人血小板膜糖蛋白VI胞外区片段的原核表达及多克隆抗体的制备  

Prokaryotic expression and polyclonal antibody preparation of human platelet glycoprotein Ⅵ extracellular domain

作　　者：屈晨雪[1] 李传保[1] 王建中[1] 武淑兰[1] 万文徽[1] 

机构地区：[1]北京大学第一医院检验科,100034

出　　处：《中华检验医学杂志》2005年第10期1068-1071,共4页Chinese Journal of Laboratory Medicine

摘　　要：目的探讨在原核细胞中表达人血小板膜糖蛋白Ⅵ(GPVI)胞外区片段,制备其多克隆抗体。方法采用PCR技术扩增人血小板GPVI胞外区片段123aa^268aa的基因序列,构建其原核表达载体,在大肠杆菌中诱导融合蛋白表达。表达产物经亲和层析法纯化及鉴定后,免疫家兔制备多克隆抗体。采用已知单克隆抗体进行ELISA双抗夹心法、Westernblot和流式细胞术鉴定其特异性。结果构建的重组质粒经酶切和测序证实序列正确。经诱导表达在相对分子量为4200处有一条明显融合蛋白条带,Westernblot分析证实与预期值一致。所得的抗血清效价为1∶128,ELISA双抗夹心法检测表明,所制抗体识别血小板GPVI分子。Westernblot和流式细胞术检测结果显示所制抗体可与血小板裂解液中及血小板膜表面GPVI分子发生特异性免疫反应。结论利用原核细胞表达的人血小板GPVI分子胞外区片段能有效地诱发动物产生多克隆抗体,所得抗体与人血小板上GPVI分子特异性结合,为深入研究GPVI分子提供了工具。Objective To express and purify recombinant human platelet glycoprotein Ⅵ extracellular domain and prepare the polyclonal antibody against it. Methods Human platelet glycoprotein Ⅵ extracellular domain fragment （123 - 268 residues ） was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-3x. The recombinant plasmid was constructed and expressed in E. coli after induction with isopropyl-β-D-thiogalactopyranoside （IPTG）. The fusion protein was identified by Western blot analysis after purification by affinity chromatography. Rabbits were immunized with the purified fusion protein, and the collected rabbit antiserum was evaluated by sandwich ELISA, Western blot and flow cytometry. Results The coding sequence of GPVI extracellular domain was successfully inserted into pGEX-3x. Sequencing result showed that the cloned gene was identical as reported. After induction, a Mr 42kd fusion protein was expressed and confirmed by Western blot, which was identical to that expected. The titers of the antisera were up to 1 ： 128. Sandwich ELISA result demonstrated that the prepared antibody recognized GPVI in human platelet. Western blot and flow cytometry revealed that the prepared antibody reacted with GPVI of platelet lysate and the native GPVI on human platelet surface. Conclusions Using purified prokaryotic expressed the fusion protein as antigen, the specific antibody was elicited in the immunized animals. The prepared polyclonal antibodies react specially with GPVI on human platelet surface and can be used for further studies of GPVI.

关 键 词：血小板 糖蛋白Ⅵ 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]