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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CMLPBMC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1872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。
Objective To understand the regularity of gene expression profile of chronic myelogenous leukemia (CML) and to clone, analyze and identify a new gene with high specificity in CML. Methods Gene expression profiling was used to compare the expression of peripheral blood mononuclear cells (PBMCs) between CML patients and normal controls. GenBank and SwissProt databases were searched, and the differentially expressed genes were analyzed by bioinformatics and compared with known functional gene sequences. According to the Kozak rule of gene start codon and the polyadenylation signal sequence conserved downstream of the stop codon, a new gene sequence was determined, and specific primers of the gene sequence were designed and synthesized. The total RNA of CMLPBMC was extracted, and RT -PCR technique to amplify the full-length sequence of the new gene and analyze the sequence of the cloned gene and its encoded product. Results A novel gene was cloned from PBMC of CML patients. The full-length cDNA was 1872 nucleotides (nt) in length and the coding sequence was 624 amino acids (aa), named CMLAP. Registered in GenBank with registration number AY762229. Conclusion The combination of gene expression microarray and bioinformatics technology has found and identified and cloned a novel gene CMLAP with high expression in CML, which lays the foundation for further study on the molecular biological mechanism of CML.