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目的分析晚期黄疸新生儿人巨细胞病毒(HCMV)感染患儿临床株US3基因序列,预测HCMV US3基因HLA-A*0201限制性CTL表位。方法应用巢式PCR法检测20例HCMV感染新生儿标本US3基因,结果进行双向DNA测序。SYFPEITHI和NetCTL方案预测HCMV临床株US3基因HLA-A*0201限制性CTL表位。结果①以Towne作为参考株,序列比对分析显示,20株临床分离株US3的ORF长度均与参考株相同,为561 bp,编码186个氨基酸蛋白。US3氨基酸序列高度保守,仅几个位点在少数分离株中存在变异,变异主要集中在序列的N端,大部分是同义突变。②通过预测获得5个HCMV US3基因HLA-A*0201限制性CTL表位:FTEKHFVSV、RMDYSSQTI、RMDYSSHTI、SIRDDNWGL和SMRDDNWGL。不同临床株US3基因HLA-A*0201限制性CTL表位存在差异。结论初步筛选出人巨细胞病毒临床株US3基因HLA-A*0201限制性CTL表位,为进一步研究HCMV感染的免疫机制提供实验依据。
Objective To analyze the sequence of US3 gene of clinical isolates of neonatal human cytomegalovirus (HCMV) infection in patients with advanced jaundice and predict the HLA-A * 0201-restricted CTL epitope of HCMV US3 gene. Methods Nested PCR was used to detect the expression of US3 gene in 20 newborns infected with HCMV. The results of bi-directional DNA sequencing. The SYFPEITHI and NetCTL regimens predict the HLA-A * 0201-restricted CTL epitope of the HCMV clinical strain US3 gene. Results ① Towne was used as a reference strain. Sequence alignment showed that the ORF length of the 20 clinical isolates was the same as that of the reference strain, which was 561 bp, encoding a protein of 186 amino acids. The amino acid sequence of US3 is highly conserved. Only a few loci have variation in a few isolates. The variation mainly concentrates on the N terminus of the sequence, most of which are synonymous mutations. ② Five HLA-A * 0201-restricted CTL epitopes of HCMV US3 gene were obtained by prediction: FTEKHFVSV, RMDYSSQTI, RMDYSSHTI, SIRDDNWGL and SMRDDNWGL. There are differences in the HLA-A * 0201-restricted CTL epitopes of the different clinical strains of US3. Conclusion The preliminary screening of HLA-A * 0201 restricted CTL epitopes of human cytomegalovirus clinical strain US3 gene provides an experimental basis for further study on the immune mechanism of HCMV infection.