中文摘要：

目的：构建突变型CD59重组质粒,建立高效真核表达系统,探讨W40位点的生物学活性。方法：采用基因点突变技术使CD5 9W40基因位置缺失,各设计两条互补突变引物和两条常规引物,以已构建的CD59 cDNA为模板,三重PCR（Overlap PCR）定点诱变扩增突变基因,重组入克隆载体PMD18-T-Vector,EcoRⅠ单酶切野生型和突变型CD59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（Chinese hamster ovary,CHO）进行表达。结果：通过PCR定点诱变成功获得目的CD59突变基因MCD59,酶切鉴定及序列测定证实成功构建了pIRES-MCD59,转染CHO细胞,G418筛选出了稳定细胞克隆,检测筛选MCD59蛋白高表达株。免疫组化、免疫荧光、SDS-PAGE、ELISA验证CD59的表达;荧光染料释放试验研究显示与野生型CD59相比,突变型CD59失去对补体的抑制功能。结论：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：

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.