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目的制备卵巢癌相关抗原CA125R单克隆抗体,并进行鉴定。方法将CA125一段串联重复序列(CA125R)基因克隆至原核表达载体pGEX-6P-1,构建重组表达质粒pGEX-CA125R,转化大肠埃希菌(E.coli)BL21(DE3),IPTG诱导表达GST-CA125R融合蛋白。表达的融合蛋白经GST亲和层析柱纯化后,免疫BALB/c小鼠,采用杂交瘤技术制备CA125单克隆抗体,Western blot法鉴定抗体的特异性,单克隆抗体亚类测定试剂盒分析单抗的亚类。腹水诱生法大量制备单抗,SDS-PAGE分析单抗的纯度,间接ELISA法检测单抗的效价。结果重组原核表达质粒pGEX-CA125R经双酶切及测序,证实构建正确;纯化的GST-CA125R融合蛋白相对分子质量约44 000,纯度约为95%,可与小鼠抗GST单克隆抗体发生特异性反应;获得1株可稳定分泌抗CA125单克隆抗体的杂交瘤细胞株3-B2,其分泌的单抗能特异识别GST-CA125R融合蛋白和天然CA125糖蛋白,其抗体亚型为IgG2a型;纯化的腹水单抗纯度约为90%,效价达1×106以上。结论成功获得1株能特异性识别天然CA125糖蛋白的单克隆抗体细胞株,并经腹水诱生法大量制备了抗体,为CA125临床检测试剂盒的研发奠定了基础。
Objective To prepare ovarian cancer-associated antigen CA125R monoclonal antibody and identify it. Methods A CA125R gene of CA125 was cloned into the prokaryotic expression vector pGEX-6P-1. The recombinant plasmid pGEX-CA125R was constructed and transformed into E. coli BL21 (DE3). The expression of GST- CA125R fusion protein. The expressed fusion protein was purified by GST affinity chromatography and then immunized BALB / c mice. The monoclonal antibody against CA125 was prepared by hybridoma technique. The specificity of the antibody was identified by Western blot. The monoclonal antibody subtype assay kit analysis single Anti-subclass. Ascites induction method for the preparation of monoclonal antibody, SDS-PAGE analysis of the purity of monoclonal antibody, indirect ELISA method for the detection of monoclonal antibody titer. Results The recombinant plasmid pGEX-CA125R was confirmed by double enzyme digestion and sequencing. The purified GST-CA125R fusion protein had a relative molecular mass of about 44,000 with a purity of about 95%, which was specific to mouse anti-GST monoclonal antibody The monoclonal antibody secreted by the monoclonal antibody can specifically recognize the GST-CA125R fusion protein and the native CA125 glycoprotein, and the antibody subtype is IgG2a. Purified ascites monoclonal antibody purity of about 90%, the titer of more than 1 × 106. Conclusion A monoclonal antibody cell line that can specifically recognize natural CA125 glycoprotein was successfully obtained. The antibody was prepared in large quantities by ascitic fluid induction, which laid the foundation for the development of CA125 clinical test kit.