鹅Toll样受体分子克隆及其介导的宿主抗NGVEV感染机制研究

项目名称： 鹅Toll样受体分子克隆及其介导的宿主抗NGVEV感染机制研究

项目编号： No.31201891

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 畜牧学与草地科学、兽医学、水产学

项目作者： 陈舜

作者单位： 四川农业大学

项目金额： 23万元

中文摘要： 哺乳动物Toll样受体(Toll like receptors,TLR)在宿主先天抗感染免疫中发挥重要作用，而鹅TLRs的分子特征和抗病毒感染的免疫功能仍不清楚。本项目利用RT-PCR和RACE技术从鹅组织中克隆出TLRs的cDNA全序列，经生物信息学分析明确其分子结构并构建分子遗传进化树。利用基因表达定量和免疫组织化学染色，明确TLRs在健康鹅体及NGVEV感染鹅体内的细胞定位、组织表达谱及其因感染而发生变化的规律。进一步以原代鹅免疫细胞为模型，利用基因表达定量鉴定鹅TLRs的激动剂，并明确下游细胞因子的表达情况。在此基础上，通过共转染报告基因和目标基因、SiRNA基因沉默技术及免疫中和技术明确NGVEV与TLRs的识别、结合及其相互作用的分子机制。本研究将阐明鹅TLRs的生物学特性并明确其介导的宿主抵抗NGVEV感染的分子机制，研究结果将丰富水禽先天抗病毒免疫学基础知识。

中文关键词： 鹅；病毒感染；抗病毒细胞免疫；Toll样受体；干扰素

英文摘要： The essential role of Toll-like receptors (TLRs) in innate immune response to microbial infection is well known. Interestingly, little is known about goose TLRs and the anti-viral mechanisms involved have not been elucidated yet. The cDNA sequence of goose TLRs (goTLRs) is supposed to be cloned by using Reverse Transcription Polymease Chain Reaction (RT-PCR) and Rapid Amplification of cDNA Ends (RACE) methods, the sequence identity and the phylogenetic analysis of goTLRs will be reported. Expression levels of goTLRs mRNA and cellular location of goTLRs in the tissues of health goose and Gosling New Type Viral Enteritis virus (NGVEV) infected goose will be investigated by real-time PCR and immunohistochemical (IHC) staining, respectively. Using the primary cultured peripheral blood lymphocytes (PBLs) and splenocytes as cell model, real-time PCRs will be performed to investigate the mRNA expression of goTLRs and the downstream pro-inflammatory cytokines after treatment with viral-TLR agonists. The infectivity titer of NGVEV is determined after the goTLRs was blocked by the specific antibody. The activation NF-кв in response to NGVEV infection is measured after co-transfected mammalian HEK293 cells with goTLRs and reporter. To further understand the interaction between the goTLRs and NGVEV,the NGVEV-stimulated cyto

英文关键词： goose；viral infection；antiviral immuology；toll like receptor；IFNs