Analysis of autoimmune bullous diseases using recombinant proteins of BP230 and BP180

使用 BP230 和 BP180 重组蛋白分析自身免疫性大疱性疾病

• 批准号: 11670857
• 负责人: KOMAI Ayako
• 金额: $ 2.24万
• 依托单位: Kurume University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670857/
• 关键词: Autoimmune disease; Bullous pemphigoid; hemidesmosome; Recombinant protein; Immunoblotting; ELISA; BP230; BP180; 自己免疫性天疱瘡; 自己抗体; 腫瘍随伴性天疱瘡

We have obtained 3 cDNAs covering entire BP230 by PCR amplification using appropriate primers and cDNA library prepared from mRNA of cultured keratinocytes. We inserted these cDNAs into bacterial expression vector, pGEX, and produced recombinant fusion proteins to GST.Using immunoblotting of these recombinant proteins, we confirmed that bullous pemphigoid (BP) sera react with multiple epitopes on the BP230 molecule, particularly on the C-terminal region. In addition, to examine the specific reactivity of BP sera with BP230, we also examined the reactivity of non-BP sera with BP230 recombinant proteins by the same method. The 3 recombinant proteins were reacted very weakly by very few sera of non-BP patients. This result suggests that BP sera specifically react with BP230, although the pathogenic role of autoantibodies with BP 230 is still unclear.By the similar method, we also obtained 3 recombinant GST fusion proteins, which covered the entire extracellular domain of BP180 as well as … More a recombinant protein of BP180 NC16a domain. Using immunoblotting of these recombinant proteins, we first confirmed that BP sera specifically react with NC16a domain in the N-terminal region of BP180. We also found that cicatricial pemphigoid sera contain IgG and IgA reactive with C-terminal region of BP180. In addition, the sera of linear IgA bullous dermatosis (LAD) of lamina lucida type seem to react with epitopes on the N-terminal region of BP180, which is a little distal to C-terminal, if compared to the epitope for BP.Therefore, we prepared an additional recombinant protein covering the 15th collagenous domain (C15) of BP180. Nearly half of LAD sera specifically reacted with this recombinant protein. This result suggests that at least some LAD sera contain IgA antibodies reactive with epitopes within C15 domain.In addition, we established a novel ELISA using the recombinant protein of BP180 NC16a domain, and we are now examining many sera from various types of autoimmune bullous diseases to confirm the specificity of tthis new ELISA.These immunoblot and ELISA analyses using various different recombinant proteins of both BP180 and BP230 should be very important methods to obtain definite diagnoses for various autoimmune bullous dermatosis with anti-basement menbrane zone antibodies. Less

我们用合适的引物和培养的角质形成细胞的mRNA制备的cDNA文库通过PCR扩增获得了3个覆盖整个BP230的cDNA。我们将这些cDNA插入细菌表达载体pGEX中，制备了与GST融合的重组融合蛋白，用免疫印迹法证实了大疱性类天疱疮（BP）血清可与BP230分子上的多个表位反应，特别是在C端区域。此外，为了检测BP血清与BP230的特异性反应性，我们还通过相同的方法检测了非BP血清与BP230重组蛋白的反应性。这3种重组蛋白与极少数非BP患者血清的反应非常弱。本实验还利用类似的方法获得了3个GST融合蛋白，它们分别覆盖了BP 180的整个胞外区和BP 230的胞外区，并与BP 180的胞外区融合。 ...更多信息 BP180 NC16a结构域的重组蛋白。利用这些重组蛋白的免疫印迹，我们首次证实BP血清特异性地与BP 180的N-末端区域的NC16a结构域反应。我们还发现，瘢痕性类天疱疮血清中含有与BP 180的C-末端区域反应的IgG和IgA。另外，透明层型线状IgA大疱性皮肤病（LAD）患者血清似乎与BP 180的N端区域的表位反应，如果与BP的表位相比，该N端区域稍微远离C端。近一半的LAD血清与该重组蛋白特异性反应。本研究还利用BP180NC16a结构域重组蛋白建立了一种新的ELISA方法，我们现在正在检测来自各种类型的自身免疫性大疱病的许多血清，以证实这种新的ELISA的特异性。BP230对各种抗基底膜带抗体的自身免疫性大疱性皮肤病的确诊具有重要意义。少

项目成果

Komai A, Hashimoto T et al: "Anti-desmoglein autoantibodies in silicosis patients with no bullous diseases"Dermatology. (in press).

Komai A、Hashimoto T 等人：“无大疱性疾病的矽肺患者中的抗桥粒芯糖蛋白自身抗体”皮肤科。

Hashimoto T et al: "Clinical significance of enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid"J Dermatol Sci. (in press).

Hashimoto T 等人：“酶联免疫吸附测定法检测大疱性类天疱疮患者循环抗 BP180 自身抗体的临床意义”J Dermatol Sci。

Komai A, Hashimoto T et al: "The clinical transition between pemphigus foliaceus and pemphigus vulgaris correlates well with the changes in autoantibody profile assessed by an enzyme-linked immunosorbent assay"Br J Dermatol. (in press).

Komai A、Hashimoto T 等人：“落叶型天疱疮和寻常型天疱疮之间的临床转变与通过酶联免疫吸附测定评估的自身抗体谱的变化密切相关”Br J Dermatol。

Hashimoto T et al: "Bullous pemphigoid sera react specifically with various domains of BP230, most frequently with C-terminal domain, by immunoblot analysis using bacterial recombinant proteins covering the entire molecule"Exp Dermatol. (in press).

Hashimoto T 等人：“通过使用覆盖整个分子的细菌重组蛋白进行免疫印迹分析，大疱性类天疱疮血清与 BP230 的各个结构域（最常见的是 C 末端结构域）发生特异性反应”Exp Dermatol。

Chen LS et al: "Epitope spreading: lessons from autoimmune skin diseases"J Invest Dermatol. 110. 103-109 (1998)

Chen LS 等人：“表位传播：自身免疫性皮肤病的教训”J Invest Dermatol。