Preparation and identification of specific monoclonal antibody against Erbin

Objective:To prepare the monoclonal antibody against Erbin. Methods:Molecular cloning technique was used to construct the prokaryotic expression plasmid containing Erbin PDZ cDNA.The plasmid was transformed to E.coli BL21(DE3) to obtain the expression of recombinant protein that was to be purified by Ni-NTA column chromatography and identified by MALDI-TOF-MS analysis. The purified protein was used to immunize BALB/c mice and the monoclonal antibody was prepared by hybridoma technique.Results:Prokaryotic expression plasmid pET-28a(+)-Erbin PDZ was constructed. The recombinant protein was expressed in E.coli efficiently,isolated in inclusion bodies,and purified by refolding and Ni-NTA column chromatography. The purity of the recombinant protein was 90%.MALDI-TOF-MS confirmed that the protein was Erbin PDZ. The monoclonal antibody was obtained by immunizing mice with the recombinant protein.Monoclonal antibody at high titer was selected and identified.Conclusion:The monoclonal antibody can recognize natural Erbin protein. It can be used in ELASA,immuno-fluorescence and immuno-precipitation. The monoclonal antibody against Erbin will be an important tool for further exploration of Erbin functions.