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Sequence analysis and expression of a cDNA clone encoding a 98‐kDa allergen in Dermatophagoides farinae
Author(s) -
Tsai L.C.,
Sun Y.C.,
Chao P.L.,
Ng H.P.,
Hung M.W.,
Hsieh K.H.,
Liaw S.H.,
Chua K.Y.
Publication year - 1999
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1046/j.1365-2222.1999.00603.x
Subject(s) - complementary dna , allergen , clone (java method) , sequence (biology) , sequence analysis , biology , microbiology and biotechnology , immunology , genetics , gene , allergy
Background The important dust mite allergens identified to date are of molecular weights ranging from 14 to 60 kDa. Our previous protein study indicated that the 98‐kDa native paramyosin in Dermatophagoides farinae mite showed IgE reactivity with 82% of the mite‐sensitive asthmatic patients suggesting that it is a novel major mite allergen. This study described the isolation and characterization of the cDNA clone encoding the 98‐kDa mite allergen. Methods A Dermatophagoides farinae cDNA library was constructed in lambda ZAPII vector and the library was immunoscreened with a monoclonal antibody 642. The cDNA insert was sub‐cloned into M13 sequencing vector for single‐stranded sequencing. The whole cDNA insert was expressed in pGEX‐2T Escherichia coli expression system as a fusion protein with GST. The allergenicity of the recombinant peptides was tested by skin tests and IgE immunoassay. The IgE and IgG immunoassays were performed with sera from 20 mite‐allergic patients. Results The cDNA clone Df642 was 2134 bp long, coding for a polypeptide of 711 amino acid residues. Protein sequence analysis and alignment confirmed that the deduced polypeptide is a mite paramyosin which is truncated slightly at the N‐ and C‐terminuses. In vivo skin tests and in vitro IgE‐binding study showed that 62% (13/21) and 50% (10/20) of the mite‐sensitive asthmatic patients reacted positively with the recombinant Dermatophagoides farinae paramyosin, respectively. Conclusion The study indicated that 98‐kDa mite paramyosin is an important allergen.