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作 者:徐祥[1] 梁华平[1] 刘昕[2] 李生茂[1] 史海水[1] 刘东擘[1] 吴强[1] 王正国[1]
机构地区:[1]第三军医大学大坪医院野战外科研究所第一研究室创伤烧伤与复合伤国家重点实验室,重庆400042 [2]第三军医大学基础医学部分子遗传学教研室,重庆400038
出 处:《免疫学杂志》2006年第2期132-136,共5页Immunological Journal
基 金:国家重点基础研究发展规划(G1999054203);国家自然科学基金(30080009;30200270);全军"十五"课题基金项目(2001-2005)资助
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pGBKT7DNA-BD载体上,与GAL4DNA-BD融合,形成GAL4DNA-BD/p65BD融合蛋白;再以GAL4DNA-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pGBKT7DNA-BD/p65BD载体。自1.28×107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。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.
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