抗人MIF单克隆抗体的制备及其活性的初步鉴定

Preparation and characterization of monoclonal antibodies against hMIF

目的构建人巨噬细胞移动抑制因子(hum an m acrophage m igration inh ib itory factor,hM IF)原核表达系统并制备其单克隆抗体。方法采用RT-PCR从乳腺癌细胞株MDA-MB453细胞克隆hM IF cDNA,测序后构建pET11b/hM IF重组表达质粒,转化入大肠杆菌BL21(DE3),诱导表达hM IF;重组hM IF经纯化和鉴定后,作为抗原常规免疫和CpG ODN加强免疫BALB/c小鼠,制备单克隆抗体并鉴定活性。结果构建pET11b/hM IF重组表达质粒,表达出预期hM IF蛋白,表达率30%,纯化后纯度达95%。W estern b lotting鉴定为hM IF,NO释放实验证实具有生物学活性。两种免疫方案共获得9株分泌抗hM IF抗体的杂交瘤细胞株,其中一株经W estern b lotting和活性鉴定证明具有特异性和生物学活性。结论获得hM IF重组蛋白和抗hM IF单克隆抗体,为其基础性研究和在脓毒症的治疗等临床应用研究奠定基础。

Objective To construct a prokaryotie expression system of human macrophage migration inhibitory factor （hMIF） and prepare its monoclonal antibodies （MAb）. Methods After hMIF eDNA was amplified from human breast cancer cell line MDA-MB453 and cloned into T vector for DNA sequencing, the confirmed hMIF eDNA was inserted into expression vector pETI lb. The recombinant was transformed into BL21 （DE3） and hMIF expression was induced with IPTG. The obtained hMIF protein was verified with Western blotting, and its biological activity was measured by NO release in cultured RAW264.7 cells. CpG ODN was introduced as the adjuvant for immunizing BALB/c mice. The generated MAbs were determined with Western blotting and in vitro NO releasing experiment. Results hMIF was obtained and purified to 95% and showed certain biological activity. CpG ODN was proved to enhance the immunization. Among the 9 stains of hybridoma obtained, 2D10 was identified with MIF antigen specificity and the ability to decrease hMIF induced NO release in the cultured RAW264.7 cells. Conclusion The obtained hMIF protein and its MAbs found a basis for basic study of this cytokine and potential clinical applications of the antibodies.