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

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

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。

Objective：To prepare the monoclonal antibodies against human F1-FO ATP synthase beta subunit （hATPSB） and to study their specificities as well as anti-tumor activity. Methods： BALB/c mice were immunized with hATPSB 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 sepharose CL-4B and was identified by Western blot and cell immunofluoreseence analysis. Cell surface ATP synthesis determination and cytotoxicity to MCF-7 as well as MCF-7/Adr cells in combination with doxorubicin were used to analyze the anti-tumor activity. Results：A hybridoma cell Mab3B8 stably secreting anti- hATP5B monoclonal antibody was obtained. The purity of the McAb was higher than 95 %. Mab3B8 was showed high specificity to the antigen of MCF-7 as well as MCF-7/Adr by Western blot and immunofluorescenee. Mab3B8 was shown obvious inhibition of cell surface ATP generation on MCF-7 and MCF-7/Adr. The combination of Mab3B8 and doxorubicin exhibited synergistic effect on MCF-7 and reversal on the resistance of MCF-7/Adr to ADM. Conclusion： A hybridoma cell hne which can secret a novel functional mouse anti-human F1-F0 ATP synthase beta subunit has been developed successfully. The mAb can effectively inhibit cell surface ATP generation and exhibited synergistic effect on cancer cell lines. It may be of significant value to cancer therapy, especially to overcoming drug resistance.