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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 investigate a series of genes that control cell differentiation and cell matrix formation and to differentiate myocardial cells in different spatial and temporal distributions. To investigate the relationship between KRAB-ZFPs and cardiac development and cardiovascular diseases . Methods: Nested PCR and 5 ’end cDNA amplification (5’ACE) were used to carry out PCR reaction using the embryonic heart library as a template to obtain a new zinc finger protein gene of KRAB box, which was named by International Gene Nomenclature Committee For ZNF649. Northern blot analysis of ZNF649 gene expression in human embryos and adult tissues. We coded the ZNF649 open reading frame through the BamHI and SaII restriction sites into the TGG codon in place of the TAG terminator in the pEGFP-NI vector starting from the start codon AUG 269 bp to the stop codon 1796 bp 505 amino acid residues of protein. The protein has a molecular mass of about 64K Daltons and is located on chromosome 19q13.41 The entire ZNF649 protein contains a KRAB box (amino acids 8-68) and ten C2H2 zinc finger motifs. Northem blot analysis yielded an approximately 3.2 kb transcript in the heart, brain, lung, liver, muscle, kidney and pancreas of 20-week-old human embryonic tissue whereas in adult tissues ZNF649 transcripts were found in heart, muscle and brain There is a stronger expression, but less expression in other organizations. CONCLUSION: The above results suggest that the successful construction of human cardiac development candidate genes ZNF649 and ZNF649 in various tissues of human embryos suggests that the gene is probably related to the maintenance of cardiac development and normal cardiac function.