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。以前，我们报道了从人冠状动脉（SMC）平滑肌细胞来源的70 kDa蛋白的自身抗体检测KD患者的血清中。在本研究中，我们试图通过cDNA表达文库的免疫筛选来鉴定与针对SMC的循环自身抗体反应的特异性抗原。1）cDNA表达文库的免疫筛选：来自SMC cDNA文库的约3×10^5个噬斑（Uni-ZAP cDNA文库，Stratagene）用于筛选显示与来自四名KD患者的血清的免疫反应性的克隆，其产生的自身抗体在初步的Western免疫印迹中是强阳性的。使用第二抗体分别用辣根过氧化物偶联的兔抗人伊加和IgM（Dako）检测结合的自身抗体。对cDNA文库进行三次筛选，筛选出阳性克隆 ...更多信息 D. 2)DNA序列分析：采用体内切除法将克隆的噬菌体DNA转化为质粒DNA。以这些质粒DNA为模板，以T3和T7为特异性引物进行PCR反应，然后用自动测序仪ABI Prism 310 Genetic Analyzer分析每个克隆的cDNA插入的核苷酸序列。利用GenBank序列数据库通过Blast分析搜索相应的蛋白。结果：筛选出8个阳性克隆。分别用伊加和IgM作为第二抗体分离了7个克隆和2个克隆。一个克隆通过伊加和IgM分离。虽然所鉴定的蛋白质的大小变化很大，但有三种蛋白质在70 kDa左右。另外，从不同KD患者血清中分离到两种蛋白，其中一种分子量约为70 kDa。结论：这些蛋白质可能是KD患者血清中自身抗体的特异性抗原，对了解自身抗体引起KD系统性血管炎的机制具有重要意义。川崎病循环自身抗体识别的特异性抗原有待进一步研究。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