Partial cDNA cloning of the 230-kD mouse bullous pemphigoid antigen by use of a human monoclonal anti-basement membrane zone antibody.
A cDNA clone with the coding sequence for the 230-kD bullous pemphioid (BP) antigen was isolated from a mouse cDNA expression library by using an anti-basement membrane zone human monoclonal antibody (MoAb-5E). The lambda gt11 cDNA expression library was constructed from poly(A)+RNA from the mouse epidermal cell line, Pam cells, by random priming. 1.5 X 10(5) recombinant clones were screened by immunostaining with MoAb-5E and one positive clone (BPM1) was obtained. All of the ten BP sera but none of the five normal or seven pemphigus sera tested reacted with the fusion protein produced by BPM1. The size of the cDNA was 3.2 kb. Northern blot analysis indicated that BPM1 cDNA hybridized to a mRNA of about 9 kb, which is large enough to encode for a 230-kD protein. DNA sequencing demonstrated a 2,991-bp open reading frame encoding a peptide of 115 kD. Sequence comparison between mouse and human cDNA clones revealed that the 230-kD BP antigen was well conserved during evolution except for the carboxyl terminus. Highly conserved and hydrophilic regions in the molecule were considered to be good candidates for antigenic determinants. This cDNA clone will be useful not only for diagnosis of BP, e.g., enzyme-linked immunosorbent assay using recombinant proteins or synthetic peptides as antigens, but also for pathophysiologic study in which mouse models of BP might be used.
Department of Dermatology, Keio University School of Medicine, Tokyo, Japan.
M
M
Amagai
T
T
Hashimoto
S
S
Tajima
Y
Y
Inokuchi
N
N
Shimizu
M
M
Saito
K
K
Miki
T
T
Nishikawa
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000276:immunology; D001483:Base Sequence; D001485:Basement Membrane / Q000276:immunology*; D015152:Blotting, Northern; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D010391:Pemphigoid, Bullous / Q000276:immunology*; D015183:Restriction Mapping; D012872:Skin Diseases, Vesiculobullous / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens