Construction and expression of mutant CD59 deleting W40 site and study on its biological function

Objective:To construct mutant CD59 eukaryotic expression system and investigate their biological function on CHO cell.Methods:Site-directed mutagenesis with deleted residue W40 site by overlap PCR.Mutant was successfully constructed and confirmed by sequence analysis.Wild-type and mutant CD59 DNAs were cloned into the eukaryotic expression vector pIRES and transfected into CHO by Lipfectamine2000.Results:Recombinant plasmids of pIRES-WTCD59 and pIRES-MCD59 have been successfully constructed according to sequence and enzyme digestion analysis.The mutant gene is about 670 bp.Stable transfectants were screened by the addition of G418.Stable populations of CHO cells of expressing relatively high levels of recombinant protein were sorted by immunological fluorescence,immunohistochemistry,ELISA and SDS-PAGE.Dye release assays suggests mutant CD59 lost its protection role of CD59 against human complement.Conclusion:The W40 of human CD59 is important to its activity,prohibition of this site may be a potential way to raise complement activity and treat tumor.