Abstract: 【Objective】 The objective of this study was to clone the mouse ATP5B gene, and construct its prokaryotic and eukaryotic expression vectors, in order to provide a foundation for studying the function of ATP5B protein and its role mechanism in the process of viral infection. 【Method】 The coding region（CDS） of mouse ATP5B gene was cloned. Bioinformatics analysis of ATP5B protein was performed using ProtParam, ProtScale, and SignalP-5.0. Construction of prokaryotic expression vector pGEX-4T-1-ATP5B^-Flag and eukaryotic expression vector pcDNA3.0-ATP5B^-Flag was carried out by homologous recombination. The prokaryotic expression vector pGEX-4T-1-ATP5B^-Flag was transformed into Escherichia coli BL21 competent cells, and IPTG was used for induction expression, the optimal induction temperature and IPTG concentration was analyzed. And the eukaryotic expression vector pcDNA3.0-ATP5B^-Flag was transfected into HEK-293T cells. The expression and distribution of ATP5B protein in cells were detected by Western blotting and indirect immunofluorescence assay（IFA）. 【Result】 The CDS of mouse ATP5B gene was 1590 bp, and the mouse ATP5B protein was composed of 529 amino acids residues, with a molecular weight of 55 kD. The theoretical isoelectric point（pI） was 5.21, it was a stable hydrophobic protein without transmembrane domains and signal peptides, and it was not a secretory protein. The secondary structure of mouse ATP5B protein included 43.67% α-helix, 32.51% random coil, 9.45% β-turn and 14.37% extended strand. After IPTG induction of the prokaryotic expression vector pGEX-4T-1-ATP5B^-Flag, the fusion protein was successfully detected at 81 kD by Coomassie brilliant blue staining and Western blotting, mainly expressed in the form of inclusion bodies, with better induction conditions at 30 ℃ and 0.5 mmol/L IPTG. After transfection of HEK-293T cells with the eukaryotic expression vector pcDNA3.0-ATP5B^-Flag, a Flag specific band was detected at 55 kD, confirming successful expression of the ATP5B protein in HEK-293T cells. The results from the IFA detection indicated that the protein was mainly located in the cytoplasm. 【Conclusion】 The prokaryotic and eukaryotic expression vectors of the mouse ATP5B gene are successfully constructed. The fusion protein expressed by the prokaryotic expression vector mainly exists in the form of inclusion bodies, with only a small amount of soluble protein. The eukaryotically expressed ATP5B protein is mainly localized in the cytoplasm. The mouse ATP5B protein is a stable hydrophobic protein with stable physicochemical properties, without signal peptide site, and is not a secretory protein.