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目的了解引起新生儿晚期黄疸的人巨细胞病毒(HCMV)UL144基因多态性,预测UL144基因各型编码蛋白的B细胞优势表位。方法应用荧光定量PCR法检测晚期黄疸新生儿标本HCMV-DNA含量,采用巢式聚合酶链反应扩增阳性标本HCMV UL144基因开放读码框(ORF),结果进行双向DNA测序,绘制种系进化树对HCMV感染患儿UL144基因进行分型。结合亲水性参数、可及性参数、抗原性参数、柔韧性参数及二级结构方案对HCMV UL144基因编码各型蛋白的B细胞表位进行预测,参照已建立的预测方法综合评价B细胞优势表位。结果①30例晚期黄疸新生儿荧光定量PCR检测阳性,其中28例UL144基因扩增阳性。②28株UL144基因与Toledo株进行同源性比较,核苷酸水平为80.4%~99.2%,氨基酸水平为78.9%~98.8%。种系进化树分析显示感染患儿标本UL144基因可分为3个基因型,G1型7株(25%),G2型7株(25%),G3型14株(50%)。③HCMV UL144基因G1型和G2型B细胞表位均位于编码蛋白N段109-119位,G3型于氨基酸116位缺失一个谷氨酰胺,B细胞表位位于编码蛋白N段109-118位。结论①HCMV UL144基因的DNA序列呈高度多态性,但B细胞表位预测高度保守;②UL144 G3型氨基酸N段116位(Q)位点缺失可能改变该抗原表位的抗原性。
Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL144 gene in neonates with advanced jaundice and to predict the predominant epitopes of B cells of various types of UL144 gene encoding proteins. Methods Fluorescent quantitative PCR was used to detect the content of HCMV-DNA in neonatal jaundice neonates. Nested polymerase chain reaction was used to amplify the open reading frame (ORF) of HCMV UL144 gene. Two-way DNA sequencing was performed to screen the phylogenetic tree The UL144 gene was genotyped in children with HCMV infection. The B cell epitopes encoding various types of HCMV UL144 genes were predicted by the combination of hydrophilicity parameters, accessibility parameters, antigenicity parameters, flexibility parameters and secondary structure programs. The B cell epitopes were evaluated according to the established prediction methods gauge. Results ① Thirty neonates with late jaundice were detected by real-time PCR, of which 28 cases were positive for UL144. ② The homology of 28 strains of UL144 with Toledo was 80.4% -99.2% at nucleotide level and 78.9% -98.8% at amino acid level. Phylogenetic tree analysis showed that the UL144 gene was divided into 3 genotypes in 7 children (25%), 7 (25%) of G2 and 14 (50%) of G3 in infected children. HCCV UL144 gene G1 and G2 type B cell epitopes are located in the coding protein N segment 109-119, G3 type in the amino acid 116 a glutamine, B cell epitopes encoded protein N segment 109-118. Conclusion ① The DNA sequence of HCMV UL144 gene is highly polymorphic, but the prediction of B cell epitopes is highly conserved. ② The deletion of the 116th (Q) site of the amino acid sequence of UL144 G3 may change the antigenicity of this epitope.