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目的制备抗人颗粒蛋白前体F片段(GrnF)的单克隆抗体(mAb),并初步鉴定其生物学特性。方法通过分子生物学技术构建含人GrnF编码序列的酵母表达载体,表达并纯化酵母来源的融合人血清白蛋白(HSA)的GrnF片段HSA-GrnF。用纯化的HSA-GrnF免疫BALB/c小鼠,然后将免疫小鼠的脾细胞与小鼠骨髓瘤细胞Sp2/0进行融合,用ELISA筛选阳性克隆,并采用有限稀释法进行亚克隆;采用免疫荧光染色及Westernblot法对所获得的mAb的特异性进行鉴定,并用抗体亚类测定试剂盒检测所获得单克隆抗体亚类。结果成功获得2株可分泌特异性抗GrnF的杂交瘤细胞株(1G6及4E8),经鉴定这2株抗体重链亚类均为IgG1亚类。结论成功制备了抗人GrnF片段结构域的单克隆抗体。
Objective To prepare a monoclonal antibody (mAb) against human granulocyte precursor F fragment (GrnF) and to identify its biological characteristics. Methods A yeast expression vector containing human GrnF coding sequence was constructed by molecular biology technique. The GmF fragment HSA-GrnF of yeast-derived fused human serum albumin (HSA) was expressed and purified. BALB / c mice were immunized with purified HSA-GrnF, then spleen cells of immunized mice were fused with mouse myeloma cells Sp2 / 0, positive clones were screened by ELISA and subcloned by limiting dilution method; Fluorescent staining and Western blotting were used to identify the specificity of the obtained mAb. The subclass of monoclonal antibody was detected by using the antibody subclass assay kit. Results Two hybridoma cell lines secreting specific anti-GrnF (1G6 and 4E8) were successfully obtained. The two heavy chain subclasses were identified as IgG1 subclasses. Conclusion Monoclonal antibodies against human GrnF fragment were successfully prepared.