CD59活性位点相关性基因突变的构建与表达

构建了野生型和突变型CD59重组质粒，建立了高效真核表达系统，探讨了W40位点的生物学活性。采用基因点突变技术使CD59W40基因位置缺失（突变1，M1）及C39W40K41→W39W40W41（突变2，M2），重叠延伸PCR（overlap extension PCR）定点诱变扩增突变基因，重组入真核表达质粒pIRES，利用阳离子脂质体（Lipfectamine2000）将重组质粒转染中国仓鼠卵巢细胞（CHO）进行表达。酶切鉴定及序列测定证实成功构建了pIRES-MICD59、pIRES—M2CD59和pIRES-WTCD59，突变基因约500bp。G418筛选出了CHO转染细胞的稳定细胞克隆，免疫荧光、ELISA检测筛选MICD59、M2CD59和WTCD59蛋白高表达株，连续传代30代有高表达；补体溶细胞反应显示与野生型CD59相比，突变型M1CD59失去对补体的抑制功能，而M2CD59抗补体活性略增高。证实CD59的W40位点对其功能具有重要作用，封闭此位点可提高补体活性，有望用于肿瘤治疗。

Construction and expression of active site relative mutant on human CD59

To construct wild type and mutant CD59 eukaryotic expression systems and investigate their biological function on Chinese hamster ovary (CHO) cells,site-directed mutagenesis with deleting residue W40 site (mutant 1,M1) and changing C39W40K41 to W39W40W41 (mutant 2,M2) were performed by overlap extension PCR.Wild-type and mu- tant CD59 DNAs were cloned into pIRES vectors and transfected into CHO ceils by Lipfectamine2000.Recombinant plas- mids of pIRES-WTCD59,pIRES-M1CD59 and pIRES-M2CD59 were successfully c...