抗血小板膜糖蛋白Ibα胞内段多肽多克隆抗体的制备及初步应用  

Preparation and preliminary application of rabbit polyclonal antibodies against intraceHular peptides of human platelet glycoprotein GPIbα

作　　者：元艳宏[1,2] 康佳蕊[3] 闫荣[1] 程虹[1] 庄逢源[1] 王月丹[3] 戴克胜[1] 

机构地区：[1]北京航空航天大学生物与医学工程学院血液与细胞生物学实验室,100083 [2]中国航天员科研训练中心分子和细胞研究室 [3]北京大学医学部免疫系

出　　处：《中华医学杂志》2009年第12期826-830,共5页National Medical Journal of China

基　　金：国家自然科学基金（30770795）;教育部新世纪优秀人才资助计划基金（NCET-06-0167）;全国优秀博士论文获得者专项基金（FANEDD200560）

摘　　要：目的制备兔抗人血小板膜糖蛋白（GP）IbαC端557～561氨基酸序列多肽的多克隆抗体，并初步用于人血小板GPIbαC端559位丝氨酸磷酸化状态的检测。方法应用化学方法合成C—R—G-S—L-P（559位丝氨酸非磷酸化，Ser559）和C—R—G—S（P）-L-P（559位丝氨酸磷酸化，pSer559）多肽。将2种多肽分别与钥孔蜮血蓝蛋白交联后，以皮下注射法分别免疫新西兰大白兔，分离获得2种抗血清（抗Ser559多抗和抗pSer559多抗）。应用斑点印迹和酶联免疫吸附试验（ELISA）方法对抗血清进行鉴定并检测效价。从人血小板裂解液中分离纯化血小板GPIbα，利用抗Ser559多抗和抗pSer559多抗、采用ELISA方法检测人血小板GPIbαC端559位丝氨酸磷酸化状况。结果所制备的2种多抗分别特异性识别各自抗原，效价分别为1：32000和1：64000。2种多抗均可与纯化的人血小板GPIbα特异结合，表明人血小板GPIbα559位点丝氨酸存在磷酸化与非磷酸化两种状态。结论应用人工合成多肽成功制备出2种可特异性识别丝氨酸磷酸化状态的兔抗人血小板GPIbα胞内段多肽多抗，并证明人血小板GPIbcαC端559位丝氨酸存在磷酸化状态。Objective To prepare rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbα. Methods Two peptides corresponding to human platelet GPIbα C- terminus were synthesized and purified by high-performance liquid chromatography （HPLC）. The peptides were cross-linked with keyhole limpet hemocyanin （KLH）. Two New Zealand white rabbits were immunized with conjugated peptides for 3 times. The polyclonal antibodies were purified by Ammonium Sulfate Precipitation and identified by dot blotting and ELISA. GPIbα intracellular peptides phosphorylation was tested with these polyclonal antibodies by ELISA. Results The titers of the two polyclonal antibodies against the GPIbα C-terminus peptides were 1 ： 3：2 000 and 1 ： 64 000 respectively and both of these antibodies reacted with purified GPIbα. Conclusions Two rabbit polyclonal antibodies against C-terminal peptides of human platelet GPIbα have been prepared successfully, providing a way for the preparation of these kinds of antibody. Both phosphorylation and dephosphorylation states exist in the intracellular peptide of human platelets.

关 键 词：血小板 血小板糖蛋白GPIb—IX复合物 磷酸丝氨酸 抗体 

分 类 号：R686[医药卫生—骨科学]