广州地区HCMV临床病毒株UL137基因的结构特征与多态性

Characterization of Human Cytomegalovirus (HCMV) UL137 Gene in Low-passage Clinical Isolates in Guangzhou

【目的】研究广州地区新生儿感染人巨细胞病毒(HCMV)临床低传代分离株UL137基因的结构特征与基因多态性。【方法】从62份被证实为临床HCMV感染的新生儿尿液标本中分离获得3株低传代临床病毒株,经多重PCR鉴定后分别命名为HCMVD2、D3和D52。对3株临床分离株进行HCMVUL137基因全序列PCR扩增,PCR产物纯化后进行基因克隆,构建HCMVUL137-pMD18-T重组质粒;基因测序;将HCMVUL137基因序列呈报GenBank,并进行基因生物信息学分析。【结果】成功从广州地区新生儿感染HCMV患儿体内分离3株HCMV临床病毒株。克隆测序后呈报GenBank并被收录,收录序列号分别为:DQ180388、DQ180376、DQ180360。通过序列分析,结果显示低传代分离株UL137基因DNA序列高度保守,同源性在96.91%～100%之间,其变异均为碱基替换;编码蛋白均由96个氨基酸残基组成,同源性在90.63%～100%之间,超半数的变异发生在第61～81位氨基酸残基之间;编码蛋白翻译后修饰位点包括PKS、MYS、cAMPs三类,其中4个PKS和44～49位的MYS高度保守,cAMPs位点仅在5株病毒出现;编码蛋白等电点在11.50～12.00之间,呈强碱性。【结论】广州地区临床低传代分离株HCMVUL137基因核苷酸序列及其编码蛋白的氨基酸序列极为保守,但仍存在一定多态性。提示HCMVUL137ORF可能是一个具有重要功能的基因。

[Objective] To investigate the characterization and sequence polymorphism of human cytomegalovirus UL137 gene in low passage clinical isolates in Guangzhou. [Methods] Three clinical low-passage strain was segregated from 62 samples of infant urine who has been proved positive HCMV infection. After multiplex PCR assessment, they were designated D2,D3, and D52, respectively. PCR was performed to amplify the entire HCMV UL137 gene region of 3 clinical isolates. The amplification products were cloned into pMD18-T-Vector and subjected to sequencing, and the sequences were analyzed together with the published homologous sequences in GenBank from 22 clinical isolates. [Results] The DNA sequences of UL137 gene from Guangzhou 3 low-passage clinical isolates and the other 22 isolates showed high conservation, homologies ranged from 96.91% to 100%, all variabilities were base substitution. The UL137 protein from 25 isolates all consisted of 96 residues,homologies ranged from 90.63% to 100%, almost over a half of variabilities occurred between amino acid residues 61 and 81 sites. There were three kinds of posttranslational modification sites in HCMV UL137 protein： protein kinase C phosphorylation site（PKS）, N-myristoylation site （MYS） as well as cAMP and cGMP dependent protein kinase phosphorylation site （cAMPs）, all four PKS and one MYS locating between amino acid residues 44 and 49 were conserved highly, cAMPs site only exists in 5 isolates, the isoelectric point of UL 137 protein ranged between 11.50 and 12.00, thus the protein was a strong alkaline. [Conclusion] All DNA and deduced amino acid sequences of ULl37 gene shared great similarity among HCMV clinical strains regardless of their polymorphism. It implied that UL137 ORF of HCMV maybe a gene playing an important role in viral infection.