Ag43嵌合疫苗通过IgE/FcɛRI通路防治变应性鼻炎的机制研究

Allergic rhinitis is an allergic diseases in which IgE mediated, eosinophils as a main reaction and a lot of inflammatory cells and cytokines participate. Targeting IgE/FcɛRI as a signaling pathway drug design has become a new targets and hot spot in the drug research of allergic disease treatment.There is a Ag43/Fcs chimeric bacterium surface antigen protein which can break of the immune tolerance against IgE antibody, blocked IgE/FcɛRI signal pathway, intervene allergic disease bridging reaction through the further preparation as protein vaccine. We intend to analyze the potential therapeutic utility of this vaccine in allergic rhinitis, examine the correlation of nasal mucosal epithelial cells in the immune response and inflammatory response as well as tissue remodeling，then evaluate the efficacy.The effect of the vaccinationis is searched in allergic rhinitis, through testing IgE and FcɛRI expression of receptors prove that it blocked IgE/FcɛRI signal pathway, analyzing the change of mastocyte degranulation of nasal mucous samples, testing balance changes of related cell inflammatory mediator factor by means of detecting downstream that mastocyte`s secretion is lowered, proving that effect of reducing of IgE production, further studying on this vaccine subsequent mechanism of action and design more perfect vaccine or combined treatment to allergic rhinitis and a set of more effective treatment strategy will be established.

变应性鼻炎是以IgE介导的，嗜酸性粒细胞反应为主的，由多种炎症细胞及细胞因子共同参与的I型变态反应性疾病。以IgE/FcɛRI信号通路为靶点设计药物，已成为治疗变态反应性疾病的一个新的热点。现有一种细菌表面抗原Ag43和Fcɛ嵌合蛋白能被进一步加工为蛋白疫苗，打破免疫耐受产生自身抗IgE抗体，阻断IgE/FcɛRI信号通路，干预变应性疾病桥联反应。我们拟分析此疫苗在变应性鼻炎中的潜在的治疗效用，检测鼻粘膜上皮细胞在免疫、炎症反应和组织重塑相关指标，客观评价Ag43嵌合细菌表面抗原蛋白疫苗对变应性鼻炎的疗效和后续应用价值。检测IgE/FcɛRI通路中IgE、FcɛRI受体的表达，分析鼻粘膜组织上肥大细胞的脱颗粒的改变，探索下游肥大细胞分泌降低后相关细胞炎性介质因子平衡的改变与IgE生成降低的关系，研究此抗体在桥联环节中的缺失及其突破免疫耐受的具体机制，并以此建立一套更有效的防治策略。

变应性鼻炎是以IgE介导的，嗜酸性粒细胞反应为主的，由多种炎症细胞及细胞因子共同参与的I型变态反应性疾病。变应性鼻炎的易感个体初次接触变应原并产生特异性IgE分子，变应原被APC处理后提呈给Th细胞，Th细胞可引起B细胞分化为浆细胞。本课题新合成的特异性IgE会被浆细胞分泌，它与位于肥大细胞、嗜碱性粒细胞等细胞表面的高亲和力受体（FcεRⅠ）结合，还与位于单核细胞、嗜酸性粒细胞等细胞表面的低亲和力受体（FcεRⅡ/CD23）结合，以阻断IgE/FcRI信号通路为出发点，通过构建一个大肠杆菌Ag43表达系统，并将外源基因成功表达于大肠杆菌表面，利用Red重组系统敲除大肠杆菌JM109的Ag43序列，并利用基因工程技术在质粒pET-28b中插入大部分Ag43的基因序列，将小鼠IgE Fcε3的基因序列插入到表达载体Ag43中，成功获得嵌合蛋白疫苗表达载体Ag43/IgE Fcε3，测序结果显示该载体的构建正确。克隆菌株经IPTG诱导后，SDS-PAGE电泳检测Ag43/Fcɛ3嵌合细菌表面抗原蛋白疫苗在菌液上清中有很明显的表达。蛋白纯化后通过变应性鼻炎小鼠模型证实Ag43/Fcɛ3嵌合细菌表面抗原蛋白疫苗对小鼠体内IgE抗体水平有变化，血清以及鼻腔灌洗液中相关炎症细胞因子在变应性鼻炎小鼠模型中无特异性表达，Ag43/Fcɛ3嵌合细菌表面抗原蛋白疫苗在变应性鼻炎小鼠模型中能下调IgE的表达，嵌合疫苗蛋白Ag43/IgE Fcε3具有针对小鼠IgE的抗原性，western blot实验结果表明嵌合蛋白Ag43/ Fcε3能特异性下调小鼠体内IgE水平，为应性鼻炎的防治奠定了重要的基础。