抗人生长转化因子15单克隆抗体制备及鉴定

Preparation and identification of monoclonal antibody against human growth differentiation factor 15

目的:制备抗人生长转化因子15(GDF15)单克隆抗体(mAb)并鉴定其特性。方法:构建GDF15原核表达载体pGEX-4T-2-gdf15,诱导表达并纯化GST-GDF15融合蛋白。以该融合蛋白免疫BALB/c小鼠,取小鼠的脾细胞与同系小鼠Sp2/0骨髓瘤细胞常规融合,依次进行阳性杂交瘤细胞株的筛选及亚克隆,最终获得稳定分泌抗GDF15 mAb杂交瘤细胞株。以间接ELISA法检测抗体效价;Western blot鉴定抗体特异性;免疫共沉淀法初步检测在肝癌患者血清中GDF15表达水平。结果:获得1株稳定分泌抗人GDF15 mAb杂交瘤细胞株,命名为9G3;抗体免疫球蛋白亚类为IgG1,轻链为κ型;免疫共沉淀及质谱分析证实抗GDF15 mAb9G3能与肝癌患者血清中GDF15蛋白特异性结合并且GDF15水平在肝癌患者血清中较正常人显著升高。结论:成功制备了抗人GDF15mAb,为后期肝癌早期诊断试剂盒的开发奠定了良好的基础。

AIM： To prepare the monoclonal antibody（mAb） against human growth differentiation factor 15（GDF15）.METHODS： The expression vector pGEX-4T-2-gdf15 was constructed and transformed into E.coli Top10F＇ for expression.Then the purified fusion protein was used to immunize the BALB/c mice.The mouse myeloma cells（Sp2/0） were fused with spleen cells from the BALB/c immunized by the purified protein.Subsequently,limited dilution method was used three times to screen hybridoma cell lines.The titer of the mAb was detected by ELISA and its specificity was analyzed by Western blot.The serum level of GDF15 in hepatocellular carcinoma（HCC） and health people was measured by co-immunoprecipitation（IP）method.RESULTS： The GST-GDF15 fusion protein was successfully expressed and purified.One hybridoma cell line designated 9G3 against GDF15 was obtained.IP and mass spectrometric analysis revealed that the mAb recognized GDF15 in human sera with high specificity.The level of GDF15 in HCC patients was much higher than that in health people.CONCLUSION： The success in mouse anti-GDF15 mAb preparation provides the basis for further developing HCC diagnose kit.