Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies

Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A+) RNA derived from total RNA of cultured k...

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Published inThe Journal of clinical investigation Vol. 82; no. 6; pp. 1864 - 1870
Main Authors STANLEY, J. R, TANAKA, T, MUELLER, S, KLAUS-KOVTUN, V, ROOP, D
Format Journal Article
LanguageEnglish
Published Ann Arbor, MI American Society for Clinical Investigation 01.12.1988
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Abstract Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A+) RNA derived from total RNA of cultured keratinocytes was used, with oligo-dT priming, to construct a cDNA library in the lambda gt11 expression vector, which was screened by the immunoperoxidase method with one BP serum. One darkly stained clone, called here the BP clone, was further characterized. 9 of 9 BP sera, but none of 6 normal and 11 pemphigus sera, bound the plaques of this BP clone. Furthermore, BP IgG affinity purified on plaques of this clone, but not unrelated clones, bound the epidermal basement membrane by immunofluorescence and immunoprecipitated the 230-kD BP antigen from extracts of cultured keratinocytes. Eco RI digestion of the BP clone's cDNA insert demonstrated a 680- and 1,500-bp fragment. Northern blots of total keratinocyte RNA showed that complementary riboprobes transcribed from both fragments hybridized to a 9-kb RNA. Dideoxy DNA sequencing from the 5' end of the BP cDNA demonstrated a 1,992-bp open reading frame, encoding a peptide of 76 kD. This BP cDNA clone will be valuable for understanding the protein structure, expression, and gene organization of BP antigen.
AbstractList Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A+) RNA derived from total RNA of cultured keratinocytes was used, with oligo-dT priming, to construct a cDNA library in the lambda gt11 expression vector, which was screened by the immunoperoxidase method with one BP serum. One darkly stained clone, called here the BP clone, was further characterized. 9 of 9 BP sera, but none of 6 normal and 11 pemphigus sera, bound the plaques of this BP clone. Furthermore, BP IgG affinity purified on plaques of this clone, but not unrelated clones, bound the epidermal basement membrane by immunofluorescence and immunoprecipitated the 230-kD BP antigen from extracts of cultured keratinocytes. Eco RI digestion of the BP clone's cDNA insert demonstrated a 680- and 1,500-bp fragment. Northern blots of total keratinocyte RNA showed that complementary riboprobes transcribed from both fragments hybridized to a 9-kb RNA. Dideoxy DNA sequencing from the 5' end of the BP cDNA demonstrated a 1,992-bp open reading frame, encoding a peptide of 76 kD. This BP cDNA clone will be valuable for understanding the protein structure, expression, and gene organization of BP antigen.
Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A super(+)) RNA derived from total RNA of cultured keratinocytes was used, with oligo-dT priming, to construct a cDNA library in the lambda gt11 expression vector, which was screened by the immunoperoxidase method with one BP serum. This BP cDNA clone will be valuable for understanding the protein structure, expression, and gene organization of BP antigen.
Author MUELLER, S
KLAUS-KOVTUN, V
STANLEY, J. R
TANAKA, T
ROOP, D
AuthorAffiliation Dermatology Branch, National Cancer Institute, Bethesda, Maryland 20892
AuthorAffiliation_xml – name: Dermatology Branch, National Cancer Institute, Bethesda, Maryland 20892
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  surname: STANLEY
  fullname: STANLEY, J. R
  organization: NIH, national cancer inst., Bethesda MD 20892, United States
– sequence: 2
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  surname: TANAKA
  fullname: TANAKA, T
  organization: NIH, national cancer inst., Bethesda MD 20892, United States
– sequence: 3
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  surname: MUELLER
  fullname: MUELLER, S
  organization: NIH, national cancer inst., Bethesda MD 20892, United States
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  surname: KLAUS-KOVTUN
  fullname: KLAUS-KOVTUN, V
  organization: NIH, national cancer inst., Bethesda MD 20892, United States
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  surname: ROOP
  fullname: ROOP, D
  organization: NIH, national cancer inst., Bethesda MD 20892, United States
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Issue 6
Keywords Bullous dermatosis
Human
Immunopathology
Skin disease
Nucleotide sequence
Autoantibody
Autoimmune disease
Gene library
Open reading frame
Antigen
Complementary DNA
Bullous pemphigoid
Genetic engineering
Molecular cloning
Aminoacid sequence
Language English
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PMID 2461961
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PublicationDate 1988-12-01
PublicationDateYYYYMMDD 1988-12-01
PublicationDate_xml – month: 12
  year: 1988
  text: 1988-12-01
  day: 01
PublicationDecade 1980
PublicationPlace Ann Arbor, MI
PublicationPlace_xml – name: Ann Arbor, MI
– name: United States
PublicationTitle The Journal of clinical investigation
PublicationTitleAlternate J Clin Invest
PublicationYear 1988
Publisher American Society for Clinical Investigation
Publisher_xml – name: American Society for Clinical Investigation
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Snippet Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of...
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StartPage 1864
SubjectTerms Amino Acid Sequence
Autoantibodies
Autoantigens - genetics
Autoantigens - immunology
Base Sequence
Biological and medical sciences
Bullous diseases of the skin
Carrier Proteins
Collagen
Collagen Type XVII
Cytoskeletal Proteins
Dermatology
DNA - isolation & purification
Dystonin
Humans
Immunoenzyme Techniques
Medical sciences
Molecular Sequence Data
Nerve Tissue Proteins
Non-Fibrillar Collagens
Pemphigoid, Bullous - genetics
Pemphigoid, Bullous - immunology
RNA - analysis
Skin Diseases, Vesiculobullous - genetics
Title Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies
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Volume 82
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