Prokaryotic expression of ATP5B and preparation and identification of its monoclonal antibody / 吉林大学学报(医学版)

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 K. 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 tlie antibody was detected by SDS-PAGE. The antibody subtype was detected by ELISA. Results: After PCR amplification, a specific band of 1 455 bp 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 tlie antibody was 1 5 240 000. The subtype of the monoclonal antibody produced by the hybridoma cells was IgGl. Conclusion: The monoclonal antibody against ATP5B protein is successfully prepared by cloning, expressing and purifying the recombinant protein.

Prokaryotic expression of ATP5B and preparation and identification of its monoclonal antibody / 吉林大学学报(医学版)

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 ATP5Bgene was amplified by PCR and cloned into the pET28avector 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 ATP5Bantibody by indirect enzyme-linked immunosorbent assay (ELISA) .The spleen cells from the immunized mice with the highest serum titer were mixed with the SP2/0cells 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 12weeks 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 pET28aempty vector was ligated to obtain a recombinant pET28a/ATP5Bvector.The target protein was expressed in the IPTG-induced bacteria solution;the SDS-PAGE results showed that the protein band was found at51 000.The indirect ELISA results showed that the serum titer of the venous blood of immunized mice was up to1:64 000.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 ATP5Bprotein is successfully prepared by cloning, expressing and purifying the recombinant protein.

Preliminary identification of the outer membrane protein H receptors of avian Pasteurella multocida / 中国人兽共患病学报

To identify the receptors for the outer membrane protein H (OmpH) of avian P.multocida,the membrane proteins of chicken embryo fibroblast (CEF) cells were separated by SDS-PAGE and analyzed by Ligand blot.The OmpH-binding protein was identified by MALDI-TOF mass spectrometry,and its distribution in the membrane proteins of different host esophageal mucosal cells was detected by Ligand blot,ELISA and immunofluorescence microscopy,respectively.Ligand blot analysis showed that a 49-kDa membrane protein of CEF cells bound to recombinant OmpH,and MALDI-TOF spectral results demonstrated that the OmpH-binding protein was ATP synthase β subunit.In addition,the OmpH receptor was present in the chicken and rabbit mucosal cell membranes,but was not detected in the bovine and swine mucosal cell membranes.The above results indicate that the OmpH receptor may be CEF cell-derived ATP synthase beta subunit.