p65结合肽与p65的亲和力检测及其对NF-κB DNA结合活性抑制作用的鉴定

Determination of affinities of oligopeptides binding with p65 and identification of their inhibiting effect on DNA binding activity of NF-κB

目的检测NF-κB p65亚基结合肽与p65间相互作用的亲和常数,以及其对NF-κB DNA结合活性的抑制效应。方法借助于生物传感器技术分析NF-κB p65亚基结合肽与p65相互作用的动力学,同时应用竞争性ELISA鉴定结合多肽对NF-κB DNA结合活性的抑制效应。结果生物传感器技术分析结果表明5条p65结合肽均能与p65发生相互作用,相互作用的亲和常数分别为GST-BPT1:2.67×10-7mol/L、GST-BPT2:9.02×10-6mol/L、GST-BPT3:1.07×10-6mol/L、GST-BPT4:8.03×10-6mol/L、GST-BPT5:9.83×10-7mol/L。竞争性ELISA检测结果表明5条p65结合肽均能在一定程度上抑制NF-κB与其顺式作用元件κB基序的结合,且这种抑制效应随多肽浓度的增加而增强。结论酵母双杂交筛选获得的5条p65结合多肽与p65间确实存在相互作用,且5条结合肽对NF-κB DNA结合活性均有一定的抑制效应,由此提示以这些多肽为先导物设计和开发靶向NF-κB的抗炎多肽药物将成为可能。

Objective To determine the affinities of five oligopeptides specifically binding with DNA binding domain of NF-κB 1365 subunit and identify their inhibiting effect on DNA binding activity of NF-κB. Methods By using biosensor the affinities were measured by means of kinetic analysis, and the inhibiting effects were determined by competitive ELISA. Results The results of biosensor showed that all of five oligopeptides really possessed the capability of specific interacting with NF-κB p65 subunit. The affinity constants of these oligopeptides were 2.67 ×10^-7 mol/L, 9.02 ×10^-6 mol/L, 1.07 ×10^-6 mol/L, 8.03 ×10^-6 mol/L, 9.83 ×10^-7 mol/L respectively. The results of competitive ELISA indicated that five oligopeptides could inhibit NF-κB from binding with KB motif, and their inhibiting effect depended on their concentration. Conclusion Five oligopeptides that were screened by yeast two-hybrid system method can really interact with 1365, and possess the inhibiting effect on DNA binding activity of NF-κB. So it will be possible that these oligopeptides are regarded as model to design and develop novel anti-inflammatory peptide drug targeting NF-κB.