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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 identify the UL54 gene of human cytomegalovirus (HCMV) infected clinical isolates after allohepatic hematopoietic stem cell transplantation (AlloHSCT). Methods The clinical isolates of HCMV were isolated from patients infected with HCMV after allogeneic hematopoietic stem cell transplantation. According to the DNA sequence of the standard laboratory strain HCMVAD169UL54 provided by GenBank and related literatures, UL54 Gene fragments and cloned into pEGM3Z vector. The recombinant pEGM3Z UL54 was sequenced and sequenced. Results A fragment of about 3728bp was amplified from the clinical isolates. The cloned product was identified by agarose gel electrophoresis and direct sequencing and confirmed to be HCMVUL54. The sequence of the conserved region of HCMVUL54 was consistent with that reported in GenBank. However, 723,910,1188,1641,2070,2278,2279,2442,3140 and other 11 loci base mutation, involving coding amino acids C304R, T760N, Y1047C, R1054K and other sites have changed, where T760N is UL54 conserved region change. Conclusion The UL54 gene of wild-type human cytomegalovirus was successfully cloned after AlloHSCT. Preliminary sequence analysis confirmed the presence of 11 base mutations and resulted in the alteration of 4 amino acid residues of the encoded product.