妊娠相关蛋白A的纯化及鉴定研究

Purification and identification of pregnancy associated plasma protein A

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。

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.