论文部分内容阅读
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。结论 采用本方法可获得纯化的具有生物学活性的人促衰变加速因子
Objective To obtain purified and biologically active human erythrocyte membrane decay accelerating factor. Methods Human erythrocyte membrane DAF was isolated and purified from human erythrocyte membrane by trypsin digestion, n-butanol extraction, DE3 2 ion exchange chromatography and antibody affinity chromatography. Purity and antigen specificity were detected by SDS PAGE and Western blotting. The inhibitory activity of complement was tested by in vitro assembly of C3 convertase and accelerated decay accelerating activity. Results 40 ml of purified whole blood could finally obtain about 42 0 μg of purified DAF with a recovery of 26% and a specific activity of 45 × 10 5 U / mg. The purified product showed 70 × 10 3 single protein bands in SDS PAGE Band and can specifically bind to anti-human DAF mAb in western blot experiments. In biological activity experiments, the purified product can promote the decay of C3 convertase, but also inhibit the formation of C3 convertase. Conclusion This method can be purified biologically active human accelerated decay accelerating factor