GST Pull-down实验鉴定NF-κB相互作用多肽  

Identification of polypeptide interacting with NF-κB by GST pull-down assay

作　　者：李生茂[1] 梁华平[1] 徐祥[1] 刘东擘[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所一室

出　　处：《免疫学杂志》2006年第1期94-97,共4页Immunological Journal

基　　金：国家重点基础研究发展规划项目(G1999054203);国家自然科学基金(300800009;30200270);全军十五课题基金资助项目

摘　　要：目的体外鉴定NF-κB相互作用多肽与p50和p65间的相互作用。方法首先构建GST-作用多肽融合蛋白的原核表达载体pET-42a/polypeptide及NF-κB p50和p65亚基保守结构域的原核表达载体pET22b/p50和pET22b/p65,并在大肠杆菌E.coliBL-21中诱导表达,后进行GST pull-down实验验证多肽与NF-κB p50和p65的结合效应。结果经诱导表达获得了可溶性的GST-多肽融合蛋白和具有DNA结合活性的p50p、65蛋白,GST pull-down实验证实3条多肽与p50发生特异地相互作用。结论证实3条多肽在体外能与p50发生物理性的相互作用,这为获得靶向NF-κB的功能拮抗多肽工作奠定了基础。Objective To identify the interaction between the NF-κB binding polypeptides and the proteins of p50/p65 in vitro. Methods GST-polypeptide fusion protein prokaryotic expression vector pET-42a/polypeptide was constructed. NF-κB p50/p65 protein rel homol- ogy domain expression vectors pET22b/p50 and pET22b/p65 were constructed. The Escherichia coli BL-21 containing the expression plasmids were induced by IFIG, and then analyzed by GST pull-down assay. Results The soluble GST-polypeptide fusion protein and the p50/p65 proteins with DNA binding activity were obtained. Three polypeptides could interact with p50 protein identified by GST pull-down assay. Conclusion Three polypeptides can interact strongly with p50 protein in vitro, which estabhshes a foundation for preparing the antagonist of NF-κB.

关 键 词：GST Pull—down实验 多肽 NF—κB p50/p65 蛋白间相互作用 

分 类 号：R346[医药卫生—基础医学]