人类胚胎干细胞特异表达新基因HPESCRG1的克隆和特性分析

Molecular cloning and characterization analysis of HPESCRG1, a novel gene expressed specifically in human embryonic stem cell

目的克隆一个人类胚胎干细胞特异表达的新基因并对其特性进行分析。方法从一个在人类胚胎干细胞(embryonicstem,ES)中特异表达的表达序列标签CF948547出发,应用生物信息学方法和分子生物学技术克隆一个新基因,采用逆转录-聚合酶链反应分析新基因的表达谱,并应用增强型绿色荧光蛋白真核表达系统分析新基因的亚细胞定位。结果成功克隆了一个新基因HPESCRG1,GenBank登录号为AY283672。该基因cDNA长1395bp,包含9个外显子和8个内含子,开放阅读框为250～1146bp,定位在3q13.13,预测编码297个氨基酸,预计分子量为33784,等电点为9.35。其编码蛋白有一个SAP功能域,该蛋白定位在细胞核内。该基因仅在人类ES细胞中表达,而在其分化细胞不表达,在人胚胎成纤维细胞、人间充质干细胞、成人和流产胚胎的多种正常组织中不表达。结论HPESCRG1基因是一个人类ES细胞中表达特异性新基因,很可能与人类ES细胞的自我更新和维持其未分化状态密切相关。

Objective To clone a novel gene expressed specifically in human embryonic stem cell and to analyze its characteristics. Methods Based on an expression sequence tags(EST) CF948547 which expressed specifically in human embryonic stem cell, the full length cDNA sequence of a novel gene was cloned by using bioinformatic and molecular biological technique. Its expression profile was analyzed by reverse transcription polymerase chain reaction(RT PCR), and subcellular location was determined by enhanced green fluorescent protein (EGFP)eukaryotic expression system. Results A novel gene HPESCRG1(homo sapiens pluripotent embryonic stem cell related gene) was cloned successfully. Its GenBank accession number was AY283672. Its cDNA length was 1395 bp. It comprised 9 exons and 8 introns, and its opening reading frame was 250 1146 bp. Its chromosomal mapping was located in 3q13 13, and the putative protein contained 297 amino acids. The theoretical molecular weight of the putative protein was 33 784 and the isoelectric point was 9.35. The protein primary structure of this gene contained a SAP motif and it was subcellularly located in nuclei. Expression analysis showed that this gene was expressed specifically in human ES cells,but not expressed in the adult human tissues, the multiple tissues of embryo aborted in over 5 months' pregnancy, the differentiated cells of HESC 1, and the human mesenchymal stem cells (hMSCs) and human embryonic fibrocytes (hEFCs). Conclusion HPESCRG1 was found to be a novel gene expressed specifically in human ES cell ,which might be related to self renewal of human ES cell and maintaining its undifferentiated state.