中文摘要：

目的建立表达人突变CD59的细胞模型，初步研究其活性。方法以正常的CD59基因全长cDNA序列为模板，应用重叠延伸PCR法产生突变使CD59糖基化基序K41-H44突变为K41-K42，产生突变CD59基因，将其克隆于pALTER质粒中，构建出重组真核表达载体。应用脂质体介导法，与pcDNA3质粒共转染中国仓鼠卵巢（CHO）细胞，以G418筛选阳性克隆。应用免疫化学染色及Western blot方法进一步检测突变CD59分子在转染细胞膜表面的表达。通过荧光染料释放试验，对突变CD59蛋白糖基化前后的补体限制活性进行检测。结果筛选出的阳性克隆细胞膜表面有突变CD59分子表达。将细胞的裂解物进行Western blot检测证实，在相对分子质量20000处可见1条与CD59相当的蛋白带。表达有突变CD59分子的阳性克隆细胞染料释放率低于对照组，糖基化后染料释放率明显升高。结论成功地获得了表达人突变CD59分子的细胞株。

英文摘要：

Objective To establish a cell model expressing human mutant CD59 molecular, and study its biological activities. Methods Nucleotides within glycosylation motif were changed to a human mutant CD59 gene （from K41-H44 to K41-K42） by overlap extension PCR. The mutant CD59 gene was inserted into a eukaryotic-expressing vector pALTER to form a recombinant plasmid. CHO cells were co-transfected by the recombinant plasmid and pcDNA3 via lipofectamine method. Target protein expressed on the membrane of the transfected cell lines was detected by immunocytochemistry and Western blot. Fluorescence dye was used to study the complement restriction activity of the mutant CD59 before and after glycosylation. Results Mutant CD59 was expressed on the surface of transfected CHO cells. The dye release rate of positive clone expressing mutant CD59 molecules was lower than that of the control group. However, the rate accelerated after glycosylation. Conclusion A stable transfected cell model expressing human mutant CD59 is successfully established, which might lay the basis for further study of its biologic functions.