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Comparative allergenicity studies of native and recombinant Blomia tropicalis Paramyosin
(Blo t 11)
Author(s) -
Ramos J. D. A.,
Teo A. S. M.,
Ou K. L.,
Tsai L. C.,
Lee B. W.,
Cheong N.,
Chua K. Y.
Publication year - 2003
Publication title -
allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.363
H-Index - 173
eISSN - 1398-9995
pISSN - 0105-4538
DOI - 10.1034/j.1398-9995.2003.00106.x
Subject(s) - recombinant dna , microbiology and biotechnology , biology , peptide , gel electrophoresis , immunoglobulin e , western blot , allergen , antibody , biochemistry , chemistry , allergy , immunology , gene
Background:The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously. This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms. Methods: Native Blo t 11 (nBlo t 11) was isolated from crude Bt extract by immuno‐affinity chromatography, analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot, and verified by MALDI‐TOF MS. Recombinant full‐length Blo t 11 (rFL‐Blo t 11) and its immunodominant peptide (fD) were expressed as glutathione S‐transferase (GST)‐fusion proteins in Escherichia coli . Immunoglobulin E (IgE) reactivity of the Blo t 11 allergens were determined by enzyme‐linked immunosorbent assay (ELISA) and skin prick test. The inhibition capacity of the nBlo t 11 against fD and vice versa was determined by absorption studies. Results: Affinity purified nBlo t 11 was susceptible to degradation with the major degraded product resolved at ∼66 kD. The nBlo t 11 was confirmed by immunoblot analysis and MALDI‐TOF MS that generated 13 peptides with complete identity to the deduced amino acid sequence of Blo t 11. Comparative in vitro and in vivo allergenicity tests and the cross inhibition studies between the native and recombinant Blo t 11 showed that recombinant fD, but not the rFL‐Blo t 11, has comparable IgE reactivity with the native counterpart. Conclusions: This comparative study confirmed that the recombinant peptide fD contains the main immunodominant region of Blo t 11. This recombinant peptide, instead of the full‐length protein, is a good candidate for diagnostic and therapeutics development for mite allergy.