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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 investigate the expression of extracellular domain of human platelet glycoprotein Ⅵ (GPVI) in prokaryotic cells and to prepare its polyclonal antibody. Methods The gene sequence of 123aa ~ 268aa in the extracellular domain of GPVI of human platelet was amplified by PCR and the prokaryotic expression vector was constructed. The fusion protein was induced in E. coli. After purification and identification of the expression product by affinity chromatography, the rabbit was immunized to prepare the polyclonal antibody. The specificity of the monoclonal antibody was detected by ELISA double-antibody sandwich ELISA, Western blot and flow cytometry. Results The constructed recombinant plasmid was verified by restriction enzyme digestion and sequencing. After induction of expression at a relative molecular weight of 4200 has a significant fusion protein band, Westernblot analysis confirmed with the expected value. The titer of antiserum obtained was 1:128. ELISA double antibody sandwich ELISA showed that the antibody produced platelet GPVI molecule. The results of Western blot and flow cytometry showed that the antibodies could specifically react with platelet lysate and GPVI on platelet membrane. Conclusion The use of prokaryotic expression of human platelet GPVI molecule extracellular region fragments can effectively induce the animals to produce polyclonal antibodies, the resulting antibody and human platelet GPVI molecules specific binding, providing a tool for in-depth study of GPVI molecules.