肝豆状核变性基因表达蛋白的多克隆抗体制备和纯化  

Preparation and purification of the polyclonal antibody against ATP7B.

作　　者：丰岩清[1] 王莹[1] 黄帆[1] 徐评议[1] 梁秀龄[1] 

机构地区：[1]中山大学附属第一医院神经科,广州510080

出　　处：《中国神经精神疾病杂志》2002年第4期241-244,共4页Chinese Journal of Nervous and Mental Diseases

基　　金：本课题由中山医科大学"211工程"重点建设项目;广东省自然科学基金(编号:001403);广东省自然科学基金(编号:990064)资助

摘　　要：目的　制备并纯化肝豆状核变性（WD）基因表达蛋白的多克隆抗体,为进一步研究WD蛋白的结构和功能打下基础。方法　应用RT-PCR扩增目的基因,GST基因融合载体表达并纯化融合蛋白,Thrombin酶切并收集目的蛋白,免疫家兔,凝胶过滤层析纯化抗体血清中的lgG,ELISA检测抗体滴度和western blot检测抗体特异性。结果　本方法生产的抗体滴度达到了1:12500,特异性好。结论　我们应用该方法成功制备了Wilson病蛋白的多克隆抗体,为我们进一步研究该蛋白的结构和功能奠定了基础。Objective To prepare and purify a specific antibody against ATP7B. Methods We use RT-PCR to amplify the target gene, and cloned it to pGEX-4T-l; GST gene fusion system was used to express the fusion protein which was purified. Thrombin cleave fusion protein was collected and used as the antigen. Rabbits were immunized subcutaneously. ELISA was used to measure the titer of the antibody in rabbit serum. Purified antibody reacted directly with Sephorose A columns Western blot was used to conform the specialties of the antibody. Results ELISA assay revealed that the titer of the prepared antiserum against fusion protein was as high as 1:1 2500. Western blot showed that the antiserum was able to react with the proteins. Conclusions We successfully prepared a polyclonal antibody against ATP7B.

关 键 词：肝豆状核变性 基因表达蛋白 多克隆抗体 制备 纯化 

分 类 号：R742.4[医药卫生—神经病学与精神病学]