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目的 :分离纯化猪血小板膜糖蛋白并对其进行鉴定。方法 :差速离心法分离猪血小板 ,1%TritonX 10 0溶解膜糖蛋白 ,麦胚凝集素亲和层析纯化糖蛋白 (0 .3mol/LN 乙酰葡萄糖胺洗脱 ) ,用鼠抗人GPⅠb单抗PHN89作点印迹 ,聚丙烯酰胺凝胶电泳 ,考马斯亮蓝 (Coomassieblue ,CB)和过碘酸 Schiff(PAS)染色鉴定糖蛋白 ,腺苷二磷酸钠盐(ADP)和瑞斯托菌素 (ristocetin)诱导的人血小板聚集测定其体外活性。结果 :用GPⅠb单抗PHN89为一抗的点印迹表明 0 .3mol/LN 乙酰葡萄糖胺能充分洗脱结合到凝集素上的GPⅠb。PAS法糖定性有 2条显色条带 ,SDS PAGE纯化后有 2条蛋白区带。ADP阳性对照血小板聚集率为 74 % ,纯化前后的样品血小板聚集率分别为 4 2 %和 4 % ,ristocetin阳性对照血小板聚集率为 10 0 % ,纯化前后的样品血小板聚集率分别为 2 5 %和 3% ,表明制备的血小板糖蛋白具有生物学活性。结论 :得到了较纯的血小板膜糖蛋白 ,为血小板膜糖蛋白的进一步研究及应用奠定基础
Objective: To isolate and purify porcine platelet glycoprotein and identify it. METHODS: Porcine platelets were isolated by differential centrifugation. 1% TritonX 10 0 dissolved membrane glycoprotein, wheat germ agglutinin affinity chromatography purified glycoprotein (0.3 mol / LN acetylglucosamine eluted) Anti-PHN89 was used for dot blotting, polyacrylamide gel electrophoresis, Coomassie blue (CB) and periodic acid Schiff (PAS) staining to identify glycoprotein, adenosine diphosphate (ADP) and ristocetin ristocetin induced human platelet aggregation in vitro activity. Results: Dot blot using GPIb monoclonal antibody PHN89 as primary antibody showed that 0.3 mol / L of acetylglucosamine eluted GPIb bound to lectin sufficiently. There are two color bands in PAS method and two protein bands in SDS PAGE. ADP-positive platelet aggregation rate was 74%, sample platelet aggregation rate before and after purification were 42% and 4%, ristocetin positive control platelet aggregation rate was 10%, before and after purification platelet aggregation rate were 25% and 3%, indicating that the prepared platelet glycoprotein has biological activity. Conclusion: The pure platelet membrane glycoprotein was obtained, which laid the foundation for further research and application of platelet membrane glycoprotein