Expression, cloning, and IgE-binding of the full-length dust mite allergen Der f 8.
Immunol Res
; 60(1): 60-8, 2014 Oct.
Article
en En
| MEDLINE
| ID: mdl-24838264
ABSTRACT
Dermatophagoides farinae, a domestic mite species, produces some of the most potent allergens that contribute to allergy in China and worldwide. We sought to clone and express the group 8 allergen of D. farinae (Der f 8) to investigate its IgE-binding reactivity. The full-length cDNA encoding Der f 8 was generated by using RT-PCR and 5' RACE, cloned into pCold-TF expression vector, confirmed by nucleotide sequencing, sub-cloned into pET-28b (+), and transfected into E. coli BL21 cells for expression. After purification by nickel affinity chromatography and identified by SDS-PAGE, the recombinant Der f 8 bound with sera from 40.9 % (9/22) of mite-allergic patients according to ELISA testing. Analysis of the recombinant DNA sequence revealed a 231 amino acid open reading frame encoding a protein with a derived molecular mass of 26.4 kDa and an isoelectric point of 6.84. The deduced amino acid sequence has nine phosphorylation sites, displaying strong homology with glutathione S-transferase, and its secondary structure comprises alpha helix (45.5 %), extended strand (11.3 %), and random coils (43.3 %). BLAST through the National Center for Biotechnology Information database and alignment identified similarity with group 8 allergens or glutathione S-transferases of Dermatophagoides pteronyssinus, Suidasia medanensis, Lepidoglyphus destructor, Glycyphagus domesticus, and Aleuroglyphus ovatus (64, 65, 53, 53, and 50 %, respectively). The first recombinant Der f 8 protein produced in full length successfully bound with patient IgE, demonstrating the importance of Der f 8 in mite allergy.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Alérgenos
/
Antígenos Dermatofagoides
/
Proteínas de Artrópodos
Límite:
Child
/
Child, preschool
/
Humans
Idioma:
En
Revista:
Immunol Res
Asunto de la revista:
ALERGIA E IMUNOLOGIA
Año:
2014
Tipo del documento:
Article