负调控变态反应融合分子构建及其作用机制研究

IgE/FcεRI signaling pathway has become a target for developing drugs of anti-allergic disease. A new peptide combined with IgE was acquired by phage library display technique,which named A6(antagonistic peptides 6). A6 can conjugate with both free IgE and membranes IgE on the effected cell surface, and block the IgE/FcεRI signaling pathway. The character of A6 is unlike others, which has not been reported. According to the feature, FcεRI with FcγRIIB will be enable cross-linking through A6. The IgG constant region (CHγ2-CHγ3) was selected to bind with FcγRIIB, activating the negative regulation in other studies. But the FcγRII belong to low affinity receptors, its affinity with IgG is lower and effect much later than other higher affinity receptor FcγR. Therefore, FcγRIIB binding peptides was select from phage display peptide library,which was fused with the IgE binding peptide A6.The fusion molecules can be specifically combined with FcγRIIB to cross-link FcεRI and FcγRIIB, block IgE/FcεRI signaling pathway. Otherwise A6 can be combined with membranes IgE on the effected cell surface, and thus indirectly affect the FcεRI receptor. The fusion molecules can possible only negative regulate mast cells and basophils integrateed with IgE, but not normal mast cells and basophils. The fusion protein will be constructed and its activity and mechanisms of anti-allergic at the cellular level and animal level will be studied. At the same time, A6 and IgE molecules binding epitope will be analyzed by computer modeling and deletion mutants of IgE. New negative regulator of signaling molecules for allergic diseases will be provided through genetic engineering, as well as the clues to utilize other small peptides or peptide analogs will be discovered from infoumation of the A6 and IgE complexes in this study.

阻断IgE/FcεRI信号通路己经成为抗变态反应性疾病治疗的研究热点。我们利用噬菌体肽库展示技术筛选到与IgE稳定结合的12肽分子（A6），初步试验证实结合肽A6可同时与游离IgE或效应细胞膜表面IgE相结合，阻断IgE/FcεRI信号通路。以此为契机拟构建具有双功能活性的IgE结合肽与抑制性受体FcγRIIB结合肽的融合分子，可特异性与FcγRIIB结合，同时摒弃了直接偶联FcεRI与FcγRIIB的弊端，仅负调控已结合IgE的效应细胞，达到阻断IgE/FcεRI信号通路的目的。本研究主要在细胞水平和动物水平对融合蛋白抗变态反应活性和相关机制进行研究，同时，利用计算机模建和构建缺失突变体的方法分析结合肽A6与IgE分子结合表位。本研究将为利用基因工程构建的负调控信号分子治疗变态反应性疾病提供新的方法，同时为揭示结合肽A6与IgE复合物的空间构象，开发其他小肽或肽似物提有意义的线索。

变态反应性疾病已经成为一种常见病和流行病，但目前仍没有肯定的治愈方法。以IgE/FcεRI信号通路为靶点设计药物，阻断IgE与其高亲和力受体（FcεRI）的结合及下游信号传递己经成为抗变态反应性疾病药物的研究热点。近年来，利用免疫负调控作用，抑制肥大细胞和嗜碱粒细胞释放炎性介质和细胞因子，为变态反应性疾病的免疫治疗开辟了新的途径，其中FcγRIIB受体介导的免疫负调控作用逐渐收到众多学者的关注。本项目首先成功克隆Cε3-Cε4和FcγRIIB基因，制备了重组的高纯度Fcε3-4蛋白和FcγRIIB胞外区蛋白。利用噬菌体随机多肽展示技术，采用固相筛选法，分别筛选Cε3-Cε4蛋白和FcγRIIB胞外区蛋白结合肽。通过对固相筛选法中靶分子浓度、作用温度、去污剂及淘选轮数等关键环节进行优化，建立了适合Cε3-Cε4蛋白和FcγRIIB胞外区蛋白的噬菌体随机多肽展示技术体系，获得了高特异性、高亲和力的Cε3-Cε4蛋白结合肽P20（A6）以及FcγRIIB胞外区结合肽P16，并对所获结合肽生物学活性进行了分析。同时，构建了稳定表达FcγRIIB的CHO细胞株，为评价FcγRIIB蛋白结合肽功能提供条件。为评价融合肽ε-γ功能，本实验利用计算机辅助设计并结合具体实验，对连接肽（linker）进行优化，最终选择(G-S-G)6作为linker，将IgE结合肽P20（A6）与FcγRIIB结合肽P16融合。以DNP-IgE和DNP-BSA为变应原直接诱导动物变态反应，建立快速PCA模型；以RBL-2H3细胞为基础，以DNP-IgE和DNP-BSA为变应原建立了肥大细胞脱颗粒模型。体内、外验证了融合蛋白ε-γ具有双功能活性，可抑制变态反应进程，且对正常肥大细胞不引发脱颗粒等变态反应。利用计算机辅助模建结合缺失突变体分析，发现P20可能通过氢键与Cε3-Cε4在ARG-342、SER-432、GLU-472位置结合。此外，制备了稳定表达人FcεRIα的RBL-2H3，证实了人、鼠IgE与其受体结合具有种属特异性，鼠IgE可与人IgE受体结合，而人IgE不能与鼠IgE受体结合。本项目结果将为利用基因工程构建负调控信号分子治疗变态反应性疾病提供参考，同时也为开发其他小肽或肽似物提有意义的线索。