卵巢癌相关抗原CA125单克隆抗体的制备  

Preparation of monoclonal antibodies against ovarian cancer-associated antigen CA125

作　　者：梁勤东[1] 马婷婷[1] 董晋豫[1] 李武县[1] 汪先桃[1] 李紫微[1] 王秦[1] 熊海玉[1] 涂植光[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2013年第10期1458-1462,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81172016)

摘　　要：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 and identify the monoclonal antibodies （McAbs） against ovarian cancer-associated antigen CA125. Methods The gene encoding CA125 tandem repeats （CA125R） was cloned into prokaryotic expression vector pGEX-6P-1, and the constructed recombinant plasmid pGEX-CA125R was transformed to competent E. colt BL21 （DE3） for expression under induction of IFFG. The expressed GST-CA125R fusion protein was purified by GST affinity chromatography and immunized to BALB/c mice, based on which the McAb against CA125 by hybridoma technique, then identified for specificity by Western blot, and for subclass by monoclonal antibody subclass determination kit. The McAbs were prepared in a large quantity by induction in ascites, and analyzed for purity by SDS-PAGE, and for titer by indirect ELISA. Results Restriction analysis and sequencing proved that recombinant plasmid pGEX-CA125R was constructed correctly. The purified GST-CA125R fusion protein, with a relative molecular mass of about 44 000, reached a purity of about 95% and showed specific reaction with mouse anti-GST McAb. A hybridoma cell strain 3-B2 stably secreting McAb against CA125 was obtained, by which the secreted McAb, of subclass IgG2a, recognized GST-CA125R fusion protein and natural CA125 protein, reached a purity of about 90% after purification and a titer of more than I x 106. Conclusion A hybridoma cell strain secreting McAb recognizing natural CA125 protein was obtained, and a large quantity of McAbs were prepared by induction in ascites, which laid a foundation of development of clinical detection kit for CA125.

关 键 词：卵巢癌 肿瘤相关抗原 CA125 单克隆抗体 

分 类 号：R737.31[医药卫生—肿瘤] R392-33[医药卫生—临床医学]