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

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.