【摘要】 目的:对比G200凝胶过滤、反向亲和层析、DEAE离子交换等不同的方法纯化孕妇血清中的妊娠相关蛋白A(PAPP-A),为下一步的临床应用研究奠定基础。方法:把正常男性血清过G200凝胶过滤纯化后免疫家兔得到兔抗人抗体,连接溴化氰活化G25凝胶制备反向免疫层析柱。收集足月孕妇血清,离心后依次应用G200凝胶过滤、反向亲和层析及离子交换层析柱纯化。用ELISA检测试剂盒检测各个洗脱峰的PAPP-A活性,SDS-PAGE检测纯化蛋白的纯度。结果:稀释兔抗人血清抗体至浓度为1∶16,双向扩散有明显沉淀线,可以用于制备抗体,蛋白A亲和层析洗脱pH3.0洗脱峰,G200凝胶过滤第一个洗脱峰,反向亲和层析的0.1 mol/LPBS洗脱峰,离子交换层析的0.45 mol/L NaCl洗脱峰中PAPP-A活性均较高。SDS-PAGE结果显示,孕妇血清用G200凝胶过滤处理后杂蛋白条带较多,反向亲和层析或者DEAE分别处理后同样存在杂蛋白条带,三种纯化方法结合应用杂蛋白条带明显减少,Western blot鉴定显示三种方法结合应用后纯化蛋白样品条带单一。结论:本研究用三种层析方式相结合的方法得到纯度较高的PAPP-A抗原,鉴定显示可以到达制备单克隆抗体(mAb)的要求,为mAb的制备以及酶联免疫试剂盒的建立奠定了基础,使PAPP-A在临床应用方面前景广阔。更多还原

【Abstract】 AIM: To purify pregnancy associated plasma protein A(PAPP-A) from the pregnancy serum using the methods of G200 gel filtration,reverse affinity chromatography and DEAE ion exchange.METHODS: The male sera were purified by G200 gel filtration and injected into rabbits to obtain rabbit-anti-human antibodies.The antibodies were linked to CNBr-activated G25 gel to make the reverse affinity chromatography.The full-term pregnancy sera were collected and centrifuged,followed by G200 gel filtration,reverse affinity chromatography and DEAE ion exchange.ELISA and SDS-PAGE were performed to detect the activity and the purity of PAPP-A.RESULTS: As detected by the double diffusion,the rabbit-anti-human antibodies were active in the dilution of 1∶16,and were further used for purification of PAPP-A.The PAPP-A exited in the last peak with the pH3.0 elutriant by SPA,the first peak by G200 gel filtration,the peak with 0.1 mol/L PBS elutriant by the reverse affinity chromatography,and the peak with 0.45 mol/L NaCl elutriant by the DEAE ion exchange.The SDS-PAGE results indicated that the triple treatments of the pregnancy sera by G200 gel filtration,the reverse affinity chromatography and the DEAE ion exchange effectively enhanced purity of PAPP-A,with a single protein band shown by Western blot,whereas a single purification method produced dirty bands.CONCLUSION: High purity PAPP-A has been obtained,ready for preparation of its monoclonal antibody.The study paves a way for development of PAPP-A based ELISA kits for clinical use.更多还原