EpCAM蛋白的表达、纯化及其单克隆抗体的制备

目的:原核表达EpCAM蛋白并制备抗EpCAM特异性单克隆抗体,初步鉴定相应单克隆抗体的特性。方法:PCR扩增EpCAM基因胞外区,将目的基因亚克隆至载体pET-28a(+),转化至大肠埃希菌株BL21,IPTG诱导表达,组氨酸亲和层析法纯化表达产物。纯化蛋白免疫BALB/c小鼠,将成功免疫的小鼠脾细胞与骨髓瘤SP2/0细胞融合,经ELISA筛选得到分泌特异性抗EpCAM的单克隆抗体的细胞株,免疫BALB/c小鼠进一步制备相应的单克隆抗体,并通过Western blot(蛋白质印记)和FACS(流式细胞分析)鉴定单抗的特异性及生物学活性。结果:成功构建重组表达载体pET28a-EpCAM并在大肠杆菌中获得表达,经His-tag亲和层析法获得纯化的EpCAM重组蛋白。EpCAM重组蛋白免疫的BALB/c小鼠的脾细胞与SP2/0细胞融合、筛选,获得两株稳定分泌EpCAM抗体的杂交瘤细胞株,分别命名为4B2、2F2并免疫BALB/c小鼠获得相应的单克隆抗体。Western blot结果显示4B2腹水纯化所得单抗能够识别FaDu细胞系(人咽鳞癌细胞)中的EpCAM蛋白,但2F2未能识别FaDu细胞中的变性的EpCAM蛋白。FACS结果显示两者均能和FaDu细胞中天然的EpCAM蛋白结合。讨论:成功制备了抗EpCAM的单克隆抗体,并能够识别人咽鳞癌细胞系FaDu中表达的EpCAM,为进一步研究EpCAM抗体在肿瘤治疗中的作用提供基础。

Expression and Purification of EpCAM Protein and Preparation of the Monoclonal Antibodies

Objective: Prokaryotic expression,purification of the EpCAM protein,preparation and characterization of the monoclonal antibodies against the protein.Methods:The gene was cloned into pET-28a(+) plasmid.The recombinant plasmid EpCAM/PET28a was transformed into E.coli BL21 and induced with IPTG.The recombinant protein was purified with Ni-NTA resin.The monoclonal antibodies were prepared by fusion of the purified EpCAM protein immunized BALB/c mouse’s spleen cells with SP2/0 myeloma cells,positive cells were screened with indirect ELISA and the positive cells were used to immune BALB/c mice to obtain the monoclonal antibodies against EpCAM protein.The binding specificity of the purified monoclonal antibodies were analysised by Western blot and FACS.Results: The recombinant plasmid EpCAM/PET28a and purified protein were obtained,two hybridoma cell lines secreting anti-EpCAM IgG McAbs were established and named as 4B2,2F2.FACS analysis showed that the two McAbs showed high specificity to FaDu cell line,while Western blotting analysis showed that only 4B2 can recognize the denatured EpCAM protein in FaDu cell line.Conclusions:The anti-EpCAM IgG McAbs were prepared which had high specificity to FaDu cell line,and it could be used to develop the anti-EpCAM antibody in cancer therapy.