Expression, cloning, and IgE-binding of the full-length dust mite allergen Der f 8.
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.
Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Jiefangnan Road 263, Yancheng, 224006, Jiangsu Province, People's Republic of China, ybcui1975@hotmail.com.
Yu-Bao
YB
Cui
Ying
Y
Zhou
Nan
N
Wang
Fei-Xiang
FX
Teng
Li-Li
LL
Yu
Yong-Hua
YH
Bian
Jin-Xia
JX
Song
Li
L
Yang
Cheng-Bo
CB
Zhang
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D039741:Antigens, Dermatophagoides / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D007073:Immunoglobulin E