Yu-Bao Cui1,2, Li-Li Yu2, Fei-Xiang Teng2, Nan Wang2, Ying Zhou1,2, Li Yang2, Cheng-Bo Zhang2. 1. Department of Clinical Laboratory, Affiliated Yancheng Hospital, School of Medicine, Southeast University, Yancheng, China. 2. Department of Laboratory Medicine, Yancheng Health Vocational & Technical College, Yancheng, China.
Abstract
BACKGROUND: House dust mite hypersensitivity affects millions of people worldwide, and although many allergens produced by house dust mite species have been identified, some of the less potent allergens remain to be studied. METHODS: The full-length cDNA encoding the group 4 allergen of the house dust mite species Dermatophagoides farinae (Der f 4) was generated through degenerate primer-based PCR, 5' RACE, and 3' RACE, and the cDNA fragment was cloned into an expression vector for nucleotide sequencing. Following codon optimization and removal of the signal peptide sequence, the mature gene fragment was subcloned into pET-28b (+) and transfected into E. coli BL21 cells for expression. The recombinant protein was purified by nickel affinity chromatography, identified by SDS-PAGE, Western blotting, and MALDI-TOF, and tested by ELISA for IgE reactivity with sera from individuals with asthma. Bioinformatics analyses were used to identify features of Der f 4. RESULTS: SDS-PAGE and Western blotting of the codon-optimized expression product showed a specific band. The mature recombinant Der f 4 was characterized as a stable and hydrophilic 57.9-kDa protein, and its secondary structure comprised alpha helix (25.3%), extended strand (22.51%), and random coils (52.19%). The structure of the recombinant protein was consistent with that of α-amylase. Among 27 pediatric asthma patients, 40.74% exhibited reactivity to rDer f 4 by ELISA. CONCLUSIONS: This initial cloning and characterization of the Der f 4 allergen serves as a foundation for future studies into the clinical importance and application of this protein for house dust mite allergy.
BACKGROUND: House dust mite hypersensitivity affects millions of people worldwide, and although many allergens produced by house dust mite species have been identified, some of the less potent allergens remain to be studied. METHODS: The full-length cDNA encoding the group 4 allergen of the house dust mite species Dermatophagoides farinae (Der f 4) was generated through degenerate primer-based PCR, 5' RACE, and 3' RACE, and the cDNA fragment was cloned into an expression vector for nucleotide sequencing. Following codon optimization and removal of the signal peptide sequence, the mature gene fragment was subcloned into pET-28b (+) and transfected into E. coli BL21 cells for expression. The recombinant protein was purified by nickel affinity chromatography, identified by SDS-PAGE, Western blotting, and MALDI-TOF, and tested by ELISA for IgE reactivity with sera from individuals with asthma. Bioinformatics analyses were used to identify features of Der f 4. RESULTS:SDS-PAGE and Western blotting of the codon-optimized expression product showed a specific band. The mature recombinant Der f 4 was characterized as a stable and hydrophilic 57.9-kDa protein, and its secondary structure comprised alpha helix (25.3%), extended strand (22.51%), and random coils (52.19%). The structure of the recombinant protein was consistent with that of α-amylase. Among 27 pediatric asthmapatients, 40.74% exhibited reactivity to rDer f 4 by ELISA. CONCLUSIONS: This initial cloning and characterization of the Der f 4 allergen serves as a foundation for future studies into the clinical importance and application of this protein for house dust mite allergy.