SITE-DIRECTED MUTAGENESIS OF CD59 GENE AND CONSTRUCTION OF EUKARYOTIC EXPRESSION SYSTEM

Objective To construct two CD59 active site relative mutagenesis and clone mutants into eukaryotic expression system. Methods The highly conserved W40 and its vicinity were selected for mutant,and site-directed mutagenesis with deleting residue W40 site and changing C39W40K41→W39W40W41 were performed by Overlap extention PCR.Two normal primers and two mutagenesis reverse complemented primers were designed.cDNAs as templates,tri-PCR were used to amplify the mutant genes.Mutant CD59 DNAs were subcloned into the cloning vector PMD18-T-Vector,then cloned into the eukaryotic expression vector pIRES after digested by EcoRⅠ. Results Mutants were successfully constructed and confirmed by sequence analysis.Recombinant plasmids of pIRES-m1CD59 and pIRES-m2 CD59 were successfully constructed according to sequence and enzyme digestion analysis.The mutant gene was about 500 bp. Conclusion Overlap extension PCR is economical and in reliable,and the recombinants will be applied for futher study on CD59 anticomplement activity.