克隆测序鉴定流感病毒诱导小鼠急性肺炎模型

发布时间：2018-04-08 21:16

本文选题：克隆测序　切入点：流感病毒　出处：《中国当代儿科杂志》2013年02期

【摘要】：目的结合RT-PCR病毒载量检测、克隆测序基因比对以及病理改变鉴定甲型流感病毒(influenzavirus,IV)鼠肺适应株(FM1株)诱导的小鼠急性肺炎模型,为急性肺炎模型提供科学的鉴定方案。方法以FM1株滴鼻诱导昆明小鼠急性肺炎,分别于第3、5、7天取小鼠肺脏,以RT-PCR法检测IV病毒载量,并通过质粒克隆测定cDNA序列;同时观察小鼠肺脏病理改变。结果正常组小鼠肺组织的肺泡、肺泡囊和肺泡隔形态完整,周围血管未见炎症细胞浸润;模型组在感染IV后第3、5、7天均见肺泡、肺泡囊、肺泡管、肺泡隔等结构破坏,肺泡间隔增厚,细支气管壁增厚,大量炎症细胞浸润,肺泡壁毛细血管扩张,可见大量红细胞。与正常组比较,模型组在不同时间点的病理变化程度均有显著性差异(P0.01)。不同时间点模型组小鼠肺组织中均可检测到IV病毒核酸,且不同时间点小鼠肺组织的病变程度与病毒载量呈正相关(P0.01)。经测序比对发现,IV感染小鼠肺组织中RT-PCR扩增产物与已知IV cDNA序列相应片段吻合度高达99.1%。结论克隆测序基因比对法可鉴定FM1株诱导的小鼠急性肺炎模型。
[Abstract]:Objective to identify the mouse acute pneumonia model induced by influenza A virus influenzavirusIV-1 strain combined with RT-PCR viral load detection, cloning and sequencing gene alignment and pathological changes, and to provide a scientific identification scheme for acute pneumonia model.Methods Acute pneumonia of Kunming mice was induced by nasal drip of FM1 strain. The lungs of mice were collected on the 7th day of the 3rd day, the viral load of IV virus was detected by RT-PCR method, the cDNA sequence was determined by plasmid clone, and the pathological changes of mouse lungs were observed at the same time.The alveolar septum thickened, the bronchiole wall thickened, a large number of inflammatory cells infiltrated, the alveolar wall capillary dilated, a large number of red blood cells were observed.Compared with the normal group, the pathological changes of the model group were significantly different at different time points (P 0.01).IV virus nucleic acid could be detected in the lung tissue of mice in different time point groups, and the pathological degree of lung tissue was positively correlated with the viral load at different time points (P 0.01).The results of sequencing showed that the RT-PCR amplification products in the lung tissues of mice infected with IV were up to 99.1% consistent with the corresponding fragments of the known IV cDNA sequences.Conclusion cloning and sequencing gene alignment method can be used to identify mouse acute pneumonia model induced by FM1 strain.
【作者单位】：辽宁中医药大学附属医院;
【基金】：国家自然科学基金(81072975)
【分类号】：R-332

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