H5亚型禽流感病毒感染和疫苗接种诱导动物免疫反应差异性研究

Highly pathogenic avian influenza virus (HPAIV) can spread rapidly in poultry and cause large-scale outbreak, this virus is categorized by OIE as a "list A" disease. Vaccination can reduce clinical symptoms and increase resistance to avian influenza viruses (AIV) infection, but may not completely protect the animal from infection and shedding of the virus. In outbreak situations, it is therefore important to be able to detect active infection in vaccinated animals in order to avoid spread of the disease. The purpose of this project is to carry out the study on difference naturally infected and vaccinated-only animals (DIVA) of H5 subtype AIV. Specific epitopes of NS1 protein of H5N1 avian influenza virus as a diagnostic potential in DIVA of H5 subtype AI will be identified with using phage display technology. A combined approach of two-dimensional gel electrophoresis (2-DE) with mass spectrometry (MS) will be used to identify the differentiation protein between naturally infected and vaccinated animals of H5 AIV. Phage library combined protein microarray will be used to identify the difference peptide of DIVA of H5 AIV. Moreover, a visual protein chip that can differentiate the naturally infected and vaccinated-only animals of H5 subtype AI will be developed. This study will provide the new database for DIAV of avian influenza, and it is useful for the prevention and monitory of AI.

疫苗免疫接种是预防H5 亚型禽流感病毒（AIV）传播行之有效的措施，但是疫苗的广范使用，使自然病毒感染血清和免疫鸡群血清学阳转容易混淆，现有技术难以鉴别病毒感染和疫苗接种,给禽流感的早期预警和监控工作带来困扰，严重威胁公共卫生安全。本研究将利用差异蛋白质组学方法，筛选H5亚型AIV感染和疫苗免疫宿主差异蛋白分子，研究感染和免疫相关的重要宿主蛋白及其信号通路，同时筛选AIV NS1 蛋白抗原表位。为进一步做好禽流感的监测和早期预警提供理论依据和技术平台。

制备了抗禽流感病毒（AIV）NS1蛋白的单克隆抗体D7，单克隆抗体为IgG1亚类/κ型亚类，该单抗可特异性的识别DAPF基序，是该单抗的抗原表位，是AIV NS1蛋白识别的最短的表位。利用该单抗进行免疫沉淀反应筛选与其结合的宿细胞蛋白,并经质谱、免疫共沉淀和Pull-down试验鉴定证明膜联蛋白A2(ANXA2)与NS1蛋白之间可能存在相互作用关系。此外,激光共聚焦试验表明NS1和ANXA2共定位于胞浆中。首次鉴定AIV NS1蛋白与宿主蛋白ANXA2间存在相互作用，为进一步研究AIV NS1蛋白的功能奠定了基础。.利用鸡脾感染组织，采用诸多技术手段对感染、对照和免疫组织的mRNA转录水平和蛋白表达水平进行进一步验证分析。.1、获得蛋白可信度在95%以上的蛋白点33个，表达27种蛋白。利用基因本体学（GO）注释显示AIV感染组与对照组差异表达蛋白可分为34个功能类别，多数差异表达蛋白参与转运、代谢和免疫系统反应。.2、获得AIV感染组与对照组差异表达有信息注释的基因2457条，其中1284个基因表现上调，1173个基因下调。GO分析表明2457个差异表达的基因基于生物学过程分类，可以归为24个类别。共发现137个信号通路。.3、最终选择16个参与感染和免疫反应相关的差异表达基因及蛋白进行验证。发现组织TGM2和DES基因表达水平与蛋白表达水平趋势一致，ACTR3、YWHAB、RPSA和B2M蛋白基因水平也与2-DE结果一致，而LY86表达变化呈现相反趋势。TGM2、IL1、IL6、IFN-γ、IFN-β、MMP1、SAMD3、PCSK5和IL2基因表达水平与基因组芯片检测的表达趋势相同。.首次进行了AIV感染鸡脾脏蛋白质组和转录组表达的完整分析，将增加我们对AI致病机制和病毒与宿主相互作用的了解，为筛选蛋白进行进一步研究和筛选抗病毒药物宿主靶标奠定基础。.