人工诱骗子的入核分布对核因子-κB活性的调控作用  

Nuclear localization of oligonucleotides decoy effect on nuclear factor-κB activity

作　　者：刘颖勋[1,2] 权富生[1] 王进科[2] 白学尧[1] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100 [2]东南大学生物电子学国家重点实验室,南京210096

出　　处：《生物工程学报》2010年第12期1683-1689,共7页Chinese Journal of Biotechnology

基　　金：国家重大科学研究计划(No.2006CB933205);国家自然科学基金(No.60871014)资助~~

摘　　要：探讨了由核定位信号(NLS)多肽介导的核因子-κB(NF-κB)寡核苷酸诱骗子(ODNs decoy)进入HeLa细胞核的效率,以及对细胞核内NF-κB活性的调控作用。利用双功能交联剂(Sulfo-SMCC)共价交联末端氨基修饰的ODNs decoy和末端巯基修饰的NLS多肽,形成NLS多肽共价连接的ODNs decoy。依靠TransME转染试剂的辅助转染NLS-ODNs decoy进入HeLa细胞,用荧光显微镜观察荧光标记的NLS-ODNs在细胞内的分布。用MTT法检测HeLa细胞的活力,以凝胶迁移实验(EMSA)检测TNF-α诱导的HeLa细胞核抽提物中NF-κB的活性。结果表明,NLS多肽成功地连接到ODNs decoy上,NLS-ODNs可高效入核,入核率达到17.9%。转染NLS-ODNs进入HeLa细胞,对细胞活力无明显影响,而显著抑制核内NF-κB的活性。结果表明NLS多肽可提高ODNs decoy的入核效率,显著增强诱骗子对NF-κB活性的抑制效果。To investigate the effect of the localization of oligonucleotides decoy（ODNs decoy） on the activation of nuclear factor-κB（NF-κB） in TNF-α induced HeLa cells.The mercapto group-modified nuclear localization signal（NLS） peptide was covalently conjugated to amino group-modified NF-κB ODNs decoy by Sulfo-SMCC cross-linker.The NLS-ODNs decoy was transfected into HeLa cells by TransME transfection reagent.The intracellular distribution of fluorescent labeled NLS-ODNs decoy was detected with a microscope.The cell viability was detected by MTT assay,and then the activity of NF-κB in cell nuclear extract was assayed by electrophoretic mobility shift assay（EMSA）.The results showed that NLS peptide was successfully conjugated to ODNs decoy by Sulfo-SMCC cross-linker.The NLS-ODNs decoy effectively entered into nucleus with high rate of 17.9%.It was observed that the cell viability of HeLa cell was not significantly affected by the transfection of NLS-ODNs decoy,while NLS-ODNs decoy significantly inhibited the activation of NF-κB in TNF-α induced HeLa cells nuclear extracts.This experiment can provide a new covalent conjugation of NLS peptide to ODNs can effectively drive decoy into nucleus,and thus improve its inhibitory effects on the activation a transcription factor.

关 键 词：核因子-ΚB 寡核苷酸诱骗子 Sulfo-SMCC交联试剂 核定位信号多肽 

分 类 号：Q75[生物学—分子生物学]