Cloning of mouse eIF4A1 gene and its prokaryotic/eukaryotic expression identification

【Objective】To clone the Kunming mouse eukaryotic translation initiation factor 4A1 gene(e IF4A1) and construct its prokaryotic/eukaryotic expression vector to explore its structure and biological characteristics, and lay a foundation for the identification of eIF4A1 interacting protein network in vitro and in vivo. 【Method】Using the cDNA synthesized by reverse transcription of total RNA of Kunming mouse brain tissue as template, mouse e IF4A1 gene coding region(CDS) sequence was amplified by PCR, and then cloned into pGEX-4T-1 and pcDNA3.0 vectors to construct prokaryotic/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 expression vector, and the expression and distribution of eIF4A1 protein in HEK-293T cells were detected by Western blotting and indirect immunofluorescence(IFA). The biological information of mouse eIF4A1 protein was analyzed by ProtParam, 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 constructed by cloning pGEX-4T-1 vector. Fusion protein eIF4A1^-Flag could be expressed in large quantities under the induction of 0.5 mmol/L IPTG at 25 and 30 ℃, and it mainly existed in the form of inclusion bodies. The mouse e IF4A1 gene was cloned into pcDNA3.0 vector and the eukaryotic expression vector pcDNA3.0-eIF4A1^-Flag was successfully constructed. After transfecting HEK-293T cells with pcDNA3.0-e IF4A1^-Flag, e IF4A1 protein was successfully expressed in HEK-293T cells and was mainly distributed in the cytoplasm. The results of bioinformatics analysis showed that the relative molecular weight of eIF4A1 protein was 46.15408 kD, the theoretical isoelectric point(pI) was 5.267, and it contained 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 transmembrane structure and signal peptide. The fusion protein eIF4A1^-Flag obtained by constructing prokaryotic/eukaryotic expression vector can be used to screen and identify eIF4A1 interacting proteins. To provide technical support for further investigation of the structure and biological function of eIFs.