目的：制备和鉴定人卵巢癌cDNA文库筛选到的肿瘤相关抗原OVA66的单克隆抗体，为研究其生物学功能提供手段。方法：采用基因重组方法构建pET32bOVA66重组表达质粒，经大肠杆菌诱导表达OVA66融合蛋白，利用NiTED亲和层析技术分离纯化HisOVA66融合蛋白；采用经典的杂交瘤技术制备OVA66单克隆抗体；ELISA和Western blotting鉴定单克隆抗体的生物学和免疫学特性，并对OVA66单克隆抗体在细胞免疫荧光法、流式细胞术和免疫组化等方法中进行了初步应用。结果：成功构建重组表达载体pET32bOVA66，并诱导表达和纯化OVA66融合蛋白。制备获得两株小鼠OVA66单克隆抗体杂交廇细胞株5F4和4G9。两株细胞分泌的抗体亚类均为IgG1，轻链为κ型，亲和力常数Ka分别为2.96×1010和0.4×1010L/mol，初步表位分析结果显示两者针对不同的抗原表位；两株抗体均可以采用细胞免疫荧光和流式细胞术检测OVA66的表达，5F4还可通过免疫组织化学检测肿瘤组织中OVA66的表达。结论：成功制备两株特异性的小鼠OVA66单克隆抗体杂交瘤细胞株，为进一步研究OVA66的生物学特性和临床应用奠定了基础。

Objective:To prepare and characterize monoclonal antibody against tumorassociated antigen OVA66, so as to provide effective method for further studying the biological function of OVA66. Methods: Recombinant pET32bOVA66 plasmid was constructed and transfected into E.coli BL21 (DE3). HisOVA66 fusion protein was induced by IPTG and purified through NiTED affinity chromatography. Hybridoma cells stably secreting antiOVA66 monoclonal antibodies were prepared by hybridoma technology. Biological and immunologic properties of monoclonal antibodies were determined by ELISA and Western blotting. The prepared antibody was applied in immunofluorescence, flow cytometry and immunohistochemistry procedures. Results: Recombinant pET32bOVA66 plasmid was successfully constructed, and OVA66 fusion protein was induced and further purified. Two hybridoma cell strains secreting OVA66 monoclonal antibodies were established by hybridoma technology and named as 5F4 and 4G9, respectively. These monoclonal antibodies were characterized as IgG1 subclass and kappa subtype with affinity constant ka being 2.96×1010 and 0.4×1010L/mol, respectively. Preliminary data showed that these antibodies acted against different antigenic epitopes. These antibodies could be used for the detection of OVA66 expression in immunofluorescence and flow cytometry assay, whereas 5F4 could also be used in immunohistochemistry assay. Conclusion: Two hybridoma cell strains secreting monoclonal antibodies against OVA66 have been successfully established, which facilitates further studies on biological functions of OVA66 and its potential in clinical application.

上海市教委科研创新项目（No. 08YZ39）；上海市免疫学研究所项目（No. 08A03）