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摘要：树突状细胞是目前所知的功能最强的抗原递呈细胞，是唯一一种沟通先天性免疫和获得性免疫的桥梁。禽流感病毒中H9N2在鸡树突状细胞上的复制受到显著的抑制。非编码RNA中的miRNA在调控树突状细胞的发育和抗原递呈能力的同时，也可以调控禽流感病毒的复制。因此可以从miRNA的层面入手研究鸡的树突状细胞抑制禽流感病毒复制的分子机制。通过miRNA过表达载体的构建、流式细胞、荧光定量PCR、miRNA对应的靶基因野生型和突变型表达载体的构建、双萤光素酶报告基因法验证互作、证明了miRNA对树突状细胞在免疫功能的影响极有可能是通过与树突状细胞上的miRNA的靶基因结合位点结合，抑制靶基因的转录和表达，并且相互作用产生一系列效应，来增强树突状细胞免疫功能，进而抑制禽流感病毒的复制，具体机制需要深入研究，但为合理的开发新型非编码RNA预防禽流感病毒提供了重要的理论依据。38755
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毕业论文关键词：H9N2禽流感病毒；鸡树突状细胞；miRNA
Immune Function of Chicken Dendritic Cells Activated by Gga - miRNA in Chicken
Abstract: Dendritic cells are currently known as the strongest antigen presenting cells and are the only bridges to communicate with innate immunity and acquired immunity. The replication of H9N2 on chicken dendritic cells in avian influenza virus was significantly inhibited. Non-coding RNA in the miRNA in the regulation of dendritic cell development and antigen presentation ability, but also can control the replication of avian influenza virus. Therefore, we can study the molecular mechanism of chicken dendritic cells to inhibit the replication of avian influenza virus from the level of miRNA. Construction of miRNA overexpression vector, flow cytometry, fluorescence quantitative PCR, construction of wild-type and mutant expression vectors corresponding to miRNAs, double-luciferase reporter assay, demonstrated that miRNAs were involved in dendritic The effect of cells on immune function is most likely to be enhanced by dendritic cell immunity by binding to target gene binding sites of miRNAs on dendritic cells, inhibiting transcription and expression of target genes, and interacting to produce a series of effects Function, and thus inhibit the replication of avian influenza virus, the specific mechanism needs in-depth study, but for the rational development of new non-coding RNA to provide an important theoretical basis for the prevention of avian influenza virus.
Key words: H9N2 avian influenza virus; chicken dendritic cells; miRNA
目 录
摘要1
关键词1
Abstract1
Key words 1
引言（或绪论）1
1 材料与方法2
1.1 实验材料2
1.1.1 试剂及耗材2
1.1.2 主要仪器3
1.2 实验方法4
1.2.1构建产生显著差异的miRNA的表达载体 4
1.2.2通过利用流式细胞技术验证筛选MicroRNAs与树突细胞表型的相关性 4
1.2.3通过荧光定量PCR方法检测禽流感病毒的复制是否受到影响 5
1.2.4筛选显著差异的miRNA对应的靶基因 5
1,2.5构建筛选显著差异的miRNA对应的靶基因野生型的表达载体 5
1.2.6利用双萤光素酶报告基因法验证软件预测的MicroRNAs与禽流感病毒靶基因的互作相关性 7
2. 结果与分析 7
2.1 构建产生显著差异的miRNA的表达载体 7
2.2 流式细胞技术结果9
2.3 荧光定量PCR实验结果9
2.4 筛选显著差异的miRNA对应的靶基因 11
2.5 构建筛选显著差异的miRNA对应的靶基因突变体的表达载体 11
2.6 构建靶基因突变型的表达载体 13
2.7 双萤光素酶报告基因法验证结果 14
3.讨论 14
致谢 16
参考文献 16
鸡Gga-miRNA调控激活鸡树突状细胞的免疫功能的研究
引言：禽流感是通过A型流感病毒引起的禽类烈性的传染病。这两年来，禽流感频繁爆发肆虐全球，给我国甚至世界上许多国家养禽业造成了极大的经济损失[1]。H9N2亚型禽流感病毒是一株低致病性的禽流感病毒，具高度变异性和易与其他亚型流感病毒发生重组等的特点，往往会造成毒力增强，甚至跨物种传播，从而造成人际间的流感爆发或大流行[2,3,4,5]。目前H9N2亚型禽流感病毒已引起我国政府和有关科学家们的高度支持，而如何有效控制和预防该病毒的传播和蔓延就成为国内外学者研究的重中之重。