Purification Of Human Mutant CD59 Protein And Preparation Of Monoclonal Antibody

Objective To establish a CHO cell model transfected with hmCD59-pALTER-MAXrecombinant plasmid. To purify recombinant human mutan CD59 protein by ANTI-FLAGM2 affinity chromatography. To prepare the polyclonal and monoclonal antibody againsthmCD59 by immunize Balb/c mice with purified recombinant hmCD59 protein. Providethe stability theoretic basis for devising the diagnostic and therapeutic measures based onthe anti-hmCD59 monoclonal antibody. Help to supply new thought for further studyingof vascular proliferative complications of diabetes.Methods The recombinant PALTER-MAX plasmid containing human mutant CD59cDNA and pcDNA3 plasmid were co-transfected into CHO cell by cation lipoidmediating method. Stably transfected clones were then selected by G418 and fluorescentantibody technique. Expression of hmCD59 was detected by SDS-PAGE. The expressiveproduct was purified by Anti-FLAG M2 Affinity Gel chromatography and identified bySDS-PAGE,western-blot and enzyme linked immunosorbent assay(ELISA). ImmunizeBalb/c mice with the purified hrnCD59 to prepare the polyclonal and monoclonalantibody. The titer and immune activity of antibody was detected by ELISA.Results Detection by fluorescent antibody technique and SDS-PAGE indicated thatstably transfected CHO cell line expressed hmCD59 was established;SDS-PAGE assayshow the hmCD59 protein have high purity after purification by ANTI-FLAG M2 affinitygel affinity chromatography, the concentration of protein was 0.6mg/ml; the result ofwestern-blot and ELISA indicated that the purified hmCD59 was combined with anti-hmCD59 antibody specially; polyclonal antibody against hmCD59 was generated byimmunizing Balb/c mice with purified hmCD59, the titer of antiserum is about 1: 100;onehybridoma clones secreting specificity monoclonal antibody was established byhybridoma technique,titer of cultivation supematant was 1: 16.Conclusions CHO cell model transfected with hmCD59-pALTER-MAXrecombinant plasmid was constructed successfully. The recombinant hmCD59 expressedon CHO cells stably. Purified hmCD59 with immunity activity had been obtainedsuccessfully by affinity chromatography. Furthermore, polyclonal and monoclonalantibody had been obtained by immunizing Balb/c mice with the purified hmCD59,which lays the foundation for further functional study and clinic application ofanti-hmCD59 antibody.