Molecular biological studies of the mechanism of the development of vasculitis in Kawasaki disease.

川崎病(KD)的病因和发病机制尚不清楚。此前，我们曾报道在KD患者的血清中检测到抗人冠状动脉(SMC)平滑肌细胞70 kDa蛋白的自身抗体。在本研究中，我们试图通过免疫筛选表达文库来鉴定与循环中抗SMC自身抗体反应的特异性抗原(S)。1)表达文库的免疫筛选：从来源于SMC的文库(Uni-ZAP cDNA文库，Stratagene)中约3×105个斑块用于筛选与4例KD患者血清具有免疫反应的克隆，这些克隆产生的自身抗体在初步的Western免疫印迹中呈强阳性。用马萝卜过氧化氢偶联的兔抗人IgA和IgM(DAKO)分别用第二抗体检测结合的自身抗体。对文库进行了3次筛选，阳性克隆为…更多的DNA序列分析：通过体内切除的方法将克隆的噬菌体DNA转化为质粒DNA。以这些质粒DNA为模板，以T3和T7为特异性引物，用ABI Prism 310型全自动测序仪对克隆的每个插入片段的核苷酸序列进行分析。使用GenBank序列数据库进行BLAST分析，搜索相应的蛋白质。结果：经筛选获得8个阳性克隆。以免疫球蛋白A和免疫球蛋白M作为第二抗体，分别分离到7个克隆和2个克隆。免疫球蛋白A和免疫球蛋白M均分离到1个克隆。虽然鉴定出的蛋白质大小差异很大，但有三个蛋白质的大小约为70 kDa。此外，从不同KD患者的血清中独立分离出两种蛋白质，其中一种大小约为70 kDa。结论：这些蛋白中的一部分可能是KD患者血清中抗自身抗体的特异性抗原(S)，这可能有助于了解自身抗体导致KD患者系统性血管炎的机制。需要进一步的研究来确定KD中循环自身抗体所识别的特异性抗原。较少

The etiology and pathogenesis of Kawasaki disease (KD) remain unknown. Previously we reported that auto-antibodies against a 70 kDa protein from smooth muscle cells derived from human coronary artery (SMC) were detected in the sera of patients with KD. In this study, we tried to identify the specific antigen(s) that react with circulating auto-antibodies against SMC by immunoscreening of the cDNA expression library.1)Immunoscreening of a cDNA expression library : Approximately 3×10^5 plaques from the cDNA library derived from SMC (Uni-ZAP cDNA library, Stratagene) were used to screen for clones showing immunoreactivity with sera from four patients with KD, which yielded auto-antibodies that were strongly positive in preliminary Western immunoblots. Bound auto-antibodies were detected using a second antibody separately with horse-radish peroxide-conjugated rabbit anti-human IgA and IgM (Dako). The screening of the cDNA library were performed three times, and positive clones were isolate … More d. 2)DNA sequence analysis : Cloned phage DNAs were converted into plasmid DNAs using the methods of the in vivo excision. PCR reactions were performed using these plasmid DNAs as templates and both T3 and T7 as specific primers, and then the nucleotide sequences of each cloned cDNA insertion were analyzed with an automated sequencer ABI Prism 310 Genetic Analyzer. Corresponding proteins were searched by Blast analysis using the GenBank sequence database. Results : Eight positive clones were identified by the screenings. Seven clones and two clones were isolated using IgA and IgM as the second antibody, respectively. One clone was isolated by both IgA and IgM. Although the sizes of the identified proteins varied widely, three proteins were around 70kDa. In addition, two proteins including one around 70kDa in size were independently isolated from the sera of different patients with KD. Conclusions : Our results suggest that some of these proteins may be a specific antigen(s) against the auto-antibodies in the sera of KD patients and it may be useful for understanding the mechanisms by which auto-antibodies may cause the systemic vasculitis in KD. Further studies will be required to definite the specific antigen recognized by the circulating auto-antibodies in KD. Less