摘要
目的制备和鉴定抗人CD47分子胞外区蛋白单克隆抗体。方法聚合酶链反应扩增人CD47胞外区基因序列,重组至原核表达载体pET-32a(+)中,利用大肠埃希菌表达系统表达CD47胞外区蛋白。用纯化的CD47胞外区蛋白免疫小鼠,采用常规方法进行细胞融合,经克隆化培养后,筛选能够特异性分泌鼠抗人CD47单克隆抗体的杂交瘤细胞株。采用酶联免疫吸附法、Western blot和流式技术检测抗体效价及特异性。结果成功构建人CD47原核表达载体,在BL21(DE3)中获得表达,经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳、Western blot鉴定后,采用层析柱法获得纯化的CD47胞外区蛋白。以纯化蛋白为免疫原,成功制备具有较强亲和力的单克隆抗体。结论成功制备了针对CD47分子胞外区蛋白的高亲和力单克隆抗体,为进一步研究CD47的生物学功能奠定了基础。
Objective To prepare and identify anti-CD47 monoclonal antibodies.Methods The gene fragment of CD47 was amplified by polymerase chain reaction and cloned into prokaryotic expressing vector pET-32a（＋）.Purified reconstructed protein was used to immunize BALB/c mice.The immunized spleen cells were isolated and fused with Sp2/0cells.After screened,hybridomas secreting anti-CD47 monoclonal antibody were acquired.Biological activities of antibodies were investigated by Western blot and flow cytometry.Results The recombinant CD47 extracellular domain protein was successfully expressed in BL21,and certificated by sodium dodecyl sulfate polyacrylamide gel electropheresis and Western blot.Data of flow cytometry detection demonstrated that the antiserum had high affinity to CD47 protein.Conclusion Recombinant CD47 and its monoclonal antibody,with high affinity,were successfully prepared,which could provide reliable tools for the future study of CD47.
出处
《国际检验医学杂志》
CAS
2015年第21期3125-3127,共3页
International Journal of Laboratory Medicine
