DR4死亡结构域结合蛋白的初步研究  

The Primary Study on a Novel Protein Binding to the Death Domain of the Death Receptor 4

作　　者：李晓玲[1] 刘彦信[1] 刘士廉[1] 郑德先[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所医学分子生物学国家重点实验室,北京100005

出　　处：《中国医学科学院学报》2002年第3期310-314,共5页Acta Academiae Medicinae Sinicae

基　　金：国家重点基础性研究973项目基金(G19990539);国家自然科学基金(30070295)资助

摘　　要：目的克隆DR4死亡结构域的结合蛋白,为进一步研究DR4诱导细胞凋亡的信号传递途径提供条件。方法以TRAIL(TNF-relatedapoptosisinducingligand)死亡受体DR4的死亡结构域(DR4DD)为钓饵,采用酵母双杂交系统筛选人白细胞MATCHMAKERcDNA文库。以DNA自动分析仪测定核苷酸序列,采用计算机软件分析核苷酸的同源性及功能结构域。并用免疫共沉淀方法分析人类甲酰肽受体1(formylpeptidereceptor-like1,FPRL1)在哺乳动物细胞内是否与DR4CD(thecytoplsmicdomainofDR4)特异性地结合。结果获得了两个阳性克隆pADB1和pADB2。DNA序列分析及同源性比较表明,pADB1的插入片段与人FPRL1的mRNA序列具有97%的同源性。pADB2的插入片段与GeneBank中的已知序列无同源性。免疫共沉淀结果表明FPRL1在哺乳动物细胞内可与DR4CD特异性地结合。结论FPRL1可与DR4的死亡结构域特异性结合,推测FPRL1与DR4的结合可能在DR4介导的细胞凋亡信号传导途径中发挥一定的作用。Objective To clone and identify novel proteins binding to the death domain of the death receptor4(DR4).Methods The yeast two -hybrid system was used for this study.Automatic sequencing was carried out for DNA sequencing.The sequence homology and the functional domains were analyzed by BLAST and the ScanProsite Tool softwares,respectively.Co -immuno-precipitate method was used to confirm human formyl peptide receptor-like1(FPRL1)binding specifically with DR4CD(the cytoplsmic domain of DR4)in HEK293T cells.Results Two positive clones,named as pADB1and pADB2,were obtained.BLAST searching showed that the homology of the insert sequence of pADB1with the mRNA of FPRL1was97%.The insert of pADB2shared no homology with any known peptides in GeneBank.Co -immunopreci-pitate analysis further confirmed that FPRL1could bind to DR4CD in vivo specifically.Conclusions FPRL1may associate with DR4CD in vivo specifically.The functional studies of FPRL1in signaling pathway mediated by TNF-related apoptosis inducing ligand(TRAIL)are in active progress in our laboratory.

关 键 词：类甲酰肽受体1 死亡受体4 TRAIL 酵母双杂交系统 

分 类 号：Q513[生物学—生物化学]