Cd59 On Gene W40 Locus Mutation, Eukaryotic Expression And Biological Activity

Objective To construct two mutant CD59 genes by side-directed mutagenesis and recombine them into eukaryotic expression vectors,establish eukaryotic expression systems and detect the expression of CD59 on transfected CHO cells and investigate their biological function for supplying the theory on turnout immune treatment.Methods The highly conserved W40 and its vicinity genes were selected for mutant.Here two Site-directed mutagenesis were performed by overlap extension PCR,to delete residue W40(Mutant 1,M1) or to change C39W40K41 to W39W40W41(Mutant 2,M2). Two normal primers and two mutagenesis reverse complemented primers were designed, cDNAs as template,tri-PCR were used to amplify the mutant genes.Mutant CD59 DNAs were subcloned into cloning vector PMD18-T-Vector,and were cloned into the eukaryotic expression vector pIRES after digested by EcoRâ… .Then the recombinants were transfected into CHO cells by Lipfectamine2000.Stable transfectants were screened by the addition of G418.Stable positive cell clones were screened by CD59 mRNA in situ hybridization,and stable populations of CHO cells expressing relatively high levels of recombinant proteins were sorted by immunological fluorescence,ELISA and Westernblot immunotecnique.A functional dye release assay and a trypan blue stain were used to measure protection role of CD59 against human complement.Results Mutants were successfully constructed and confirmed by sequence analysis. Recombinant plasmids of pIRES-M1CD59 and pIRES-M2CD59 have been successfully constructed according to sequence and enzyme digestion analysis.The mutant gene is about 500bp.Stable populations of CHO cells of expressing relatively high levels of recombinant protein were screened by immunoteenique.After 30 passages culturing, proteins can be tested.Dye release assays and Trypan Blue stain suggest that M1CD59 loses activity against complement,while M2CD59 increases the anti-complement activity slightly.Conclusion 1.The recombinant eukaryotic expression systems containing mutant CD59 were successfully constructed.2.M1CD59 and M2CD59 are stablely expressed on CHO cells.3.Results indicate that W40 of human CD59 is important to its activity,prohibition of this site may be a potential way to increase complement activity and to treat tumors.