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为探究人巨细胞病毒(HCMV)无症状性感染者UL144基因多态性,本研究采用聚合酶链反应扩增HCMVDNA阳性无症状性感染者尿沉渣标本UL144基因开放读码框(ORF),阳性结果进行双向DNA测序,通过生物信息学工具BioEdit、DNAstar、Mega5.0、GeneDoc等软件进行序列特征分析。结果显示,在50例HCMV-DNA阳性无症状性感染者尿沉渣标本中,21例标本UL144基因扩增阳性,阳性率为42%,并完成测序。同源性比较显示核苷酸同源性为80.2%~100%,氨基酸同源性为77.8%~100%。种系进化树分析显示21例无症状性感染者DNA序列主要分为2个基因型,A型(10例,47.61%)和B型(11例,52.38%),未见其他基因型。Expasy数据库预测分析UL144基因编码产物重要功能基团,结果显示ASN、PKC、TNFR、NCD3G等翻译后修饰位点高度保守。与UL144A型相比,UL144B型在1-16和30-96氨基酸残基之间各增加了一个PROKAR_LIPOPROTEIN位点和ZF_CTCHY位点。与Toledo标准病毒株比较,UL144A型的CRD1和CRD2区域高度保守,UL144B型的CRD1和CRD2功能区变异较大,但跨膜区(transmembrane domain)和胞浆区(cytoplasmic domain)在UL144A型和B型中均高度保守。UL144A型和B型碱基变异未对UL144蛋白的预测等电点和二级结构造成较大改变。总之与HCMV显性感染者不同,HCMV无症状性感染者UL144基因型为A型和B型。
In order to explore the polymorphism of UL144 gene in human asymptomatic HCMV infection, polymerase chain reaction (PCR) was used to amplify the open reading frame (ORF) of urinary sediment samples from patients with HCMVDNA positive asymptomatic infection and positive Results Two-dimensional DNA sequencing was carried out and the sequence characteristics were analyzed by bioinformatics tool BioEdit, DNAstar, Mega5.0 and GeneDoc. The results showed that in 50 cases of HCMV-DNA positive asymptomatic infection in urine sediment specimens, 21 cases of specimens UL144 gene amplification positive, the positive rate was 42%, and complete the sequencing. Homology comparison showed nucleotide homology was 80.2% ~ 100%, amino acid homology was 77.8% ~ 100%. Phylogenetic tree analysis showed that the DNA sequences of 21 asymptomatic cases were divided into two genotypes: A (10 cases, 47.61%) and B (11 cases, 52.38%). No other genotypes were found. Expasy database predictive analysis of UL144 gene coding products important functional groups, the results showed ASN, PKC, TNFR, NCD3G post-translational modification sites such as highly conserved. Compared to the UL144A type, the UL144B type has a PROKAR_LIPOPROTEIN site and a ZF_CTCHY site each added between 1-16 and 30-96 amino acid residues. The CRD1 and CRD2 regions of UL144A are highly conserved compared to the Toledo standard strains, while the CRD1 and CRD2 functional regions of UL144B are highly variable, but the transmembrane and cytoplasmic domains differ between UL144A and B Type are highly conservative. UL144A and B base variations do not significantly alter the predicted isoelectric point and secondary structure of UL144 protein. In conclusion, different from HCMV dominant infection, HCMV asymptomatic infected UL144 genotype A and B type.