摘
要
目的
了解慢性髓细胞白血病（CML）基因表达谱的规律，并对在CML中特异性高表达的一个新基因进行克隆、分析与鉴定。
方法
应用基因表达谱芯片技术，比较CML患者和正常人外周血单个核细胞（PBMC）基因的表达差异。检索核苷酸序列数据库（GenBank）和蛋白质一级结构序列数据库（SwissProt），对差异表达的基因进行生物信息学分析，与已知功能基因序列进行同源性比较。根据基因起始密码子的Kozak规则和终止密码子下游保守的多聚腺苷酸信号序列，确定新基因序列，据此设计并合成该基因序列的特异性引物，提取CML PBMC的总RNA，以RT-PCR技术扩增获得该新基因的全长序列，并对克隆的基因及其编码产物的序列进行分析。
结果
在CML患者PBMC中克隆一个新的基因，经测序证实，其编码序列全长为1 872个核苷酸（nt），编码产物由624个氨基酸残基（aa）组成，命名为CMLAP。在GenBank中注册，注册号为AY762229。
结论
基因表达谱芯片技术与生物信息学技术相结合，发现并鉴定、克隆了在CML中高表达的新基因CMLAP，为进一步研究CML发生发展的分子生物学机制奠定基础。
关键词
白血病，髓样，慢性；基因表达谱芯片；基因克隆化
Cloning and identification of a novel human gene, CMLAP, involved in chronic myelogenous leukemia.
Li Mianyang, Liu Yuan et al. Department of Clinical Laboratory, General Hospital of PLA, Beijing 100853, China
Abstract Objective
To study the regulation of gene expression profiles of chronic myelogenous leukemia (CML) and to find new sensitive molecules expressed specifically in peripheral blood mononuclear cells（PBMC） of CML patients for further elucidating the potential molecular biological mechanisms leading to CML.
Methods
The expression of mRNA from PBMC of CML patients was compared with that of normal controls using a cDNA microarray . The bioinformatics analysis was used for every up- or down-regulated gene in CML. The new gene with unknown function and no homology to known genes in the database was confirmed and electric polymerase chain reaction was conducted for the cloning of its full-length DNA in conjunction with Kozak role and the exist of polyadenyl signal sequence. Sequence specific primers were designed to amplify the new gene from the mRNA of CML PBMC with the reverse transcription PCR (RT-PCR).
Results
Many genes commonly up- or down-regulated in CML cells were identified. Of these, we found a novel gene with unknown function expressed highly in the patients cells. Its nucleotide sequence and corresponding protein-encoding amino acid had been determined, which contained 1872 nt and 624 aa respectively. We named the new gene as human chronic myelogegous leukemia associated protein gene (CMLAP) and deposited the sequence of the CMLAP gene into GenBank, and the accession number is AY762229.
Conclusion
CMLAP gene expressed highly in CML PBMC was cloned and identified successfully by combining DNA chip techenology and bioinformational techenology. The gene is likely to play an important role in the disease and may serve as a molecular marker for CML. The findings will pave the way for the study of the molecular mechanism of CML.
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