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目的:制备抗果蝇ECP蛋白的单克隆抗体(mAb)并进行特性鉴定。方法:用大肠杆菌表达并纯化的GST-ECP融合蛋白,免疫6~8wk的雌性BALB/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合,经间接ELISA筛选和克隆化培养制备杂交瘤细胞系。用间接ELISA及Western blot等方法对mAb的Ig亚类(型)、腹水效价及特异性进行鉴定。结果:获得1株杂交瘤细胞株,命名为8G9。Ig亚类(型)鉴定表明,此株mAb为IgG1(κ型)。腹水mAb的效价为1∶1×105。Western blot分析表明,该株mAb可与果蝇的幼虫、蛹及成虫组织中表达的ECP特异性结合,可特异性的识别ECP抗原的231~300aa区段。结论:获得1株能稳定分泌抗ECP mAb的细胞株,为进一步研究ECP的功能奠定了基础。
OBJECTIVE: To prepare and characterize monoclonal antibodies against Drosophila ECP protein (mAb). METHODS: Female BALB / c mice were immunized with GST-ECP fusion protein expressed and purified by E. coli. The spleen cells of 6-8 wk were immunized with Sp2 / 0 myeloma cells, and then hybridized by ELISA and cloning Tumor cell line. Indirect ELISA and Western blot were used to identify the Ig subclass (mAb), ascites titer and specificity. Results: One hybridoma cell line was obtained and named as 8G9. Ig subclass (type) identification showed that this strain mAb is IgG1 (κ type). Ascites mAb titer 1: 1 × 105. Western blot analysis showed that this mAb specifically binds to ECP expressed in larval, pupal and adult tissues of Drosophila and specifically recognizes the 231-300aa segment of ECP antigen. Conclusion: Obtaining a cell strain that can stably secrete anti-ECP mAb lays the foundation for further study on the function of ECP.