IL-4Rα胞外段的真核表达及鼠源单克隆抗体的制备与鉴定  

Eukaryotic expression of IL-4Rα extracellular domain and preparation and identification of murine monoclonal antibody

作　　者：陈莹 詹珊珊 吴小丽 宋刚 桂芳 潘勇兵 CHEN Ying;ZHAN Shan-shan;WU Xiao-li;SONG Gang;GUI Fang;PAN Yong-bing(Antibody Laboratory of Wuhan Institute of Biological Products Co.,Ltd.,National Engineering Technology Research Center for Combined Vaccines,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司抗体研究室,国家联合疫苗工程技术研究中心,湖北武汉430207

出　　处：《微生物学免疫学进展》2021年第2期28-34,共7页Progress In Microbiology and Immunology

摘　　要：目的表达IL-4Rα胞外段蛋白(IL-4 receptor α extracellular domain, IL-4Rα ECD),并制备对IL-4及IL-13与IL-4R结合具有阻断能力的抗IL-4Rα鼠源单克隆抗体(单抗)。方法构建含IL-4Rα ECD DNA序列的真核表达质粒,转染Expi293F细胞,对上清中的IL-4Rα ECD进行纯化。采用SDS-PAGE及Western blot对该蛋白进行纯度分析及鉴别;将其免疫小鼠后,筛选相应的鼠源单抗;分别采用SDS-PAGE、Western blot、ELISA、流式细胞法及细胞抑制法对单抗的纯度、特异性、结合活性、阻断活性进行鉴定。结果酶切及测序结果表明,IL-4Rα ECD重组表达质粒构建正确;蛋白成功表达,相对分子质量为43 000~48 000,纯度在95%以上;免疫后小鼠血清效价在107以上;制备的1株鼠源单抗6-G2与IL-4Rα的ELISA半最大效应质量浓度(concentration for 50%of maximal effect, EC50)为10.4 ng/mL;竞争ELISA检测结果显示,6-G2单抗对IL-4与IL-4Rα结合的最大抑制率为27.3%;流式细胞法检测结果显示,6-G2单抗能阻断IL-13与相应受体的结合;细胞增殖抑制法检测结果显示,6-G2单抗几乎能完全抑制细胞的增殖。结论成功表达并纯化了IL-4Rα ECD,制备的抗IL-4Rα鼠源单抗6-G2单抗能同时阻断IL-4及IL-13与IL-4R的结合,为后续开发针对抗IL-4Rα的治疗性抗体药物奠定了基础。Objective To express IL-4 Rα extracellular domain(IL-4 Rα ECD) and to prepare an anti-IL-4 Rα mouse monoclonal antibody with the ability to block IL-4 and IL-13 binding to IL-4 R. Methods A eukaryotic expression plasmid containing IL-4 Rα ECD DNA sequence of Expi293 F cells was constructed. The purity of the recombinant protein was analyzed and identified by SDS-PAGE and Western blot. Mice were immunized with the recombinant protein, and the corresponding mouse monoclonal antibodies were screened. The purity, specificity, binding activity and blocking activity of the monoclonal antibody were determined by SDS-PAGE, Western blot, ELISA, flow cytometry and cell inhibition. Results The recombinant expression plasmid of IL-4 Rα was constructed correctly which proved by the enzyme digestion and sequencing. The protein was successfully expressed with a relative molecular weight of 43 000-48 000 and a purity of more than 95%. The serum titer of immunized mice was above 107. The ELISA result showed that the concentration for 50% of maximal effect of the mouse monoclonal antibody 6-G2 to IL-4 Rα was 10.4 ng/mL. The result of competitive ELISA showed that the maximum inhibition rate of 6-G2 monoclonal antibody on the binding of IL-4 and IL-4 Rα was 27.3%. Flow cytometry experiment showed that 6-G2 monoclonal antibody could block the binding of IL-13 to the corresponding receptor. Cell proliferation assay showed that 6-G2 monoclonal antibody could almost completely inhibit cell proliferation. Conclusion The IL-4 Rα protein was successfully expressed and purified. The 6-G2 monoclonal antibody against IL-4 Rα could simultaneously block the binding of IL-4 and IL-13 to IL-4 R, which laying a foundation for the development of therapeutic antibody drugs against IL-4 Rα in the future.

关 键 词：IL-4Rα胞外段蛋白 真核表达 单克隆抗体 结合活性 阻断活性 

分 类 号：Q782[生物学—分子生物学]