【摘要】 用制备并纯化的鸡p15INK4B蛋白免疫BALB/c小鼠,制备了p15INK4B蛋白的单克隆抗体,并进行了鉴定。结果表明:2株分泌单克隆抗体的杂交瘤细胞株1C6和3F5分别属于IgG1和IgM抗体亚类;培养上清的效价分别为1∶820和1∶680,腹水的效价分别为1∶4×106和1∶7×105;2株单克隆抗体亲和力常数分别为3.13×109L/mol(1C6)和1.24×108L/mol(3F5),相对亲和力1C6>3F5,且具有不同的抗原识别位点;Western-blot鉴定表明,1C6和3F5这2株单克隆抗体均能与原核表达纯化的和杆状病毒表达的p15INK4B蛋白结合,而不与其他蛋白结合,说明本研究成功制备了鸡p15INK4B的单克隆抗体,可作为深入研究鸡p15INK4B蛋白的功能提供特异的抗体材料。更多还原

【Abstract】 Preparation of monoclonal antibody(McAb) against chicken p15INK4B(p15) protein was carried out for the function research of p15.The purified p15 protein as antigen immunized BALB/c mice.Two hybridoma,1C6 and 3F5,stably secreting McAb were produced by fusion of SP2/0 myeloma cells and spleen cells of syngeneic mice which had been previously immunized by p15 protein.Their titers in cell culture supernatant were 1∶820 and 1∶680 respectively,titers in ascites were 1∶4×106 and 1∶7×105 respectively,and affinity constants were 3.13×109 and 1.24×108 L/mol respectively.The monoclonal antibodies were of high affinity and different antigen binding epitope,and they could specifically bind to p15 protein purified from prokaryotic expressing product and expressed in BAC-TO-BAC Baculovirus Expression Systems but not other cell proteins.All of these show that the McAb against p15 was successfully prepared.更多还原