抗人F1-F0 ATP合成酶beta亚基单抗的制备及其抗肿瘤活性研究

目的：制备鼠抗人F1-F0ATP合成酶beta亚基（hATP5B）单抗,并对其特异性和抗肿瘤活性进行研究。方法：以原核表达人hATP5B免疫BALB/c小鼠,通过杂交瘤技术,筛选分泌抗hATP5B单抗的杂交瘤细胞株。采用Protein A亲和层析法纯化抗体腹水,SDS-PAGE检测纯化产物纯度。利用Western blot、细胞免疫荧光对其特异性进行鉴定,并通过抑制乳腺癌细胞MCF-7及其耐药株MCF-7/Adr表面ATP生成,细胞毒性实验进行抗肿瘤活性研究。结果：获得一株稳定表达抗hATP5B单抗的杂交瘤细胞株Mab3B8,Western blot和细胞免疫荧光结果表明,该抗体与乳腺癌MCF-7及其耐药细胞MCF-7/Adr细胞天然抗原结合；可明显抑制MCF-7及其耐药株MCF-7/Adr细胞膜表面ATP合成；与化疗药物多柔比星（曾用名：阿霉素）联合作用,可降低化疗药物多柔比星对肿瘤细胞的IC50。结论：成功建立了一株可特异性识别天然hATP5B的单抗,有阻断肿瘤细胞膜表面ATP合成活性并可明显增强MCF-7及其耐药株MCF-7/Adr细胞对多柔比星敏感性的作用。此项研究对膜表达F1-F0ATP合成酶功能探讨及肿瘤治疗研究都具有重要意义。

Preparation and anti-tumor activity of mouse monoclonal antibody against human F1-F0 ATP synthase beta subunit

Objective:To prepare the monoclonal antibodies against human F1-F0 ATP synthase beta subunit (hATP5B) and to study their specificities as well as anti-tumor activity.Methods:BALB/c mice were immunized with hATP5B expressed by prokaryotic system.Spleen lymphocytes were fused with SP2/0 myeloma cells.Hybridoma cells secreting monoclonal antibodies (McAb) were produced by three-time of limited dilution method.The monoclonal antibody in ascites was purified by using affinity chromatography with protein A sephar...