白塞病新自身抗原的筛选与应用

发布时间：2018-09-14 07:42

【摘要】：白塞病(Behcet's disease, BD)是一种可能与遗传因素、环境微生物感染、以及其他病人生活环境因素等相关的系统性自身免疫性疾病。目前已有证据表明该疾病在相对高发的亚洲人群中具有一些特殊遗传位点,但与环境微生物感染方面的相关性仍需要更多的实验证据。本课题采用五种人源细胞系对BD患者的血清自身抗体谱进行测定,通过分析不同细胞免疫荧光强度的差异,选取人脐静脉内皮细胞(HUVEC)为可用于特异性抗原分子筛选的靶细胞。然后分别采用一维分离技术(免疫印迹和免疫沉淀)和二维分离技术(双向电泳联合个体全蛋白质组生物芯片)对HUVEC细胞裂解液中的能够与BD患者血清抗体特异性识别的靶蛋白进行筛选。实验结果表明,在本课题构建的一维和二维实验体系中,分别成功筛选出一个新的疾病相关自身抗原。经过MALDI-TOF/TOF质谱进行序列鉴定,确定其分别为抗增殖蛋白Prohibitin和微管蛋白β-tubulin。然后,通过分子克隆和发酵表达,分别获得大量纯化的重组蛋白。接着制备对应的酶免检测试剂盒,进行临床验证和疾病症状关联性分析。结果表明,抗Prohibitin抗体和抗β-tubulin抗体在我国汉族BD患者的血清水平均较健康组有显著性差异(p0.0001)。本课题首次在我国汉族BD患者血清中发现抗Prohibitin抗体和抗β-tubulin抗体的存在,且上述两种新发现自身抗体所对应的抗原与微生物热激蛋白HSP65相关；提示病菌感染、病人的生活环境等的确都可能参与BD发病进程,为该疾病的发病机制和分子诊断研究提供了一定有益信息。
[Abstract]:Behcet's disease (Behcet's disease, BD) is a systemic autoimmune disease that may be associated with genetic factors, environmental microbial infections, and other environmental factors in patients' lives. There is some evidence that the disease has some special genetic loci in the relatively high incidence Asian population, but more experimental evidence is needed for the correlation between the disease and the environmental microorganism infection. In this study, five human cell lines were used to detect the serum autoantibodies of patients with BD. By analyzing the difference of immunofluorescence intensity between different cells, human umbilical vein endothelial cells (HUVEC) were selected as target cells for screening specific antigen molecules. One-dimensional separation (Western blotting and immunoprecipitation) and two-dimensional separation (two-dimensional electrophoresis combined with individual whole proteome biochip) were used to detect specific antibodies in HUVEC cell lysate to patients with BD. Target proteins for sex recognition were screened. The experimental results show that a new disease-related autoantigen was successfully screened out in the one-dimensional and two-dimensional experimental systems constructed in this paper. The antiproliferative protein Prohibitin and tubulin 尾-tubulin were identified by MALDI-TOF/TOF mass spectrometry. Then, a large number of purified recombinant proteins were obtained by molecular cloning and fermentation. Then the corresponding Elisa kit was prepared for clinical verification and disease symptom correlation analysis. The results showed that the serum levels of anti Prohibitin antibody and anti 尾 -tubulin antibody were significantly different from those of healthy controls in Chinese Han nationality patients with BD (p0.0001). In this study, the presence of anti Prohibitin antibody and anti 尾 tubulin antibody was first found in the serum of BD patients of Han nationality in China, and the antigens corresponding to these two new autoantibodies were associated with microbial heat shock protein HSP65. The living environment of the patients may be involved in the pathogenesis of BD, which provides useful information for the pathogenesis and molecular diagnosis of the disease.
【学位授予单位】：北京科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R597.9;R446.6

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