人巨细胞病毒临床分离株的鉴定及UL148基因分析

目的:研究广州地区先天性感染患儿体内人巨细胞病毒(HCMV)临床低传代分离株的鉴定及其UL148基因的序列特征分析。方法:收集广州地区感染HCMV的新生儿尿液标本,从中分离低传代临床病毒株,应用多重PCR方法鉴定,对其进行UL148基因全序列扩增,扩增产物克隆到pMD18-T载体后进行序列测定及分析。结果:成功分离出3株HCMV低传代病毒株(D2、D3、D52),经鉴定后的UL148基因序列被GenBank收录,序列号为:DQ180380,DQ180363和DQ180354。序列分析显示,3株临床分离株中UL148序列高度保守;在UL148全基因序列951个核苷酸中,所有变异均为碱基替换,无插入及缺失突变;编码蛋白由316个氨基酸残基组成,氨基酸序列也比较保守,各分离株中变异率为0.3%～1.9%;编码蛋白翻译后修饰位点包括细胞黏附相关的特征序列、PKC磷酸化位点、酪氨酸激酶II磷酸化位点、cAMP和cGMP依赖性蛋白激酶磷酸化位点等;其编码蛋白等电点为9.17～9.37。结论:广州地区HCMV UL148基因核苷酸序列及其编码的氨基酸序列极为保守,但仍具有一定的多态性,提示UL148 ORF可能是一个重要的功能基因。

Analysis on identification of clinical isolated strains of human cytomegalovirus and UL148 gene

Objective:To study the identification of clinical low-passage isolated strains of human cytomegalovirus(HCMV) and the characteristics of UL148 gene sequence in children with congenital infection of HCMV in Guangzhou.Methods:The urine samples of neonates with HCMV infection in Guangzhou were collected,then the clinical low-passage strains were isolated,multiplex PCR was used to identify the strains,the complete sequence of UL148 gene was amplified,the PCR products were cloned into PMD-18 vector,gene sequencing and analysis were performed.Results:Three low-passage HCMV strains(D2,D3,and D52) were isolated successfully,UL148 gene sequence after identification was submitted to GenBank,the GenBank serial numbers were DQ180380,DQ180363,and DQ180354,respectively.Sequencing analysis showed that UL148 sequences of three clinical isolated strains were highly conversative;among 951 nucleotides in complete sequence of ULl48 gene,all the variations were base substitution,without base insertion and base deletion;encoding protein was consisted of 316 amino acid residues,the sequence of amino acid was conversative,the variation rate of isolated strains was 0.3%-1.9%.The modification sites of encoding protein after translation included characteristic sequence related to cell adhesion,PKC phosphorylation site,tyrosine kinase Ⅱ phosphorylation site,cAMP and cGMP dependent protein kinase phosphorylation site;the isoelectric point of encoding protein ranged from 9.17 to 9.37.Conclusion:The nucleotide sequence of HCMV ULl48 gene and the amino acid sequence were conservative in Guangzhou,but they still had a certain polymorphisms,which indicate that UL148 ORF of HCMV may be one of the important functional genes.