应用噬菌体展示肽技术筛选交链孢霉过敏原B细胞表位的实验研究

The prevalence of allergic diseases has increased in recent years worldwide.Alternaria is an important airborne allergen and can induce asthma, allergic rhinitis and other allergic diseases. Alternaria vaccine immunotherapy has been applicated widely in clinical practice as a disease-modified treatment, however,various factors such as allergen materials and extract technique makes it difficult for allergen vaccines to be stardardized, which had an adverse impact on the efficacy and safety of immunotherapy. Nowadays, standardized Alternaria vaccine is not yet available both in US and Europe.The aim of our study is to analysis the B cell epitopes of Alternaria antibody by phage display technique for further personalized epitope vaccine immunotherapy. In our study, Alternaria specific IgE from allergic patients and non-specific IgE from healthy subjects were utilized for positive screening and negative screening respectively. Alternaria B cell epitopes(both linear and conformational epitope) will be mapped. Before immunotherapy, the peptides from random peptide libraries will be used as probes to figure out the personalized B cell epitope. Our study is expected to provide basic data for further epitope vaccine development and personalized epitope vaccine immunotherapy.

近年来过敏性疾病发病率快速上升。交链孢霉是引起过敏性疾病的重要过敏原，用交链孢霉提取物进行脱敏治疗在临床上应用广泛。然而交链孢霉提取物受原材料和提取技术等因素的影响，在蛋白成分、含量等方面差异性大，直接影响了脱敏疗效。欧美国家目前均未实现交链孢霉疫苗的标准化。我们拟抛开对过敏原本身的研究，用交链孢霉过敏患者血清中针对交链孢霉的特异性IgE抗体为钓饵，进行由果到因的反推。通过噬菌体展示随机肽技术，用患者血清中IgE阳性筛选出能特异结合的肽，再用健康人血清中IgE进行阴性筛选排除IgE保守的重链结合肽，得到特异性和患者血清中IgE抗体高变区结合的交链孢霉B细胞线性以及构象表位肽库，绘制交链孢霉B细胞表位图谱。在脱敏治疗前先用肽库中的肽作为探针进行检测，再用相应短肽进行针对性脱敏治疗，能在过敏反应速发相中封闭IgE，阻止效应细胞活化。本研究为交链孢霉表位疫苗的研究和临床表位个性化治疗提供新思路。

交链孢霉是引起过敏性疾病的重要过敏原，用交链孢霉提取物进行脱敏治疗在临床上应用广泛。然而交链孢霉提取物标准化难度较大，直接影响了脱敏疗效。我们抛开对过敏原本身的研究，用交链孢霉过敏患者血清中针对交链孢霉的特异性IgE抗体为钓饵，进行由果到因的反推。通过噬菌体展示随机肽技术，用患者血清中IgE阳性筛选出能特异结合的肽，再用健康人血清中IgE进行阴性筛选排除IgE保守的重链结合肽，得到特异性和患者血清中IgE抗体高变区结合的交链孢霉B细胞线性以及构象表位肽库。然后体外合成交链孢霉的主要B细胞表位，在小鼠动物模型中进行肽段免疫治疗，探讨B细胞肽段治疗的疗效。我们建立了交链孢霉阳性IgE血清池，采用Ph.D.-7噬菌体展示肽技术，共筛选出52个B细胞表位，其中有2个B细胞表位在多轮次的筛选过程中均有出现，其核酸序列为ATGATCAGCACGAGTCGCAAA和CAAAAACGGAATACGATTACT，对应的氨基酸序列分别MISTSRK和QKRNTIT，相应的两种合成肽与人交链孢霉IgE有较强结合活性。同时，我们成功建立了交链孢霉致敏的Balb/c哮喘小鼠模型，并证实两个合成肽段亦可与小鼠IgE结合，继而在小鼠哮喘模型中进一步探讨了肽段免疫治疗的疗效。我们的研究结果显示肽段免疫治疗可显著减少哮喘小鼠气道及支气管肺泡灌洗液（BALF）中炎症细胞如嗜酸性粒细胞的浸润，BALF中IL-4减少，而IFN-γ增多，肺组织中IL-23 mRNA表达亦减少，外周血中IgE水平也明显下降，提示B细胞表位肽段治疗显著抑制了哮喘小鼠气道过敏性炎症，促进了哮喘小鼠炎症反应由Th2向Th1转化。本研究结果提示基于噬菌体展示肽筛选的B细胞肽段免疫治疗可以实现个体化精准化免疫治疗，并有效避免天然过敏原提取物可能出现的引起严重过敏反应的风险。本研究亦可以用于其他过敏原包括尘螨、花粉乃至食物过敏原的B细胞表位免疫治疗，有良好的应用前景。