LncRNA介导的快慢羽鸡抗ALV-J天然免疫反应差异的分子机制研究

Fast (FF) and slow feathering (SF) auto-sexing was applied widely in poultry industry, however, most of the SF lines that harbored endogenous virus-21 gene (ev21) show the reduced production and slightly weaker disease resistance than FF lines. It is worth studying whether the host innate immune mediated by long non coding RNA (lncRNA) caused such differences between SF and FF chicken lines. From the perspective of the host, this project intends to study the molecular mechanism of antiviral innate immunity and immune evasion through cultivating the primary monocyte-derived macrophages of the Specific pathogen free (SPF) chickens infected with subgroup J avian leukosis viruses (ALV-J), and to identify which pattern recognition receptors (PRRs) of macrophages would be involved in sensing ALV-J and leading to subsequent key protein of innate immune signaling pathways and cytokines and type I interferon expression. The differential innate immune responses in ALV-J infected FF and SF chickens will be studied by transcriptomic approach through next generation sequencing, lncRNA involved in chicken innate immune responses will be identified, and the regulatory mechanism involved in host antiviral innate immune response will be studied in detailed and validated with genome-wide association study and in vitro reporter gene assay. This project would elucidate the molecular mechanisms of the differences anti-ALV-J innate immune responses between SF and FF chickens. It is beneficial to overcome severe economic losses caused by the widespread of ALV-J and to genetic improvement of layers and broilers.

养鸡业中常用的快慢羽自别雌雄因慢羽鸡含有内源性禽白血病病毒（avian leukemia virus, ALV）基因座ev21，而使其生长及抗病性能受到不利影响，这是否由于长链非编码RNA（long non-coding RNA, lncRNA）介导的宿主天然免疫出现差异引起值得研究。本项目拟从宿主角度出发，通过无特定病原鸡原代巨噬细胞的分离培养与攻毒，鉴定巨噬细胞识别ALV-J的模式识别受体、相关细胞因子表达及天然免疫信号通路关键蛋白的表达来分析宿主抗病毒天然免疫和病毒免疫逃逸机制；以转录组测序分析快慢羽鸡天然免疫反应的差异，并找到参与鸡天然免疫反应的lncRNA，研究其调控天然免疫反应的机制。旨在阐明lncRNA介导的快慢羽鸡抗ALV-J天然免疫反应差异的分子机制。这对解决近年来J亚群禽白血病普遍流行给我国的养禽业造成的严重经济损失，培育健康蛋鸡、肉鸡品种均有积极意义。

针对快、慢羽鸡抗ALV-J天然免疫反应的差异，本项目围绕 鸡（SPF鸡和普通黄鸡）抗ALV-J天然免疫应答和ALV-J免疫逃逸机制，快慢羽鸡抗ALV-J天然免疫应答反应的差异分析以及慢羽鸡ev21基因对ALV-J免疫反应分析， 参与抗ALV-J天然免疫应答的lncRNA及其调控网络，lncRNA介导的快慢羽鸡抗ALV-J天然免疫反应差异导致快慢羽鸡生活力差异分子机制四个方面进行研究， 研究获得了SPF雏鸡人工感染J亚群禽白血病病毒(ALV-J)诱发的天然免疫反应及ALV-J感染诱发的肿瘤患鸡产生的天然免疫应答规律；通过ALV-J易感的重要免疫细胞（巨噬细胞）为载体，获得了ALV-J感染鸡原代巨噬细胞后，病毒逃逸宿主免疫打击的策略；发现了ALV-J感染快慢羽鸡后病毒感染规律以及免疫应答规律；通过鸡原代巨噬细胞为载体，构建了ALV-J感染巨噬细胞后lncRNA参与的lncRNA-gene，以及lncRNA-gene-miRNA调控网络，获得了参与抗ALV-J天然免疫反应的关键lncRNA；基于ALV-J在快慢羽鸡中的感染规律，选择差异关键感染时间点进行lncRNA高通量测序，获得了快慢羽鸡抗ALV-J天然免疫反应关键候选lncRNA，快慢羽鸡对ALV-J免疫力的差异在于重复序列的存在，并且与催乳素水平有着密切关系。项目已发表了8篇SCI收录论文，培养博士1名，硕士4名。研究结果已应用于快慢羽自别雌雄系的建立与培育实践中。