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摘要：禽流感是由禽流感病毒引起的鸟类烈性传染病。其中，H9N2亚型禽流感病毒因其致病性弱，不易感染人但病毒重组率高而成为毒株研究对象。在禽流感病毒入侵复制释放周期中，核定位信号肽直接参与病毒复制相关蛋白从细胞质入核进而在核内完成复制周期。而microRNAs近年来被报道参与流感病毒相关蛋白表达调控。本研究从miRNAs及核定位信号肽两部分入手，在细胞水平从miRNAs能否靶向病毒复制相关蛋白的核定位信号肽，miRNAs能否调控病毒复制相关蛋白含量和miRNAs能否影响细胞表型三个方面进行试验。结果表明，gga-miR-6675-3p能靶向H9N2亚型禽流感病毒PB1复制蛋白的核定位信号肽，并极显著降低PB1及NP蛋白含量，对鸡树突细胞表型有一定激活作用。38351
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毕业论文关键词：H9N2；禽流感；microRNAs；细胞
Functional verification of nuclear localization signals of H9N2 avian influenza virus and related microRNAs in cells
Student majoring in Base of Life Science and Technology
Abstract：Avian influenza, a serious pathogen, causes acute respiratory disease infected by avian influenza virus (AIV). H9N2 avian influenza virus becomes the research object because of its weak pathogenicity, infecting humans difficultly but high virus recombination rate. In the replication phase of avian influenza virus, nuclear localization signals directly involve in replication related proteins entering nucleus from cytoplasm and finishing replication cycle in nucleus. MicroRNAs have been reported to participate in regulation of expression of influenza virus proteins in recent years. This research combines nuclear localization signals with miRNAs including whether miRNAs can target at nuclear localization signals of viral replication related proteins at cellular levels, whether miRNAs can regulate viral replication related proteins content and whether miRNAs can affect cell phenotypes. The result shows that gga-miR- 6675-3p can target at nuclear localization signals of PB1 of H9N2 avian influenza virus ,significantly reduce PB1 and NP content and activate chicken dendritic cells to some extent.
Key words: H9N2；avian influenza；microRNAs；cells
目 录
摘要： 1
关键词： 1
ABSTRACT： 1
KEY WORDS: 1
引言 1
1 材料与方法 3
1．1 材料与仪器 3
1．1．1 实验材料 3
1．1．2 实验动物 3
1．1．3 实验试剂及耗材 3
1．1．4 实验器材 4
1．2 实验方法 4
1．2．1 MIRNAS过表达载体的构建 4
1．2．2 核定位信号肽过表达载体的构建 9
1．2．3 鸡骨髓源树突状细胞体外诱导培养 12
1．2．4 双萤光素酶报告基因检测 13
1．2．5 荧光定量PCR检测复制相关蛋白PB1及NP的表达量 13
2 结果与分析 14
2．1 生物信息学预测 14
2．2 MIRNAS过表达载体的构建 15
2．3 核定位信号肽过表达载体的构建 16
2．4 双萤光素酶报告基因检测 16
2．5 MIRNAS对复制相关蛋白PB1及NP表达的影响 17
2．6 MIRNAS对鸡树突细胞表型的影响 18
3 讨论 19
3．1 生物信息学预测 19
3．2 双萤光素酶报告基因检测 19
3．3 MIRNAS对复制相关蛋白PB1及NP表达的影响 19