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

以培养的角质形成细胞的mRNA为模板，用合适的引物和文库进行聚合酶链式反应，获得了覆盖BP230全长的3条cDNAs。我们将这些基因插入到细菌表达载体pGEX中，获得重组融合蛋白GST，通过免疫印迹实验证实大疱性类天疱疮(BP)血清与BP230分子上的多个表位发生反应，特别是在C末端区域。此外，为了检测BP血清与BP230的特异性反应性，我们还用同样的方法检测了非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患者血清反应很弱。通过类似的方法，我们还获得了3种重组GST融合蛋白，它们覆盖了BP180和…的整个胞外区进一步鉴定为BP180 NC16a结构域的重组蛋白。通过对这些重组蛋白的免疫印迹分析，我们首先证实了BP血清与BP180 N端的NC16a结构域发生了特异性的反应。我们还发现，瘢痕性类天疱疮患者血清中含有与BP180 C端区结合的Ig G和Ig A。此外，与BP180的表位相比，LAD的血清似乎与BP180的N端的表位发生了反应。因此，我们额外制备了一个覆盖BP180第15个胶原区(C15)的重组蛋白。近一半的LAD血清与该重组蛋白发生特异性反应。此外，我们利用BP180 NC16a结构域的重组蛋白建立了一种新的ELISA法，并检测了许多不同类型的自身免疫性大疱病患者的血清，以证实这种新的ELISA的特异性。利用BP180和BP230的各种不同的重组蛋白进行的免疫印迹和ELISA法分析对于利用抗基底膜带抗体对各种自身免疫性大疱性皮肤病进行明确诊断是非常重要的方法。较少

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