人肠三叶因子串联亲和层析表达载体的构建与表达  

Construction and expression of trefoil factor 3 expressing vector for tandem affinity purification

作　　者：黄建坤[1] 王琳[1] 裴一花 刘国彦[1] 

机构地区：[1]厦门大学附属中山医院,福建省厦门市361004

出　　处：《中国组织工程研究》2015年第24期3839-3843,共5页Chinese Journal of Tissue Engineering Research

基　　金：厦门市科技计划项目资助(3502Z20124016);课题名称:应用蛋白质学研究人肠三叶因子相互作用蛋白~~

摘　　要：背景:人肠三叶因子为新型的生长因子,可促进细胞生长与迁移,并使细胞具有很强抗凋亡能力,在维持胃肠黏膜完整性、黏膜保护与损伤修复发挥着重要作用,与胃肠道等多个肿瘤的发生发展密切相关。人肠三叶因子(三叶因子3)在黏膜修复及作为肿瘤标志物有着广阔的临床应用前景,但其相互作用蛋白及具体分子机制不明确。目的:构建和表达带串联StrepI I和6×His两个蛋白标签的三叶因子3重组蛋白,以进一步利用研究体内蛋白质相互作用的串联亲和层析技术纯化获得三叶因子3相互作用蛋白。方法:化学合成用于串联亲和层析的标签(StrepI I-TEV-6×His)基因序列,并以三叶因子3表达载体为模板,PCR扩增三叶因子3基因序列,通过Xba I位点实现标签与三叶因子3基因序列的融合,并将标签置于三叶因子3蛋白的C端,融合基因通过EcoR I+Hind III酶切,克隆于表达载体pC DNA3.0,构建了三叶因子3串联亲和层析表达载体pT FF3-C-StH。表过载体通过脂质体瞬时转染胃癌细胞AGS,采用Western Blot检测重组蛋白的表达。结果与结论:成功构建了三叶因子3串联亲和层析表达载体pT FF3-C-StH,经酶切和测序鉴定载体构建完全正确。表达载体成功转染胃癌细胞株,并获得重组蛋白的表达,通过Western Blot检测证明表达产物具有很好的抗原性和特异性,为串联亲和层析纯化三叶因子3相互作用蛋白提供重要的实验基础和工具。BACKGROUND：As a novel growth factor, human intestinal trefoil factor （TFF3） can promote cel growth and migration, and increase cel resistance to apoptosis, and it plays a great role in maintaining the mucosa integrity, mucosa protection and repairing the injured mucosa, also it has been closely related to the tumor growth and progression. With the function of mucosa repair, and as the tumor biomarker, TFF3 has a promising clinical application, but its definite interacting protein and molecular mechanism is stil unclear. OBJECTIVE：To construct and express the TFF3 recombinant protein with the tandem tag of StrepII-6＆#215;His in the target cel s for further purifying its interaction protein in the native condition based on the tandem affinity purification technique. METHODS：The DNA sequence for the tag （StrepII-TEV-6＆#215;His） and TFF3 as template was got by chemical＆amp;nbsp;synthesis and PCR amplification respectively. They were fused by the restriction enzyme XbaI site, and the tag sequence was located at the C terminus of TFF3 protein. TFF3-tag fusion gene was cloned into the pCDNA3.0 using EcoRI＋HindIII, thus the TFF3-tag expressing vector pTFF3-C-StH was constructed and transfected transiently into gastric cel AGS by lipofectin. The recombinant TFF3-tag protein was expressed and detected by western blot assay. RESULTS AND CONCLUSION：The expressing vector pTFF3-C-StH for tandem affinity purification was constructed successful y, and was confirmed further by restriction enzyme analysis and sequenced. The recombinant TFF3-C-StH protein of TFF3-tag was expressed in the AGS cel , and showed specific antigenicity by western blot assay. Thus this work provides experimental base for further purification of the TFF3 interacting proteins.

关 键 词：组织构建 组织工程 串联亲和层析纯化 TFF3 StrepII 标签 6× His标签 

分 类 号：R318[医药卫生—生物医学工程]