摘要目的 探讨H7N9流感病毒神经氨酸酶(neuraminidase,NA)基因非翻译区(untranslated region,UTR)可变区碱基多态性对NA基因转录与翻译的影响.方法 通过生物信息学方法分析H7N9流感病毒NA基因UTR的可变区碱基多态性位点,人工合成N9-UTR可变区随机突变文库.利用自主构建的RNA聚合酶I eGFP报告系统,构建N9-3'UTR-eGFP-5'UTR RNA聚合酶I随机突变荧光报告文库.从N9-UTR突变荧光报告文库中随机挑选克隆转染H1N1流感病毒辅助感染的MDCK细胞,荧光显微镜观察eGFP的荧光表达,同时用多功能酶标仪定量检测eGFP的相对荧光强度;利用 β-actin作为内参,采用实时荧光RT-PCR定量检测eGFP基因的转录变化.测序鉴定eGFP上调或下调的N9-UTR克隆并分析导致eGFP表达变化的UTR可变区SNP特征.结果 随机筛选了6个N9-UTR可变区随机突变荧光报告克隆,发现N9-2克隆的eGFP下调,而N9-7克隆eGFP上调.测序分析发现突变核苷酸位点位于5'UTR的27位,eGFP上调表现为A27G.结论 H7N9流感病毒NA基因的5'UTR可变区A27G突变可增强NA基因转录与翻译.

abstractsObjective To explore the effect of the nucleotide polymorphism in the variable region of 5' untranslated region (UTR) of neuraminidase (NA) gene on the transcription and translation of NA gene in influenza A (H7N9) virus. Methods The polymorphism sites in UTR of the NA gene of influenza A (H7N9) virus were analyzed by bioinformatics methods. The random mutation gene library containing the N9-UTR variable region was constructed. The random mutant RNA polymerase I fluorescent reporter library of N9-3'UTR- enhanced green fluorescent protein (eGFP)-5'UTR was constructed by utilizing the eGFP RNA polymerase I fluorescence reporting system. The clones randomly selected from the randomized mutagenesis library of N9-UTR were transfected into MDCK cells infected by influenza A (H1N1) virus. Fluorescence microscopy was used to observe the fluorescence expression of eGFP, and the relative fluorescence intensity was measured by a multifunctional microplate reader. The real-time RT-PCR was used to quantify transcriptional changes of eGFP with β-actin as the internal reference. The up-regulated or down-regulated clones of N9-UTR were identified by sequencing to analyze the characteristics of single nucleotide polymorphism in variable region of UTR leading to transcriptional changes of eGFP gene. Results Six randomized mutagenesis fluorescent reporter clones of N9-UTR were randomlyselected. The expression of eGFP in the N9-2 mutant clone was down-regulated, while the expression of eGFP in the N9-7 mutant clone was up-regulated. Gene sequencing revealed a nucleotide mutation at position 27 of 5'UTR, up-regulation of eGFP related to A27G mutation. Conclusions The A27G mutation in the variable region of 5'UTR enhanced transcription and translation of the NA gene.