[Objective]To clone the Kunming mouse eukaryotic translation initiation factor 4A1 gene(eIF4A1)and construct its prokaryotic/eukaryotic expression vector to explore its structure and biological characteristics,and lay a foun-dation for the identification of eIF4A1 interacting protein network in vitro and in vivo.[Method]Using the cDNA synthe-sized by reverse transcription of total RNA of Kunming mouse brain tissue as template,mouse eIF4A1 gene coding region(CDS)sequence was amplified by PCR,and then cloned into pGEX-4T-1 and pcDNA3.0 vectors to construct prokaryo-tic/eukaryotic expression vectors,respectively.The prokaryotic expression vector was induced,purified and identified by Escherichia coli BL21(DE3)receptive cells.At the same time,HEK-293T cells were transfected with eukaryotic ex-pression vector,and the expression and distribution of eIF4A1 protein in HEK-293T cells were detected by Western blot-ting and indirect immunofluorescence(IFA).The biological information of mouse eIF4A1 protein was analyzed by Prot-Param,ProtScale,TMHMM-2.0,SignalP-5.0,SOPMA and SWISS-MODEL.[Result]The CDS sequence length of mouse eIF4A1 gene was 1221 bp,and the prokaryotic expression vector pGEX-4T-eIF4A1-Flag could be successfully con-structed by cloning pGEX-4T-1 vector.Fusion protein eIF4A1-Flag could be expressed in large quantities under the induc-tion of 0.5 mmol/L IPTG at 25 and 30℃,and it mainly existed in the form of inclusion bodies.The mouse eIF4A1 gene was cloned into pcDNA3.0 vector and the eukaryotic expression vector pcDNA3.0-eIF4A1-Flag was successfully con-structed.After transfecting HEK-293T cells with pcDNA3.0-eIF4A1-Flag,eIF4A1 protein was successfully expressed in HEK-293T cells and was mainly distributed in the cytoplasm.The results of bioinformatics analysis showed that the rela-tive molecular weight of eIF4A1 protein was 46.15408 kD,the theoretical isoelectric point(pI)was 5.267,and it con-tained 407 amino acid residues.It was an unstable hydrophilic non-secretory protein with no transmembrane structure and no signal peptide,and its secondary structure was dominated by α-helix and random coil.There were 10 main interacting proteins of mouse eIF4A1 protein,including Paip1 and Pdcd4 interacting proteins in addition to eIFs family proteins.[Conclusion]eIF4A1 is an unstable hydrophilic non-secreted protein which distributes mainly in cytoplasm,without trans-membrane structure and signal peptide.The fusion protein eIF4A1-Flag obtained by constructing prokaryotic/eukaryotic ex-pression vector can be used to screen and identify eIF4A1 interacting proteins.To provide technical support for further in-vestigation of the structure and biological function of eIFs.
维普
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