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

我们使用合适的引物和从培养的角质形成细胞mRNA中制备的cDNA文库进行PCR扩增，获得了覆盖整个BP230的3个cDNA。我们将这些cdna插入细菌表达载体pGEX中，产生GST的重组融合蛋白。利用这些重组蛋白的免疫印迹，我们证实了大疱性类天疱疮（BP）血清与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，我们还采用相同的方法检测了非BP血清与BP230重组蛋白的反应性。3种重组蛋白与极少数非bp患者血清的反应非常弱。这一结果表明BP血清与BP230特异性反应，尽管自身抗体与BP230的致病作用尚不清楚。通过类似的方法，我们还获得了3个重组GST融合蛋白，它们覆盖了BP180的整个胞外结构域以及…More一个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血清与BP180 n端区域的NC16a结构域特异性反应。我们还发现，瘢痕性类天疱疮血清中含有与BP180 c端区反应的IgG和IgA。此外，与BP表位相比，透明层型线性IgA大疱性皮肤病（LAD）的血清似乎与BP180的n端区域的表位发生反应，该区域距离c端稍远。因此，我们制备了一种覆盖BP180第15胶原结构域（C15）的重组蛋白。近一半的LAD血清与该重组蛋白特异性反应。这表明至少一些LAD血清含有与C15结构域表位反应的IgA抗体。此外，我们利用BP180 NC16a结构域的重组蛋白建立了一种新的ELISA，目前我们正在检测多种自身免疫性大疱性疾病的血清，以证实该新ELISA的特异性。利用BP180和BP230的不同重组蛋白进行免疫印迹和酶联免疫吸附试验，对多种自身免疫性大疱性皮肤病基底膜带抗体的明确诊断具有重要意义。少

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