Expression of Selenium Binding Protein 2 and Their Interaction with FKBP38 Protein
Objective:This study aims to construct pcDNA3.1-6×HIS-mSELENBP2 recombinant plasmid and explore the interaction between SELENBP2 and FKBP38 proteins.Methods:Firstly,total RNA from mouse liver tissue was isolated as a template and designed the primers according to the CDS sequence of the mouse SELENBP2 gene to synthesize the SELENBP2 protein coding sequence through RT-PCR.The SELENBP2 gene sequence was recycled by restriction endonuclease and connected by ligase with pcDNA3.1-6×HIS plasmid vector,and then transformed into a JM108 competent Escherichia coli for amplification.After selecting positive monoclonal E.coli and extracting plasmids,the pcDNA3.1-6×HIS-mSELENBP2 recombinant plasmid was obtained using genetic engineering technology.Then,after sequencing analysis and identification,the recombinant plasmid was transfected into AML 12 cells using Lipofectamine 3000 transfection reagent and cultured for 30 h to detect protein expression using Western blot analysis.Using AlphaFold Multimer to predict the possibility of interaction between SELENBP2 and FKBP38 proteins,as well as the possible domains of interaction.Finally,the recombinant plasmid was transferred into AML12 cells which over expressed FKBP38 protein and after 30 h of cultivation,cell protein samples were collected for immunoprecipitation test.Results:The sequence analysis results of pcDNA3.1-6× HIS-mSELENBP2 recombinant plasmid showed that the inserted SELENBP2 protein coding sequence was in accordance with the expected sequence as the CDS sequence of the mouse SELENBP2 gene in GenBank report.After transfection of pcDNA3.1-6× HIS-mSELENBP2 recombinant plasmid into AML12 cells,the SELENBP2 HIS fusion protein can be effectively expressed.The results of AlphaFold Multimer showed that the possibility of interaction between SELENBP2 and FKBP38 proteins is high,and it acted on the TPR domain of FKBP38 protein.There is an interaction between the SELENBP2 protein and the FKBP38 protein in AML12 cells according to the co-immunoprecipitation experiment.Conclusions:The pcDNA3.1-6×HIS-mSELENBP2 recombinant plasmid has been successfully constructed and can be correctly expressed in AML 12 cells,laying the foundation for further research on the function of the SENELBP2 protein.The interaction between SELENBP2 and FKBP38 proteins further broadens the thinking of the study of SELENBP2 protein and provides a new direction for the further studying of the biological function of SELENBP2 protein in the liver.
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