中文摘要：

目的探讨2种突变CD59分子（HM3、HM4）糖基化前后抗补体活性的变化，为糖尿病血管增殖的发生机制提供理论基础。方法采用重组聚合酶链反应定点诱变技术在CD59易于糖基化的位点“41位赖氨酸-44位组氨酸”将44位的组氨酸基因位置改变，构建2种不同的cD59突变基因，克隆入真核表达质粒pALTER-MAX。利用阳离子脂质体将重组质粒和pcDNA3共转染中国仓鼠卵巢细胞。G418筛选稳定转染细胞克隆，免疫标记技术检测筛选突变CD59基因蛋白高表达株。染料释放实验检测突变CD59分子糖基化前后抗补体活性。结果序列测定及酶切鉴定均证实成功构建了pALTER-突变CD59重组真核表达载体系统。经酶免疫组织化学、荧光免疫组织化学和流式细胞术鉴定出较高表达突变CD59分子的阳性细胞克隆。免疫印迹技术检测出阳性细胞克隆裂解液中有1条相对分子质量约20000的CD59蛋白表达带。染料释放实验初步证实突变CD59分子具有抗补体活性，糖基化后抗补体活性明显降低。结论2种突变CD59分子均具有抗补体活性，高糖环境下易被糖基化，糖基化后抗补体活性明显降低，为进一步研究糖尿病血管增殖的发生机制提供了线索.

英文摘要：

Aim To investigate whether glycation could inhibit the protection role of mutant CD59s against human complement. Methods Site-directed mutagenesis to replace residue 37 or 38 with histidine （H） was performed by recombinant PCR. Mutant CD59 DNAs were inserted into the mammalian expression vector pALTER-MAX and transfected into CHO together with the selection marker peDNA3, which eonfered resistance to G418. Expression of mutant CD59s in the G418-resistant clones were tested by Western blot, immunohistochemistry and FCM. A functional dye release assay was used to measure protection role of CD59s against human complement. Results Recombinant plasmids of pALTER-HM CD59 had been successfully constructed according to sequence and enzyme digestion analysis. Stable transfeetants were selected by the addition of G418. Stable populations of CHO cell, which expressed relatively high levels of recombinant protein, were sorted by immunolabled technique. In Western blot assay, the mutant proteins expressed on CHO was about 20 kDa. Dye release assays confirmed both mutants still preserved CD59 activity of antieomplement, and glyeation of CD59 in CHO increased their sensitivity to MAC-mediated lysis. Conclusions Residues by Site-directed mutagenesis to replace residue 37 or 38 with histidine still preserved CD59 activity of antieomplement. Mutant CD59s can be glyeated in vitro and loses its most MAC-inhibitory function. The presence of this glyeation motif in human mutant CD59, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.