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目的 :鉴定 3株抗人TNF单克隆抗体 (mAb)D2、E6和F6对TNF诱导的NF κB核转位的抑制作用。方法 :将不同浓度的 3株mAb及无关mAb分别与TNF溶液孵育后 ,加入到ECV30 4细胞培养液中。孵育 1h后收获细胞 ,从中提取核蛋白并测定其含量 ,用电泳迁移率变动分析 (EMSA)检测核提取液中NF κB的核转位。结果 :3株抗TNFmAb均可抑制TNF诱导的ECV30 4细胞中NF κB的核转位。其中mAbD2的抑制作用最强 ,其次为mAbF6和E6 ,无关对照抗体也有一定的非特异性反应。抑制作用与加入的mAb呈良好的剂量依赖关系 ,在 10mg/L和 0 .1mg/L条件下 ,mAbD2的抑制率分别为 94 .2 %和75 .1% ,mAbE6分别为 6 4 .9%和 2 8.6 % ,mAbF6分别为 70 .3%和 4 9.5 % ,无关对照抗体分别为 2 0 .0 %和 11.1%。结论 :mAbD2、E6和F6可特异性地抑制TNF诱导的NF κB核转位 ,为制备对感染性疾病和自身免疫性疾病等均具有治疗作用的嵌合抗体奠定了基础
OBJECTIVE: To determine the inhibitory effects of D2, E6 and F6, three anti-human TNF monoclonal antibodies (mAbs) on NF-κB nuclear translocation induced by TNF. Methods: Three different concentrations of mAb and irrelevant mAb were incubated with TNF solution and then added to ECV30 4 cell culture medium. The cells were harvested after incubation for 1h. The nucleoprotein was extracted and assayed. The nuclear translocation of NFκB in nuclear extracts was detected by electrophoretic mobility shift assay (EMSA). Results: All three anti-TNF mAbs could inhibit NF-κB nuclear translocation induced by TNF in ECV30 4 cells. Among them mAbD2 had the strongest inhibitory effect, followed by mAbF6 and E6, and unrelated control antibody had some non-specific reaction. The inhibitory effect was in a dose-dependent manner with the addition of mAb. At 10 mg / L and 0.1 mg / L, the inhibitory rates of mAbD2 were 94.2% and 75.1%, respectively. The mAb E6 were respectively 6.49% And 2 8.6%, mAbF6 respectively 70.3% and 4 9.5%, unrelated control antibodies were 20.0% and 11.1%. CONCLUSION: mAbD2, E6 and F6 can specifically inhibit TNF-induced nuclear translocation of NF-κB, which lays the foundation for the preparation of chimeric antibodies that have therapeutic effects on infectious diseases and autoimmune diseases