鹅Toll样受体分子克隆及其介导的宿主抗NGVEV感染机制研究

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 cytokines expression is measured by real-time PCR after partical silencing of goTLRs by SiRNA. This work may reveal the interaction between the host and NGVEV through TLRs, and show the significant role of goTLRs in initiating anti-NGVEV immune responses. This research may shed light on the immunoregulatory effects and mechanisms of TLRs during avian viral infection, and may provide new insights into the innate immunity system of waterfowl.

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。

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TLR7与TLR21的cDNA全序列（首次报道），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。利用半定量PCR与定量PCR技术，明确TLRs在健康雏鹅、成年鹅及NGVEV感染鹅体内的组织表达谱及其因感染而发生变化的规律。进一步以原代鹅脾细胞或鹅外周血淋巴细胞为模型，利用定量PCR技术，择不同TLRs的激动剂及多种鹅源病毒（NGVEV, GPV, H9N2 AIV, TMUV, NDV）为刺激物，监测鹅TLR7与鹅TLR21并明确下游细胞因子（IFNA, IFNG, IL6, IL1beta）的表达情况。在此基础上，我们进一步探究鹅TLRs的重要下游分子IFNs，分别分子克隆出鹅IFNA, IFNAR1, IFNAR2, IFNG, IFNGR1, IFNGR2, IFNL, IFNLR 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。同时，制备了相应的多克隆抗体。利用定量PCR技术，免疫组化技术，免疫沉淀技术等，对鹅TLRs，IFNs,及病毒病原（GPV, H9N2 AIV, TMUV）的相互作用关系，进行了深入的讨论。.水禽在许多重要病原的携带、传播、变异中发挥重要作用（比如，禽流感病毒），但是对水禽的先天免疫系统的研究极少。鹅，作为重要的饲用水禽，在我国广泛养殖，特别是在我国南方与西南方地域。本课题的研究结果，鉴定了一批（超过10个）鹅先天抗病毒相关的免疫分子，弥补了水禽先天抗病毒免疫的许多研究空白，推动了鹅分子抗病毒免疫系统研究。由于水禽在许多重要病院的携带、传播、及变异中发挥重要作用（比如，禽流感病毒），但是对水禽的先天免疫系统的研究极少。鹅，作为重要的饲用水禽，在我国广泛养殖，特别是在我国南方与西南方地域。本课题执行期间鉴定了一批（超过10个）鹅先天抗病毒相关的免疫分子，弥补了水禽先天抗病毒免疫的许多研究空白，推动了鹅分子抗病毒免疫系统研究。

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