112 Lab, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China.
Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Key Laboratory of Rheumatology and Clinical Immunology, Ministry of Education, Beijing, China
Biosci Rep. 2018 Jan 19;38(1). doi: 10.1042/BSR20170778. Print 2018 Feb 28.
The autoantibodies profile of Behçet's disease (BD) is yet incompletely understood. Annexins are a family of highly conserved proteins which are involved in some human autoimmune diseases. Autoantibodies directed toward Annexin A1 and A2 are involved in BD pathology, but correlation in their clinical role is controversial. The aim of our study is to estimate and evaluate the expression correlation between Annexin A1 and A2 autoantibodies in BD patients. We have designed and implemented different technical approaches to prove the hypothesis. First, bioinformatics tools such as amino acid sequence alignment, epitope prediction analysis, and 3D structural comparison were performed to find out the correlation between Annexin A1 and A2. Second, amplification of the corresponding gene by RT-PCR, then cloning, and purification techniques were applied to acquire the recombinant Annexin A1. Third, the target protein band was excised from gel electrophoresis, digested with trypsin, and analyzed by MALDI-TOF/TOF. Finally, in-house ELISA was developed to determine the induced anti-Annexin A1 autoantibodies in BD patients. Obtained results demonstrated that the BD serum reactivity against recombinant Annexin A1 was significantly higher as compared with healthy control (HC) (<0.001). Moreover, bioassay results of Annexin A1 and A2 also showed the presence, absence, and independent coexistence of autoantibodies, when reacted with BD sera. In conclusion, Annexin A1 has a similar immunogenic expression and correlation with its analog Annexin A2 and their association may be a novel immune target of BD in Han Chinese population.
贝赫切特病（BD）的自身抗体谱尚未完全理解。膜联蛋白是一类高度保守的蛋白质，参与了一些人类自身免疫性疾病。针对膜联蛋白 A1 和 A2 的自身抗体参与了 BD 的病理过程，但它们在临床作用中的相关性存在争议。我们的研究旨在评估和评估 BD 患者中膜联蛋白 A1 和 A2 自身抗体的表达相关性。我们设计并实施了不同的技术方法来验证假设。首先，进行了生物信息学工具，如氨基酸序列比对、表位预测分析和 3D 结构比较，以找出膜联蛋白 A1 和 A2 之间的相关性。其次，通过 RT-PCR 扩增相应的基因，然后应用克隆和纯化技术获得重组膜联蛋白 A1。第三，从凝胶电泳中切下靶蛋白条带，用胰蛋白酶消化，并用 MALDI-TOF/TOF 进行分析。最后，开发了内部 ELISA 来确定 BD 患者中诱导的抗膜联蛋白 A1 自身抗体。获得的结果表明，BD 血清对重组膜联蛋白 A1 的反应性明显高于健康对照组（HC）（<0.001）。此外，膜联蛋白 A1 和 A2 的生物测定结果还表明，当与 BD 血清反应时，自身抗体存在、不存在和独立共存。总之，膜联蛋白 A1 具有与类似物膜联蛋白 A2 相似的免疫原性表达和相关性，其相关性可能是汉族人群中 BD 的一个新的免疫靶标。
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