CAJ | 학술논문

目的研究人脐血CD34+造血干/祖细胞来源的新基因HSPCSET的结构和生物学功能.方法采用生物信息学技术对HSPCSET蛋白进行结构分析和同源性比较.酵母双杂交实验筛选与HSPCSET蛋白相互作用的蛋白分子,测序后筛选出功能较明确的阳性克隆进一步回复性杂交,采用β-gal实验和GST拉下试验验证.免疫荧光共定位方法分析HSPCSET和相关基因细胞内定位情况.结果蛋白结构分析和同源性比对揭示HSPCSET是古老的SET基因家族的成员,可能具有非常保守而重要的生物学功能.应用酵母双杂交技术筛选能与之相互作用的蛋白,获得13个阳性克隆,分别涉及信号传导、转录调控、细胞凋亡、肿瘤发生、发育等途径.与细胞增殖、分化、凋亡及肿瘤发生密切相关.采用GST拉下试验进一步在体外水平验证了其中4个基因:GADD34(凋亡相关分子)、SIVA(凋亡相关分子)、DNAJ(分子伴侣)和PHF](转录相关因子)与HSPCSET蛋白确实存在相互作用.免疫荧光共定位方法显示HSPCSET和GADD34共转染细胞株后共同定位于细胞核内,两者之间存在强烈的相互作用.结论新基因HSPCSET可能参与凋亡途径、具有非常保守而重要的生物学功能.本研究结果为进一步研究HSPCSET在造血调控中的作用及研究肿瘤发生奠定基础.
목적 연구인제혈CD34+조혈간/조세포래원적신기인HSPCSET적결구화생물학공능.방법 채용생물신식학기술대HSPCSET단백진행결구분석화동원성비교.효모쌍잡교실험사선여HSPCSET단백상호작용적단백분자,측서후사선출공능교명학적양성극륭진일보회복성잡교,채용β-gal실험화GST랍하시험험증.면역형광공정위방법 분석HSPCSET화상관기인세포내정위정황.결과 단백결구분석화동원성비대게시HSPCSET시고로적SET기인가족적성원,가능구유비상보수이중요적생물학공능.응용효모쌍잡교기술사선능여지상호작용적단백,획득13개양성극륭,분별섭급신호전도、전록조공、세포조망、종류발생、발육등도경.여세포증식、분화、조망급종류발생밀절상관.채용GST랍하시험진일보재체외수평험증료기중4개기인:GADD34(조망상관분자)、SIVA(조망상관분자)、DNAJ(분자반려)화PHF](전록상관인자)여HSPCSET단백학실존재상호작용.면역형광공정위방법 현시HSPCSET화GADD34공전염세포주후공동정위우세포핵내,량자지간존재강렬적상호작용.결론 신기인HSPCSET가능삼여조망도경、구유비상보수이중요적생물학공능.본연구결과 위진일보연구HSPCSET재조혈조공중적작용급연구종류발생전정기출.
Objective To characterize the structural and the functional feature of a novel gene HSPCSET isolated from human CD34+hematopoietic stem/progenitor cells(HS/PCs).Methods Bioinformatic technology was used tO identify the structural features of the HSPCSET protein and perform the multiple sequence alignment.Yeast-two-hybrid system was used to identify the proteins interacting with the HSPCSET protein.After sequencing,we selected out the positive clones which had clear functions,and carried out β-gal experiment and GST pull down assay to confirm the results.The cellular location of the HSPCSET was checked by immunofluorescence assay.Results The HSPCSET protein belongs to a SET domain family,which is evolutionarily conserved across species.It implied that HSPCSET may have biologically important function.Using yeast-two-hybrid system.we showed that the protein sequence with SET domain might bind to 13 proteins,which involved in signaling transduction.transcriptional regulation,apoptosis,tumorigenesis,development,etc.And 4 proteins(GADD34,SIVA,DNAJ and PHF1)wereconfirmed by one-on-one back of the hybrid experiment,~-gal test and GST pull down assay.When GADD34 and HSPCSET were co-transfected,they co-localized in the nucleus,suggesting a strong interaction.Conclusion The novel gene HSPCSET is likely to have biologically important function.This study provides the basis for further studies of its function in hematopoiesis and tumorigenesis.