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目的制备特异性的鼠源单克隆抗DNAH2(Homo sapiens dynein,axonemal,heavy chain 2)抗体。方法首先针对人DNAH2蛋白N端1~300 aa序列,构建可表达His标签的免疫原融合表达质粒,在大肠杆菌内以IPTG诱导His-免疫原的表达并纯化;然后免疫BALB/c小鼠,分离出脾淋巴细胞,并与骨髓瘤细胞融合,形成杂交瘤细胞;最后以酶联免疫吸附测定(ELISA)、蛋白免疫印迹法(Western blot)筛选阳性杂交瘤细胞。结果利用IPTG有效诱导了大肠杆菌内DNAH2免疫原的表达;筛选的阳性杂交瘤细胞检测出DNAH2蛋白条带。结论成功制备针对人DNAH2蛋白的鼠源单克隆抗体。
Objective To prepare a specific murine monoclonal antibody against DNAH2 (axonemal, heavy chain 2). Methods Firstly, a recombinant plasmid expressing His-tag was constructed based on the sequence of 1 to 300 aa from the N-terminal end of human DNAH2 protein. His-immunogen was induced by IPTG in Escherichia coli and purified. Then BALB / c mice were immunized, Splenic lymphocytes were isolated and fused with myeloma cells to form hybridoma cells. Finally, positive hybridoma cells were screened by enzyme-linked immunosorbent assay (ELISA) and Western blot. Results IPTG induced the expression of DNAH2 in Escherichia coli. The positive hybridoma cells detected DNAH2 protein bands. Conclusion Murine monoclonal antibody against human DNAH2 protein was successfully prepared.