducible kinase 1 and the response to cell stress［J］. Cell Stress， 2018， 3（1）： 1-8.
5	LU R Q， ZHANG Y Y， ZHAO H Q， et al. SGK1， a critical regulator of immune modulation and fibrosis and a potential therapeutic target in chronic graft-versus-host disease［J］. Front Immunol， 2022， 13： 822303.
6	WEBSTER M K， GOYA L， GE Y， et al. Characterization of sgk， a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum［J］. Mol Cell Biol， 1993， 13（4）： 2031-2040.
7	ZHU M， WU G， LI Y X， et al. Serum- and glucocorticoid-inducible kinase-1 （SGK-1） plays a role in membrane trafficking in caenorhabditis elegans［J］. PLoS One， 2015， 10（6）： e0130778.
8	ARTEAGA M F， ALVAREZ DE LA ROSA D， ALVAREZ J A， et al. Multiple translational isoforms give functional specificity to serum- and glucocorticoid-induced kinase 1［J］. Mol Biol Cell， 2007， 18（6）： 2072-2080.
9	DI CRISTOFANO A. SGK1： the dark side of PI3K signaling［J］. Curr Top Dev Biol， 2017， 123： 49-71.
10	LANG F， STRUTZ-SEEBOHM N， SEEBOHM G， et al. Significance of SGK1 in the regulation of neuronal function［J］. J Physiol， 2010， 588（Pt 18）： 3349-3354.
11	VALINSKY W C， TOUYZ R M， SHRIER A. Aldosterone， SGK1， and ion channels in the kidney［J］. Clin Sci （Lond）， 2018， 132（2）： 173-183.
12	LANG F， GÖRLACH A， VALLON V. Targeting SGK1 in diabetes［J］. Expert Opin Ther Targets， 2009， 13（11）： 1303-1311.
13	DONADEI C， ANGELETTI A， CANTARELLI C， et al. Erythropoietin inhibits SGK1-dependent TH17 induction and TH17-dependent kidney disease［J］. JCI Insight， 2019， 5（10）： e127428.
14	LAI Y， LUO X Y， GUO H J， et al. PO-322 exerts potent immunosuppressive effects in vitro and in vivo by selectively inhibiting SGK1 activity［J］. Br J Pharmacol， 2020， 177（7）： 1666-1676.
15	TALARICO C， DATTILO V， D’ANTONA L， et al. SGK1， the new player in the game of resistance： chemo-radio molecular target and strategy for inhibition［J］. Cell Physiol Biochem， 2016， 39（5）： 1863-1876.
16	RITTER M， BRESGEN N， KERSCHBAUM H H. From pinocytosis to methuosis-fluid consumption as a risk factor for cell death［J］. Front Cell Dev Biol， 2021， 9： 651982.
17	张慧灵，韩迪，郭文秀，等. SGK1对Cyclin B/Cdc2通路介导小鼠G1期受精卵卵裂的调控作用及其机制［J］.吉林大学学报（医学版）， 2024， 50（3）： 628-637.
18	KWON O C， SONG J J， YANG Y， et al. SGK1 inhibition in glia ameliorates pathologies and symptoms in Parkinson disease animal models［J］. EMBO Mol Med， 2021， 13（4）： e13076.
19	HEO E J， LEE Y， HYUNG SEO M， et al. Association between SGK1 and α-synuclein in skeletal muscle in an MPTP-induced Parkinson’s disease model［J］. Neurosci Lett， 2023， 814： 137464.
20	YAO Y， JIANG Q， JIANG L， et al. Lnc-SGK1 induced by Helicobacter pylori infection and highsalt diet promote Th2 and Th17 differentiation in human gastric cancer by SGK1/Jun B signaling［J］. Oncotarget， 2016， 7（15）： 20549-20560.
21	GREENAWALT E J， EDMONDS M D， JAIN N， et al. Targeting of SGK1 by miR-576-3p inhibits lung adenocarcinoma migration and invasion［J］. Mol Cancer Res， 2019， 17（1）： 289-298.
22	GODBOLE M， TOGAR T， PATEL K， et al. Up-regulation of the kinase gene SGK1 by progesterone activates the AP-1-NDRG1 axis in both PR-positive and-negative breast cancer cells［J］. J Biol Chem， 2018， 293（50）： 19263-19276.
23	KULKARNI S， GOEL-BHATTACHARYA S， SENGUPTA S， et al. A large-scale RNAi screen identifies SGK1 as a key survival kinase for GBM stem cells［J］. Mol Cancer Res， 2018， 16（1）： 103-114.
24	LEE L Y W， WOOLLEY C， STARKEY T， et al. Serum- and glucocorticoid-induced kinase sgk1 directly promotes the differentiation of colorectal cancer cells and restrains metastasis［J］. Clin Cancer Res， 2019， 25（2）： 629-640.
25	LIU W W， WANG X C， LIU Z P， et al. SGK1 inhibition induces autophagy-dependent apoptosis via the mTOR-Foxo3a pathway［J］. Br J Cancer， 2017， 117（8）： 1139-1153.

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Construction and identification of eukaryotic expression vector of mouse SGK1 gene

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