人肿瘤坏死因子样蛋白全长cDNA的克隆及原核表达  

The Cloning and Prokaryotic Expression of Human T umor Necrosis Factor Like Protein

作　　者：胡晓燕[1] 周严[1] 代芸[1] 陈建萌[1] 张斌[1] 彭小忠[1] 袁建刚[1] 强伯勤[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246-249,共4页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金(39830070);国家高技术研究发展计划863重点项目基金(2001AA221041);国家重点基础性研究973项目基金(G1998051002)资助

摘　　要：目的分离、克隆编码人肿瘤坏死因子样蛋白(TNFLP)的全长cDNA,并对其功能进行初步研究。方法从本室构建的λZAP表达胎脑文库中分离到编码TNFLP的全长cDNA。应用多种软件对该基因及其编码蛋白进行分析,寻找其同源蛋白,分析其亲、疏水性及其存在的结构域和基序。以Northern印迹分析检测TNFLP的mRNA的组织分布,并用谷胱苷肽-S-转移酶(GST)原核表达系统分段表达TNFLP的N端和C端蛋白。结果TNFLP全长共2112bp,编码一208个氨基酸的蛋白。亲、疏水性分析显示,TNFLP有两个疏水区,且C端95个氨基酸与小鼠的TNF-α一致性为42%。基序分析显示,TNFLP的C端有一内质网膜定位信号-TYKRL,与TNF-α完全一致。人的TNFLP位于人的16号染色体上。Northern杂交显示,TNFLP在心、脑和胰腺中呈高表达,可见一个转录本。用GST原核表达系统,分段表达了TNFLP的N端和C端。结论初步分析了TNFLP的组织表达谱,并分段表达了其N端和C端,为进一步研究其功能提供了条件。Objective To identify and clone the gene encoding human TNFLP(tumor necrosis factor like protein)for some functional study on TNFLP.Methods The full-length cDNA of TNFLP was isolated from fetal brain cDNA library.Several kinds of software were used to analyze nucleotide sequence and amino acid sequence of TNFLP.TNFLP mRNA distribution was identified by Northern blot.TNFLP-C and TNFLP-N were expressed in E.coli with GST expression system.Results The cDNA of human TNFLP was2112bp,which encoded protein of208amino acid.Hydrophobili ty analysis found there were two hydrophobility regions of human TNFLP.TNFLP-C (112-207amino acid)and mouse TNF-αwere homologous.The identity of their amino acid sequence was42%.Morever,both of them had a motif-TYKRL.TNFLP was located in chromosome 16.Human TNFLP was widely expressed in various human tissues.Northern blot showed TNFLP was highly expressed in heart,brain and spleen,only one transcript can be seen.GST-TNFLP-C and GST-TNFLP-N fusion proteins were obtained.Conclusions Tissue expression spectrum of TNFLP and prokaryotic expression of TNFLP have been done,which establish the base for the functional analysis of TNFLP.

关 键 词：肿瘤坏死因子 -α 肿瘤坏死因子样蛋白 原核表达 基因克隆 

分 类 号：Q781[生物学—分子生物学]