异基因造血干细胞移植后人巨细胞病毒感染临床毒株UL54基因的克隆及序列分析  

Cloning and sequencing of UL54 gene of HCMV isolates from the recipients of Allo HSCT

作　　者：田传军[1] 李月琴[1] 王波[1] 叶铁真[2] 张纯青[2] 苏运贞[1] 何华坤[1] 周天鸿[1] 

机构地区：[1]暨南大学生命科学与技术学院生物化学与分子生物学实验室,广州510632 [2]广州医学院第一附属医院

出　　处：《临床内科杂志》2005年第4期248-251,共4页Journal of Clinical Internal Medicine

基　　金：国家自然科学基金 (项目编号 30370776 );广东省自然科学基金重大项目(项目编号 36703);广东省医学科学基金项目(项目编号A2004272)

摘　　要：目的　研究异基因造血干细胞移植(AlloHSCT)后感染人巨细胞病毒(HCMV)临床病毒株UL54基因的克隆与鉴定。方法　从异基因造血干细胞移植后感染HCMV的患者体内分离出HCMV临床毒株,根据GenBank提供的实验室标准病毒株HCMVAD169UL54基因DNA全序列及有关文献,从该临床毒株DNA中扩增分离出UL54基因片段,并克隆入pEGM3Z载体。对重组体pEGM3Z UL54进行测序鉴定,并进行初步的序列分析。结果　从临床病毒株中扩增出约3728bp的片段,克隆产物经琼脂糖凝胶电泳及直接测序鉴定,证实为HCMVUL54基因,其序列保守区域与GenBank报道AD169基本一致,但在第519、618、723、910、1188、1641、2070、2278、2279、2442、3140等11个位点发生碱基突变,涉及到编码氨基酸C304R、T760N、Y1047C、R1054K等位点发生改变,其中T760N为UL54保守区域改变。结论　成功克隆出AlloHSCT后感染野生型人巨细胞病毒的UL54基因,初步的序列分析证实其存在11个碱基发生突变,并导致编码产物4个氨基酸位点发生改变。Objective To clone and sequence analysis of HCMV UL54 gene from the recipients of Allo hematopoietic stem cell transplantation (Allo HSCT).Methods HCMV was isolated from the patients who infected HCMV after Allo HSCT.UL54 gene was amplified by PCR and cloned into pEGM3Z vector according to laboratory standard strain AD169 UL54 complete sequences provided by GeneBank and related literature.The recombinant plasmid pEGM3Z-UL54 was subsequently sequenced and analyzed primarily.Results One fragment with 3728bp was amplified.The clone was attested to HCMV UL54 gene by agarose gelatin electrophoresis and sequenced directly.The conserved regions of UL54 gene sequence were approximately correspond with AD169 reported in GeneBank.But there are mutations in the sites of 519、618、723、910、1188、1641、2070、2278、2279、2442、3140 of the clinical isolate HCMV,which resulted coding site alterations of amino acids such as C304R、T760N、Y1047C、R1054K.Conclusion HCMV UL54 gene from the recipient of Allo HSCT was successfully cloned,it has 11 site mutations,which result in alterations of 4 amino acids.

关 键 词：异基因造血干细胞移植 人巨细胞病毒 UL54 克隆 序列分析 

分 类 号：R733.71[医药卫生—肿瘤]