CAJ | 학술논문

目的以IRX1基因启动子为诱饵,在胃cDNA文库内钓取与之相互作用的蛋白质.方法构建IRX1启动子诱饵报告基因质粒并转化酵母单杂交(Y1H)Gold酵母获得诱饵基因酵母菌;抽提人胃组织总RNA,逆转得到cDNA文库,文库片段为0.3～2.5 Kb,作为猎物与线性化pGADT7-Rec文库质粒共转染诱饵酵母菌.结果对胃cDNA文库的筛选效率达到3×106,从人胃组织cDNA文库共钓取到8个阳性克隆,测序分析获得3个具有完整开放读码框(ORF)的基因,其编码蛋白均含锌指结构域.结论以酵母单杂交筛选文库方法得到与IRXI基因启动子序列相互作用蛋白质,为探讨IRX1基因的转录调控机制奠定了基础.
목적 이IRX1기인계동자위유이,재위cDNA문고내조취여지상호작용적단백질.방법 구건IRX1계동자유이보고기인질립병전화효모단잡교(Y1H)Gold효모획득유이기인효모균;추제인위조직총RNA,역전득도cDNA문고,문고편단위0.3～2.5 Kb,작위작물여선성화pGADT7-Rec문고질립공전염유이효모균.결과 대위cDNA문고적사선효솔체도3×106,종인위조직cDNA문고공조취도8개양성극륭,측서분석획득3개구유완정개방독마광(ORF)적기인,기편마단백균함자지결구역.결론 이효모단잡교사선문고방법득도여IRXI기인계동자서렬상호작용단백질,위탐토IRX1기인적전록조공궤제전정료기출.
Objective To construct bait yeast reporter plasmid with IRXI core promoter sequence and screen gastric cDNA library for characterizing the binding proteins in IRX1 transcriptional regulation.Methods Bait yeast reporter plasmid with IRX1 promoter was constructed and transfected into Gold Y1H yeast.Meanwhile,gastric cDNA library with size range of 0.3 to 2.5 kb was constructed by total RNAs extracted from normal gastric tissues.The interacting proteins were screened by yeast one-hybrid (Y1H) system.Results With screening efficiency of 3 × 106,we got eight positive colonies.By sequence analysis,3 candidate genes with complete opening reading frame were confirmed.Conclusion Characterizing the interacting proteins with IRXI core promoter by YIH system facilitates the understanding of regulatory mechanism for IRX1.