Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen

Miau-Yaun Wang, M. F. Lee, C. H. Wu

Research output: Contribution to journalArticle

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Abstract

Background: Previously, we have identified several Per a 1 (Cr-PII) allergens from a λgt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties. Methods: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE. Results: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino-acid protein (50 kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42. Conclusions: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE-reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat. The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies.

Original languageEnglish
Pages (from-to)119-127
Number of pages9
JournalAllergy: European Journal of Allergy and Clinical Immunology
Volume54
Issue number2
DOIs
Publication statusPublished - 1999 Apr 13

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Periplaneta
Allergens
Immunoglobulin E
Recombinant Proteins
Amino Acids
Epitopes
Complementary DNA
Blattellidae
Enzyme-Linked Immunosorbent Assay
Monoclonal Antibodies
Epitope Mapping
Peptides
Ion Exchange Chromatography
Sequence Homology
Gene Library
Affinity Chromatography
Immunoblotting
Digestion
Peptide Hydrolases
Nucleotides

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology

Cite this

@article{4287bc94b2174b5188fbcb87d6e2f7ac,
title = "Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen",
abstract = "Background: Previously, we have identified several Per a 1 (Cr-PII) allergens from a λgt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties. Methods: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE. Results: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino-acid protein (50 kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4{\%} (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42. Conclusions: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE-reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat. The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies.",
author = "Miau-Yaun Wang and Lee, {M. F.} and Wu, {C. H.}",
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T1 - Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen

AU - Wang, Miau-Yaun

AU - Lee, M. F.

AU - Wu, C. H.

PY - 1999/4/13

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N2 - Background: Previously, we have identified several Per a 1 (Cr-PII) allergens from a λgt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties. Methods: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE. Results: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino-acid protein (50 kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42. Conclusions: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE-reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat. The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies.

AB - Background: Previously, we have identified several Per a 1 (Cr-PII) allergens from a λgt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties. Methods: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE. Results: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino-acid protein (50 kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42. Conclusions: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE-reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat. The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies.

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