| Co-Investigator(Kenkyū-buntansha) |
MIYASATO Minoru Assistant Professor, Dermatology, Kurume University, 医学部, 講師 (50182001)
MOIRI Osamu Associate Professor, Dermatology, Kurume University, 医学部, 助教授 (10175630)
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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 reacted with multiple epitopes on the BP230 molecule, and that this reactivity is specific to BP. By the similar method, we also obtained 3 recombinant GST fusion proteins, which covered the entire extracellular domain of BP180. Using immunoblotting of these recombinant proteins, we confirmed that BP sera specifically react with NC16a domain in the N-terminal region of BP180, and that cicatricial pemphigoid sera contain IgG and IgA reactive with C-terminal region of BP180. In addition, the sera of linear IgA bulloud dermatosis of lamina lucida type seem to reacti with epitope on the N-terminal region of BP180, which is a li
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ttle distal to C-terminal, if compared to the epitope for BP. Next, we obtained recombinant proteins of NC1 and NC2 domains of type VII collagen. By immunoblotting using these recombinant proteins, we found that both epidermolysis bullosa acquista and bullous SLE react preferentially with NC1 domain, and less frequently with NC2 domain of type Vli collagen. Linear IgA bullous dermatosis of sublamina densa type never reacted with either recombinant proteins. These immunoblot analyses using various different recombinant proteins of both BP180, BP230 and type VII collagen should be very important methods to obtain a definite diagnosis for various autoimmune bullous dermatosis with anti-basement membrane zone antibodies. We are now attempting to establish ELISA using these recombinant proteins, which will be much more convenient method for the diagnosis of these disease than the conventional immunofluorescence tests. Finally, we are now attempting to identify the 2000 kDa new deraml protein, which is reacted by some patients of psoriasis showing bullous lesions vesivular pemphigoid-like patients. The studies performed so far (cDNA cloning and various biological studies) revealed that this molecule may be related with type VII collagen or lamine gamma 1, or may be a new type of collagen. We will continue to work with this protein. Less
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