大鼠抗人表皮生长因子受体3(ErbB3)二聚化界面区多克隆抗体的制备与鉴定

Preparation and identification of rat anti-human ErbB3 dimerization domain polyclonal antibody

目的制备由人表皮生长因子受体3(ErbB3)二聚化界面区第236至308位氨基酸肽段(ErbB3Ⅱ)与麻疹病毒蛋白第288至302位氨基酸肽段(MVF)连接而成的融合蛋白MVF-ErbB3Ⅱ,并制备和表征其大鼠多克隆抗体.方法化学合成法合成MVF-ErbB3Ⅱ的基因,利用DNA连接酶与pET-21b载体连接成重组质粒,转化E.coli BL21(DE3)进行MVF-ErbB3Ⅱ原核表达,MVF-ErbB3Ⅱ经镍离子亲和层析法纯化后,免疫大鼠制备多克隆抗体,采用ELISA检测抗体滴度,Western blot法、免疫共沉淀法(IP)和流式细胞术鉴定抗体特异性和靶向性.结果SDS-PAGE证实大肠杆菌成功表达了MVF-ErbB3Ⅱ蛋白,ELISA结果表明多克隆抗体具有较高的抗体效价,Western blot法、IP和流式细胞术结果表明多克隆抗体具有良好的MVF-ErbB3Ⅱ及非变性ErbB3特异性和二聚化界面靶向性.结论成功进行了MVF-ErbB3Ⅱ原核表达和分离纯化及大鼠抗MVF-ErbB3Ⅱ多克隆抗体的制备和表征.

Objective To prepare the fusion protein MVF-ErbB3Ⅱ composed of measles virus fusion(MVF)protein 288 to 302 amino acid peptide and human epidermal growth factor receptor 3(ErbB3)236 to 308 amino acid(ErbB3Ⅱ)peptide,then prepare and characterize the anti-MVF-ErbB3Ⅱ polyclonal antibody(pcAb).Methods The MVF-ErbB3Ⅱ gene was synthesized artificially and subcloned into pET-21b plasmid using DNA ligase.After transformation,the recombinant MVF-ErbB3Ⅱ protein was expressed in E.coli BL21(DE3)and purified using nickel ion affinity chromatography.Subsequently,the purified MVF-ErbB3Ⅱ protein was used as antigen to immunize rats subcutaneously for induction of anti-MVF-ErbB3Ⅱ pcAb.The titer of anti-MVF-ErbB3Ⅱ pcAb was analyzed by ELISA.The ErbB3 specificity and targeting ability of pcAb were evaluated by Western blotting,immunoprecipitation(IP)and flow cytometry(FCM).Results SDS-PAGE confirmed that MVF-ErbB3Ⅱ protein was successfully expressed and purified.ELISA showed that the titer of pcAb was 1024000.Western blotting,IP and FCM assays showed that the anti-MVF-ErbB3Ⅱ pcAb not only had good antigen specificity against purified MVF-ErbB3Ⅱ and native ErbB3 but targeted the ErbB3 dimerization interface.Conclusion The prokaryotic expression and purification of MVF-ErbB3Ⅱ is successfully achieved,rat anti-MVF-ErbB3ⅡpcAb is prepared and characterized successfully.