低致敏性蟑螂过敏原Per a 10 的研究

Specific immunotherapy (SIT) has been shown to be only disease-modifying and antigen-specific treatment of allergy. However, the use of crude allergen extracts as active components of allergy vaccines limits the broad application of SIT. Hypoallergenic allergy vaccine was contructed by genetic engineering to reduce allergenic activity. American cockroach is one of major indoor allergenic resourse. Per a 10 is a major allergen in American cockroach. We used bioinformatic analysis,ELISA,basophil activation test and T cell proliferation assay to determine the major T cell and Ig E binding epitopes of Per a 10. Per a 10 mutations were obtained either by mutation of the amino acid residues invovled in Ig E-binding, or by the disruption of the three-dimensional structure of the Per a 10. The Ig E binding activities of Per a 10 mutations were compared by immuno-blot, skin prick test, basophil activation test. Antigenicities of Per a 10 mutations were compared by T cell proliferation assays. The Per a 10 specific IgG antibody response were determined by animal immunization experiments. Combined these, we selected a Per a 10 mutation to be a potential allergen vaccine candidate.

特异性免疫治疗（脱敏治疗）被认为是当前过敏性疾病唯一的对因治疗方法。目前使用的标准化脱敏疫苗有诱导IgE介导的过敏反应的危险。通过基因操作进行过敏原改造,减少其致敏性,保留其免疫原性。这种疫苗称为低致敏过敏原疫苗。美洲大蠊是一种主要室内过敏源。Per a 10 是美洲大蠊主要过敏原。我们采用生物信息学软件分析，ELISA，嗜碱性粒细胞激发实验和T细胞增殖实验验证,明确鉴定出Per a 10的主要T细胞表位和IgE结合表位。在此基础上采用Ig E结合表位点突变和构象改变突变两个方法得到低致敏过敏原Per a 10候选重组蛋白。采用免疫印迹，ELISA，皮试，嗜碱性粒细胞激发实验比较它们的Ig E结合活性。采用T细胞增殖实验比较它们的抗原性。动物致敏实验比较它们诱导保护性抗体Ig G的能力。从而筛选出一个能保持抗原性，但是Ig E结合活性明显下降，同时表达体系能够稳定表达的突变体做为候选疫苗

Per a 10是美洲大蠊中的主要过敏原蛋白，国内外对此研究很重视。我们对Per a 10的B表位和T表位进行了深入研究，我们首先克隆出蟑螂过敏原基因，亚克隆到pET-22b(+)表达质粒中，转化到大肠杆菌Rosette-gami中，诱导表达出Per a10蛋白，纯化后再变性复性得到重组Per a10蛋白。构建了Per a 10的三级结构，通过运用DNAStar软件的Protean模块、 在线预测工具Bioinformatics Predicted Antigenic Peptides (BPAP)和BepiPred 1.0 server三种方法对Per a 10的抗原B表位进行预测，得到Per a 10B细胞表位为2-12，55-67，98-120，125-133，149-160，170-182，201-208和223-227。选择NetMHCIIpan-3.0方法对HLA-DR型的T表位进行预测，而对于HLA-DQ基因型的T表位预测，使用NetMHCII-2.2方法。预测出了2个基于HLA-DR基因型的T表位：81-92和139-147。预测基于HLA-DQ基因型的T表位为：81-92和162-170。所以最终的预测出来的T表位为：81-97，139-147和162-170。我们根据预测的表位合成多肽，正在临床验证中。本课题研究以第一或通讯作者共发表SCI收录论文6篇、已接受SCI收录2篇，修回1篇。共同作者发表SCI收录论文2篇（均标注该基金支持）。培养硕士研究生1名，在读硕士生1名。