摘要
目的 用单克隆抗体亲和层析方法从人血小板破碎液中纯化血小板因子 4和P -选择素。方法 将单克隆抗体SZ - 95 -IgG和SZ - 5 1-IgG分别与溴化氰活化的Sepharose4B凝胶连接成SZ - 95 -IgG -Sepharose4B和SZ -5 1-IgG -Sepharose4B亲层析柱 ,人血小板破碎液经此亲和层析柱上样后 ,再经FPLC系统获得的所要的产品 ,产品经SDS -聚丙烯酰胺凝胶电泳鉴定其纯度 ,用点杂交鉴定其生物学活性。结果 SZ - 95 -Sepharose4B和SZ - 5 1-Seph arose4B亲和层析柱的偶联率分别 72 %和 6 8% ,每 1ml(约 1× 10 9个血小板 )血小板破碎液中可以纯化到 18μgPF4和12 μgP -selectin ,所得产品经SDS -PAGE ,在分子量约为 12kD和 140KD处各显单一的蛋白区带 ,经点杂交印迹显示产品分别与单抗SZ - 95和SZ - 5 1反应显带。结论 用单克隆抗体亲和层析柱纯化的PF4和P -selectin产品得率高、纯度高。
Purpose:To purify human platelet factor 4(PF4)and P-selectin using monoclonal antibody affinity chromatography.MethodsAfter binding two monmclonal antibodies SZ-95-IgG and SZ-51-IgG to CNBr activated sepharose 4B,we got two affinity chromatographies SZ-95-sepharose 4B and SZ-51-sepharose 4B.Proteins,which were purified from triton X-100 solubilized platelet solution through FPLC by SZ-95-sepharose 4B,SZ-51-sepharose 4B affinity chromatography,were characterized by SDS-PAGE and Dot blot.Results The binding rate of this affinity chromatography is 72%,68%.18μg of PF4 and 12μg of P-selectin can be purified from about 1×10 9 of platelets.Our SDS-PAGE and Dot blot indicated this 12kD protein is PF4,140kD protein is P-selectin Conclusin Using SZ-95-sepharose 4B,SZ-51-sepharose 4B affinity chromatography to purify PF4,P-selectin is an efficient method to prepare human PF4,P-selectin with high biological activity.
出处
《中国血液流变学杂志》
CAS
2001年第3期179-181,185,共4页
Chinese Journal of Hemorheology
