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Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).
- Source :
-
Molecular immunology [Mol Immunol] 2000 May; Vol. 37 (7), pp. 361-75. - Publication Year :
- 2000
-
Abstract
- An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.
- Subjects :
- Alkylation
Allergens chemistry
Allergens isolation & purification
Amino Acid Sequence
Animals
Antigens, Plant
Baculoviridae
Base Sequence
Cats
Cell Line
Cloning, Molecular
DNA, Complementary
Dermatitis
Disease Models, Animal
Dogs
Escherichia coli
Gene Expression
Genetic Vectors
Immunoglobulin E blood
Mass Spectrometry methods
Molecular Sequence Data
Pichia
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins immunology
Recombinant Fusion Proteins isolation & purification
Spodoptera
Allergens genetics
Allergens immunology
Insect Proteins
Salivary Glands immunology
Siphonaptera immunology
Subjects
Details
- Language :
- English
- ISSN :
- 0161-5890
- Volume :
- 37
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Molecular immunology
- Publication Type :
- Academic Journal
- Accession number :
- 11074254
- Full Text :
- https://doi.org/10.1016/s0161-5890(00)00061-4