摘要
目的 获得纯化并具有生物学活性的人红细胞膜促衰变加速因子。方法 经胰蛋白酶消化、正丁醇抽提及DE3 2离子交换色谱和抗体亲和层析等步骤从人红细胞膜影中分离纯化人红细胞膜DAF。以SDS PAGE及免疫印迹实验检测纯度及抗原特异性 ,以C3转化酶体外组装实验及促衰变加速活性实验检测其补体抑制活性。结果 40 0ml人全血最终可得纯化DAF约 42 0 μg ,回收率达 2 6% ;比活性为 4 5× 10 5U/mg ;纯化产物在SDS PAGE中表现为 70× 10 3 的单一蛋白条带 ,并在免疫印迹实验中可与抗人DAF单抗特异性结合。在生物学活性实验中 ,纯化产物既可促进C3转化酶的衰变 ,也可抑制C3转化酶的形成。
Objective To purify decay accelerating factor (DAF) with biological activity from human erythrocyte membrane. Methods Human DAF was purified from human erythrocyte ghost by trypsin digestion, butanol extraction, sequential chromatography on DE32 and immunoaffinity chromatography. The purity of purified protein and its reactivity with antibody to human DAF was investigated by SDS PAGE and Western blotting. The effects of purified DAF on assemble and decay of C3 convertase were also tested. Results Approximately 420 μg purified DAF was obtained from 400 ml human whole blood with 26% activity recovery and 4.5×10 5 units/mg specific activity. On SDS PAGE, the purified protein showed a single band and gave an apparent Mt. about 70×10 3. Western blotting showed that monoclonal antibody to DAF could bind purified protein specifically and also inhibit the assemble and accelerate the decay of classical pathway C3 convertase. Conclusion This method can be used to purify DAF with biological activity from human erythrocyte membrane.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2002年第11期1316-1319,共4页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目 ( 30 0 80 0 32 )
重庆市科委重点攻关项目 ( 2 0 0 0 )
