Molecular cloning of canine bullous pemphigoid antigen 2 cDNA and immunomapping of NC16A domain by canine bullous pemphigoid autoantibodies.

摘要

The autoantibody-mediated subepidermal blistering skin disease bullous pemphigoid affects both humans and dogs. We previously demonstrated that canine bullous pemphigoid patient's autoantibodies targeted skin basement membrane component and a 180-kDa keratinocyte protein. We extend our works to partially isolate the cDNA encoding canine bullous pemphigoid antigen 2 (BPAg2, BP180). Total RNA extracted from a papillomavirus-immortalized canine keratinocyte cell line and a cultured canine squamous carcinoma cell line SCC 2/88 were used to isolate fragments of cDNA encoding BPAg2 by reverse transcription-PCR and 5'-rapid amplification of cDNA end. The isolated sequence included the 5'-untranslated region, the entire intracellular, transmembranous, and extracellular NC16A autoantigenic domains, plus a small segment of the collagenous domain. Sequence analyses of the isolated cDNA showed 87 and 85% identities between canine and human at the nucleotide sequence and at the deduced amino acid sequence levels, respectively. The canine BPAg2 sequence was confirmed by a rabbit antibody raised against a 18-amino acid peptide deduced from the canine NC16A nucleotide sequence. Autoantibodies from canine bullous pemphigoid patients' sera recognized epitopes within the human NC16A domain. The cloning of the cDNA encoding this disease-associated protein may allow us to develop a canine model in dissecting the immunopathologic mechanism underlying bullous pemphigoid.