| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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
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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
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