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目的: 构建人突变CD59(hmCD59)基因的真核表达系统, 深入研究hmCD59在糖尿病血管并发症中的作用。方法: 分别构建两种含有hmCD59全长cDNA序列的重组pALTER质粒, 运用脂质体介导法, 与pcDNA3质粒共转染CHO细胞, 以G418筛选阳性克隆(编号为hmCD59- 1- CHO及hmCD59 -2- CHO)。应用荧光抗体技术、免疫酶联技术及Westernblot, 进一步检测hmCD59蛋白在转染细胞膜表面的表达。通过双羧乙基碳氧荧光素四乙酰氧甲酯 (BCECF/AM)荧光染料释放试验, 对hmCD59蛋白糖基化前后的抗补体活性进行检测。结果: 筛选出的阳性克隆细胞用荧光抗体技术免疫酶联技术检测表明, 在细胞膜表面有hmCD59分子表达。将细胞的裂解物进行Westernblot证实, 在其相对分子质量(Mr)为 20 000处可见 1条与CD59的Mr相当的蛋白带。BCECF/AM荧光染料释放试验提示, 两种突变质粒的表达产物均具有抗补体活性, 糖基化后活性减弱。结论: 获得两株可稳定表达hmCD59的细胞。表达的hmCD59具有抗补体活性, 但糖基化后活性减弱。
OBJECTIVE: To construct a eukaryotic expression system of human mutant CD59 (hmCD59) gene and further investigate the role of hmCD59 in diabetic vascular complications. Methods: Two recombinant pALTER plasmids containing the full - length cDNA of hmCD59 were constructed. CHO cells were co - transfected with pcDNA3 plasmid by lipofectamine and the positive clones were screened by G418 (hmCD59 - 1 - CHO and hmCD59 - 2-CHO). The expression of hmCD59 protein on the surface of transfected cell membrane was further detected by fluorescent antibody, immunoenzyme and Western blot. The anti-complement activity of hmCD59 protein before and after glycosylation was detected by fluorescent dye release assay of dicarboxyethyl fluorescein tetraacetoxymethyl ester (BCECF / AM). Results: The positive clones screened by fluorescent immunosorbent assay showed that there was hmCD59 expression on the cell membrane surface. The cell lysate was confirmed by Western blot. One protein band corresponding to Mr59 of CD59 was found at a relative molecular mass (Mr) of 20,000. BCECF / AM fluorescence dye release test showed that the expression products of the two mutant plasmids have anti-complement activity, decreased activity after glycosylation. Conclusion: Two hmCD59-stable cells were obtained. The expressed hmCD59 has anti-complement activity, but its activity diminishes after glycosylation.