中文摘要：

目的以NF-κB p65亚基DNA结合域为诱饵蛋白，筛选能够与其发生相互作用的多肽，并鉴定这些多肽对NF-κB DNA结合活性的抑制效应。方法首先利用PCR搭桥扩增方法获取NF-κB p65亚基DNA结合域的基因片段，后将其克隆到酵母双杂交pGBKT7 DNA-BD载体上，与GAL4 DNA-BD融合，形成GAL4 DNA-BD／p65BD融合蛋白；再以GAL4 DNA-BD／p65BD融合蛋白为诱饵蛋白行酵母双杂交实验，自16肽随机肽库中筛选与NF-κB p65亚基DNA结合域相互作用的多肽；最后选择性人工合成筛选获得的多肽，并进行EMSA实验检测相互作用多肽对NF-κB DNA结合活性的抑制效应。结果扩增获得了NF-κB p65亚基DNA结合域的基因片段，并成功构建pGBKT7 DNA-BD／p65BD载体。自1．28×10^7个酵母转化子中最终筛选获得5个阳性克隆，测序结果和同源氨基酸序列比对表明5个阳性多肽为p65BD新型相互作用多肽，其中4条多肽拥有一个共同的基序。EMSA实验结果表明合成的两条多肽对NF-κB的DNA结合活性均具有一定的抑制效应。结论经初筛、复选和假阳性鉴定，自16肽库中获得5个能与NF-κB p65亚基发生相互作用的新型多肽，并已证实其中两条多肽对NF-κB的DNA结合活性具有抑制效应。

英文摘要：

Objective To screen oligopepfides interacted with DNA binding domain of NF-κB p65 sanbunit and explore the inhibiting effect of these oligopeptides on DNA binding activity of NF-κB. Methods By means of primer dimer building bridge, the gene fragment of NF-κB p65 subunit DNA binding domain was amplified, and then inserted into pGBKT7 DNA-BD vector to construct pGBKT7 DNA-BD/p65BD. The oligopeptides interacted with p65BD were screened from a random peptides library having an open reading frame for 16 amino acids by yeast two-hybrid system method. The positive oligopeptides were synthesized and purified. The inhibitory effect of the oligopeptides on DNA binding activity of NF-κB was identified by EMSA. Results The eDNA of NF-κB p65 subunit DNA binding domain was obtained, and inserted into pGBKT7 DNA-BD vector to construct pGBKT7 DNA-BD/p65BD. Five positive oligopeptides possessed capability of specific interaction with DNA binding domain of NF-κB p65 subunit were obtained from 1.28×10^7 yeast transformants. The DNA and protein sequences of the five oligopeptides were different with each other, but four of them contained a common motif. EMSA results showed that two of the five peptides could inhibit DNA probe from binding to NF-κB in a concentration-dependent manner. Conclusion Five novel oligopeptides specially interacted with DNA binding domain of NF-κB p65 subunit are found, and two of them possess the inhibiting effect on DNA binding activity of NF-κB.