title: Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae.
abstract: 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.
  
  
  CONCLUSIONS
  The study indicated that 98-kDa mite paramyosin is an important allergen.
authors.affiliation: Department of Medical Research, Veterans General Hospital, Taipei, Taiwan.
authors.forename: L
authors.initials: L
authors.lastname: Tsai
authors.forename: Y
authors.initials: Y
authors.lastname: Sun
authors.forename: P
authors.initials: P
authors.lastname: Chao
authors.forename: H
authors.initials: H
authors.lastname: Ng
authors.forename: M
authors.initials: M
authors.lastname: Hung
authors.forename: K
authors.initials: K
authors.lastname: Hsieh
authors.forename: S
authors.initials: S
authors.lastname: Liaw
authors.forename: K
authors.initials: K
authors.lastname: Chua
mesh_terms: D000293:Adolescent; D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D018076:DNA, Complementary / Q000235:genetics; D004391:Dust / Q000009:adverse effects; D015723:Gene Library; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D007118:Immunoassay; D007073:Immunoglobulin E / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D008925:Mites / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology; D017422:Sequence Analysis, DNA; D012882:Skin Tests; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
chemical_list: D039741:Antigens, Dermatophagoides; D018076:DNA, Complementary; D004391:Dust; D006023:Glycoproteins; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D014335:Tropomyosin; D007073:Immunoglobulin E