摘要
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。
AIM: To construct an eukaryotic expression system of human mutant CD59(hmCD59)gene, and investigate the effect of hmCD59 on diabetic vascular complications. METHODS: Two recombinant pALTER plasmids containing two hmCD59 cDNAs were transfected into CHO cells respectively with pcDNA3 by lipofectamine.Stably transfected clones were then selected by G418. Expression of hmCD59 on CHO cells was detected by fluorescent antibody technique, immunoenzymatic histochemistry and Western blot. 2’, 7’-bis(carboxyethyl)-5, 6-carboxyfluorescein acetoxymethylester (BCECF/AM) was used to study the anti-complement activity of hmCD59 before and after glycosylation.RESULTS: hmCD59 was expressed on stably transfected CHO cell surface. Both hmCD59 had the anti-complement activity which was weakened after glycosylation.CONCLUSION: Two stable cell lines expressing hmCD59 have been successfully established.Both hmCD59 had the anti-complement activity, which was weakened after glycosylation.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2005年第2期151-154,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No. 30170893)
