人类心脏发育候选基因ZNF649的克隆与表达研究  

Cloning and Expression Analysis of Human Heart Developmental Candidate Gene ZNF649

作　　者：杨鸿[1] 黄春霞[1] 

机构地区：[1]长沙医学院生物化学教研室,湖南长沙410219

出　　处：《长沙医学院学报》2005年第2期16-20,28,共6页Journal of Changsha Medical University

摘　　要：目的:探讨一系列控制着细胞分化、细胞基质形成的基因,在不同的时空表达构成了心肌的分化过程;研究包含有KRAB的锌指蛋白(KRAB-ZFPs)与心脏发育以及心血管疾病的关系。方法:应用巢式PCR技术和5′端快速扩增cDNA末端(5′ACE)技术,以胚胎心脏文库为模板进行PCR反应,获得一个KRAB框的锌指蛋白新基因,通过国际基因命名委员会命名为ZNF649。采用Northern blot分析ZNF649基因在人体胚胎和成体组织中的表达。我们把ZNF649开发阅读框通过BamHI和SaII酶切位点连入发TGG密码子取代了TAG终止子的pEGFP-NI载体由第269bp的起始密码子AUG开始到第1786bp左右的终止密码子编码一个505个氨基酸残基的蛋白质。蛋白质分子量大约为64K道尔顿,位于染色体19q13.41.整个ZNF649蛋白质包含了一个KRAB框(氨基酸8-68)和10个C2H2锌指模体。Northem blot分析在20周的人体胚胎组织的心脏,大脑,肺,肝脏,肌肉,肾脏和胰腺中都得到一个大约为3.2KB的转录本,而在成体组织中,ZNF649在心脏,肌肉和大脑中有较强烈的表达,而在基他组织中的表达较弱。结论:上述结果提示本研究成功的构造了人类心脏发育候选基因ZNF649,ZNF649成功的在人类胚胎各种组织表达也提示了该基因很可能与心脏发育和心脏正常功能的维持有关。Objective:To explore cardiac differentiation involves a cascade of co-ordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporo-spatial manner and to research the relationship between KRAB-zinc protein(ZFPs)and cardiac or cardiovascular diseases.Methods:Using nested PCR and 5'-rapid amplification of cDNA ends(5'RACE)technique to obtain the full-length cDNA of the novel KRAB-Zinc finger genes from human embryonic heart cDNA library.HUGO Nomenclature committee approved named'ZNF649'novel gene,we used the Norhern blot analyzed the ZNF649 expression in human embryo tissues. The ZNF649 ORF was subcloned into the BamHI and SalI sites of pMD18-T vector and was subcloned into the Xhol and SalI sites of pEGFP-N1 vector with tht TGG codon instead of the TAG stop codon.Results:The ZNF649 gene consists of an open reading frame(ORF)of 1518 bp extending from the initiation AUG codon at nu- cleotide 269 to a termination UAG at 1786.The deduced ZNF649 protein has 505 amino acids with a calculated molecular mass of 59 kDa.ZNF649 is assigned to human chromosome 19q13.41 according to the mapping infor- mation in NCBI,and spans approximately 17 on the genome.ZNF649 contains an N-terminal Kruppel-associated box(KRAB)domain(amino acids 8-68)and 10 C2H2 zinc-finger motifs that extending to the end of the protein sequence.Northern blot analysis detected an expected transcript of about 3.2 kb in the 20-week embryo tissues, including heart,brain,lung,liver,skeletal muscle,kidney,and pancreas.The expression of ZNF649 at adult was pre- dominant in heart,skeletal muscle,and brain,but weaker in other tissues.Conclusions:These findings confirm that human hear developmental candidate gene ZNF649 is successfully constructed and expressed in human em- bryo tissues.So it suggested that the ZNF649 might be involved in heart development.

关 键 词：心脏发育 锌指蛋白 ZNF649 KRAB框 

分 类 号：R346[医药卫生—基础医学]