广州地区人巨细胞病毒临床低传代株UL136基因的序列特征及多态性

Nucleotide sequence characterization and genetic polymorphism of human cytomegalovirus UL136 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）临床低传代株UL136基因的序列特征与基因多态性。方法从10例广州感染新生儿体内分离获得2株（D2、D3）临床HCMV分离株，经多重PCR鉴定后进行UL136基斟全序列扩增。PCR产物纯化后进行基因克隆，构建HCMVUL136-pMD18-T重组质粒。经基冈测序及应用生物信息学分析方法，分析其核酸序列稳定性、编码蛋白质的二级结构与特衙。结果成功分离2株HCMV临床分离株，测序结果显示，D2、1）3及与GenBank中公布的11株临床分离株（4J、51C、39J、33J、63J、22M、10J、32C、29C、27C、Toledo）中，UL136序列高度保守。同源性分析显示在UL136全基因序列1019个核苷酸中，存在30个位点变异，所有的变异均为碱基替换，无插入及缺失突变。编码蛋白的氨基酸序列也高度保守，240个氨基酸残基巾，不同临床分离株氨基酸变异率为1．6％～3．7％。不同分离株的UL136蛋白巾参与形成二级结构的氨基酸数目及等电点不同。进化树分析结果显示D2和D3均属于1a群。结论广州地区临床低传代分离株HCMVUL136基因核苷酸序列及其氨基酸序列极为保守，但仍存在一定多念性。其基因的稳定性提示HCMVUL136开放阅读框（ORF）可能是一个具有重要功能的基因。其编码后修饰位点提示UL136可能与膜受体介导的细胞信号转导通路有关。

Objective To investigate the nucleotide sequence characterization and genetic polymorphism of human cytomegalovirus （HCMV） UL136 gene in low-passage clinical isolates of intants in Guangzhou. Methods Two clinical HCMV strains （D2 and D3） were isolated from 10 infected infants in Guangzhou. After identification by multiplex PCR, the entire HCMV U1,136 gene sequence was amplified. The pured PCR products were cloned into pMDI8- T- vector to construct HCMV U1,136- pMD18-T recombinant plasmid. The sequence stahility, secondary structure and characterization of coding protein were analyzed by sequencing and biological methods. Results Two HCMV clinical strains were successlully isolated. After cloning and sequencing, UL136 genes of D2, D3 and 11 clinical isolates published in GenBank （4J, 51C, 39J, 33J, 63J, 22M, 10J, 32C, 29C, 27C, Toledo） were shown to be highly conservative. Homology analysis revealed mutations in 30 sites among 1019 base pairs in UL136 gene sequence, which were base substitutions without insertion and deletion. High conservative amino acid sequence was also seen in coded proteins. Among 240 amino acid residues, the aberration rates across various clinical isolates ranged from 1.6% to 3.7%. The UL136 properties among these isolates differed in terms of amino acid amounts involved in secondary structure and their isoelectric points. Cladogram showed that D2 and D3 were members of group la. Conclusion All nucleotide and deduced amino acid sequences of UL136 gene share great conservation among low passage HCMV clinical strains in Guangzhou regardless of their polymorphism. The gene stability implies that UL136 open reading frame of HCMV plays an important role in viral infection. The post-translation modified sites suggest that UL136 protein may be related to membrane-receptor mediated cellular signal transduction.