全人源抗Trop-2 IgG的制备及对卵巢癌细胞生物特性的影响

Preparation of a full human anti-Trop-2 IgG and its effect on ovarian cancer cells

目的 :构建全人源抗人滋养层细胞表面抗原2(human trophoblast cell surface antigen 2,Trop-2)抗体Ig G真核表达系统,表达并纯化该抗体,并检测其对卵巢癌细胞生物特性的影响。方法:构建全人源抗Trop-2抗体Ig G的重组表达载体;在293Free Style(293F)真核表达体系中表达并纯化该抗体。使用SDS-PAGE、ELISA、亲和力测定、免疫荧光等方法鉴定该抗体并分析其免疫学活性;使用cell counting kit-8(CCK-8)法、划痕实验、Transwell实验和细胞凋亡实验分析其对卵巢癌细胞特性的影响。结果:成功制备出全人源抗Trop-2 Ig G;经多种方法检测,该抗体能与Trop-2蛋白特异性结合;对表达Trop-2的卵巢癌细胞增殖、侵袭、迁移和凋亡有明显影响。当抗体浓度为400μg/m L时,卵巢癌细胞HO8910的生存率仅为55.95%(P<0.01),划痕愈合率仅为24.71%(对照组为70.80%,P<0.01),侵袭细胞数是对照组的32.11%(P<0.05),细胞凋亡率是对照组的2倍(P<0.05)。结论 :全人源抗Trop-2 Ig G可特异性识别卵巢癌细胞表面的Trop-2蛋白,对卵巢癌细胞的恶性行为具有明显的抑制作用。因此,全人源抗Trop-2 Ig G在卵巢癌的靶向治疗中有潜在应用价值。

Objective：We sought to construct a full human anti-Trop-2 Ig G expression vector,and to express and purify it,then analyze the effect of anti-Trop-2 Ig G in human ovarian cancer cells. Methods：The recombinant expression vector of full human antiTrop-2 Ig G was constructed and the Ig G was expressed in 293 Free Style（293F）cell eukaryotic expression system and then purified using AKTA system. The immune activity of the Ig G was verified and analyzed by SDS-PAGE,enzyme-linked immunosorbent assay（ELISA）,affinity assay,Western blot assay,flow cytometry method（FCM）and immunofluorescence assay. The effect on the character of ovarian cancer cells was detected by cell counting kit-8（CCK-8）assay,wound healing assay,transwell test and cell apoptosis assay.Results：The results demonstrated that the full human anti-Trop-2 Ig G was successfully produced. The Ig G specifically bound Trop-2protein,and it could play an important role in the proliferation,migration and invasion of ovarian cancer cells as well as in inducing ovarian cancer cell apoptosis. When the concentration of Ig G was at 400 μg / m L,only 55.95%（P〈0.01）of HO8910 cells survived.The amount of invasive cells in the experimental group was only 32.11% of those in the control group（P〈0.05）. The wound healingrate of the experimental group was 24.71% while that of control group being 70.80%（P〈0.01）,and the apoptosis rate was 2-folds higher than the control group（P〈0.05）. Conclusion：The full human anti-Trop-2 Ig G could specifically bind Trop-2 protein on the surface of ovarian cancer cells and restrain the malignant biological behavior of ovarian cancer cells. The Ig G has potential applications in ovarian cancer targeting therapy.