禽致病性大肠杆菌感染鸡异嗜性粒细胞时RIP2功能及其调控机制的研究

Avian pathogenic Escherichia coli (APEC) can cause serious respiratory disease, resulting in great economic losses in poultry. Currently, there is no effective method to control disease caused by this bacterium. In previous study, we found RIP2 gene and its pathway was significantly changed in response to APEC infection, indicating its important role in immunity. In human and mouse, properly inhibiting RIP2 gene expression will relieve the excessive inflammatory response in respiratory disease, contributing to prognosis treatment. However, there is almost no literature on the identification of RIP2 in chicken, as well as its regulatory mechanism on chicken immune and inflammatory responses. This project will use chicken heterophils and RIP2 gene as material to identify the function of RIP2 gene and select the binding proteins via gene over-expression and RNA interference, qRT-PCR, Western blotting, GST-pull down, CHIP-PCR, and Co-IP etc. technology, constructing the RIP2’s signal network and elucidating the mechanism of RIP2 to regulate host immune and inflammatory responses. Meanwhile, we will utilize point mutation, double luciferase reporter assay, and bisulfite sequencing PCR etc. technology to explore the factors that regulate the activity of RIP2, which will build an important theoretical foundation for improving immunity and controlling excessive inflammation in poultry.

禽致病性大肠杆菌（APEC）可引起严重呼吸道疾病，造成禽业巨大经济损失。目前该菌尚无很好控制方法。前期实验显示APEC感染时RIP2及其通路有显著性免疫应答，表明其有重要免疫作用。在人和鼠中适当抑制基因RIP2表达，可缓解呼吸系统疾病的过度炎症反应，有助于预后治疗。但在鸡中RIP2功能的研究还鲜少报道，对家禽免疫和炎症的调节机制尚不清楚。本项目将以鸡特有异嗜性粒细胞和RIP2基因为研究材料，利用RIP2基因过表达和干扰、qRT-PCR、Western blotting、GST-pull down、CHIP-PCR、Co-IP等技术验证RIP2基因功能及筛选其互作蛋白，构建其信号调控网络，解析RIP2调节异嗜性粒细胞免疫和炎症反应的分子机制；同时采用定点突变、双荧光素酶检测、亚硫酸氢盐转化测序等技术探寻调控基因RIP2活性的关键因子，为提高家禽免疫和控制过度炎症反应奠定重要理论基础。

集约化高密度家禽业的迅速发展使家禽生产面临更多细菌性感染和炎症反应。如禽致病性大肠杆菌（APEC）可引起严重的呼吸系统疾病，作为初级或继发性免疫原，经不同致病机制形成禽大肠杆菌病，造成禽业巨大的经济损失，包括增加死亡率和胴体污染率；降低生长率和饲料转化率。目前该菌尚无很好控制方法。前期转录组测序实验结果显示APEC感染时RIP2及其通路有显著性免疫应答，表明其有重要免疫作用。因此，本项目将以鸡特有异嗜性粒细胞和RIP2基因为研究材料验证RIP2基因功能并筛选其互作蛋白，构建其信号调控网络，解析RIP2调节异嗜性粒细胞免疫和炎症反应的分子机制，并探寻调控基因RIP2活性的关键因子。结果显示鸡RIP2基因可有效调节细胞免疫炎症反应，其启动子核心区域为 -2300 ~ -1839 bp，转录因子NFIB可结合于此核心区域调节RIP2基因表达活性。过表达或干扰NFIB可显著性调节RIP2及其通路活性，进而介导细胞免疫炎症反应。同时发现10 μmol/L 5-Azadc和1 μmol/L TSA均可显著性增强鸡RIP2启动子活性，联合使用这两种试剂将以数倍级别增强启动子活性。禽致病性大肠杆菌感染可显著性影响鸡RIP2基因甲基化状态。DNA甲基化抑制剂5-氮杂胞苷可降低鸡RIP2基因上游部分CpG位点甲基化水平，使其启动子区低甲基化，从而促进基因表达。而CpG甲基转移酶M.SssI使鸡RIP2基因启动子区甲基化水平升高，抑制基因表达。并筛选出鸡RIP2互作蛋白TLR4和ITIF5等，验证其与RIP2基因有相似的功能。研究结果对提高家禽免疫和控制过度性炎症反应具有十分重要的现实意义。