Abstract: Objective: To construct and identify the monoclonal antibody of ATP synthase beta subunit(ATP5B) with high purity, and to lay foundation for further study.Methods: The ATP5B gene was amplified by PCR and cloned into the pET28a vector and transformed into E. coli BL21 (DE3). The protein expression was induced by IPTG and then the fusion protein was purified by nickel affinity chromatography column. The protein purity was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Three female Balb/C mice were immunized with purified fusion protein and the tail vein blood was taken to detect the titer of ATP5B antibody by indirect enzyme-linked immunosorbent assay (ELISA). The spleen cells from the immunized mice with the highest serum titer were mixed with the SP2/0 cells to establish the hybridoma cells and the fused cells were screened by indirect ELISA and monoclonally cultured. Karyotype analysis were performed in the positive cells. The hybridoma cells were intraperitoneally injected into 12 weeks old BALB/C mice to estabilish the ascites models. The titer of ascites was detected by indirect ELISA. The purity of the antibody was detected by SDS-PAGE. The antibody subtype was detected by ELISA.Results: After PCR amplification, a specific band of 1 455bp was obtained, and the pET28a empty vector was ligated to obtain a recombinant pET28a/ATP5B vector. The target protein was expressed in the IPTG-induced bacteria solution; the SDS-PAGE results showed that the protein band was found at 51 000. The indirect ELISA results showed that the serum titer of the venous blood of immunized mice was up to 1:64 000. In karyotype analysis, the total number of chromosomes in hybridoma cells was about the sum of myeloma cells and normal mouse spleen cells. The mouse ascites was prepared with the hybridoma cell line, and the highest titer of the antibody was 1:240 000. The subtype of the monoclonal antibody produced by the hybridoma cells was IgG1.Conclusion: The monoclonal antibody against ATP5B protein is successfully prepared by cloning, expressing and purifying the recombinant protein.

Key words: ATP synthase beta subunit, monoclonal antibody, prokaryotic expression, protein purification