中文摘要：

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。

英文摘要：

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 mutant CD59 DNAs were cloned into pIRES vectors and transfected into CHO cells by Lipfectamine2000. Recombinant plasmids of plRES-WTCD59, pIRES-M1CD59 and pIRES-M2CD59 were successfully constructed according to sequencing. The mutant gene was about 500bp. Stable transfectants were screened by the addition of G418. Stable populations of CHO cells expressing relatively high levels proteins were screened by immunological fluorescence and ELISA. After culturing of 30 passages, proteins can be tested. Complement lysis assays suggests that M1CD59 losts its protection role of CD59 against human complement, while M2CD59 increases the anti-complement activity slightly. Thus WdO of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and treat tumors.