title: Dermatophagoides farinae allergen Der f 9: Cloning, expression, purification, characterization and IgE-binding in children with atopic asthma.
abstract: The house dust mite species Dermatophagoides farinae releases allergens that cause allergies and asthma worldwide. This study sought to clone and express the full-length cDNA encoding the group 9 allergen of D. farinae (Der f 9).
  
  
  
  The published sequence of Der f 9 was used to design primers for RT-PCR and RACE to obtain the full-length cDNA encoding Der f 9. After removal of signal peptide sequence, Der f 9 was then sub-cloned into plasmid pET-28b (+), and the plasmid was transformed into Escherichia coli BL21 (DE3) cells for expression. The recombinant protein was purified by Nickel affinity chromatography, identified by SDS-PAGE, Western blotting, dot blotting, and MALDI-TOF, and tested by ELISA for IgE reactivity with sera from children with asthma. Bioinformatics analyses were used to identify features of Der f 9.
  
  
  
  By RT-PCR, 3'-RACE, and 5'-RACE, the full-length sequence of Der f 9 was generated, which was confirmed by nucleotide sequencing. The mature Der f 9 was expressed successfully in E. coli, which was identified by SDS-PAGE. The recombinant allergen was purified by chromatography and confirmed by SDS-PAGE, Western blotting, dot blotting, and MALDI-TOF. Sera from 56.7% (17/30) of mite-allergic patients reacted with the purified recombinant Der f 9.
  
  
  
  The successful production of recombinant Der f 9 protein revealed the importance of Der f 9 in mite allergy, and provides a foundation for further study of this allergen in diagnosis and treatment of symptoms. Pediatr Pulmonol. 2017;52:282-292. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Clinical Laboratory, Affiliated Yancheng Hospital, School of Medicine, Southeast University, Yancheng, 224001, Jiangsu Province, P. R. China.|Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, 224006, Jiangsu Province, P. R. China.
authors.forename: Yubao
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, 224006, Jiangsu Province, P. R. China.
authors.forename: Feixiang
authors.initials: F
authors.lastname: Teng
authors.affiliation: Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, 224006, Jiangsu Province, P. R. China.
authors.forename: LiLi
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Clinical Laboratory, Affiliated Yancheng Hospital, School of Medicine, Southeast University, Yancheng, 224001, Jiangsu Province, P. R. China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, 224006, Jiangsu Province, P. R. China.
authors.forename: Chengbo
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, 224006, Jiangsu Province, P. R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
mesh_terms: D000818:Animals; D039741:Antigens, Dermatophagoides / Q000235:genetics* / Q000276:immunology*; D060829:Arthropod Proteins / Q000235:genetics* / Q000276:immunology*; D001249:Asthma / Q000276:immunology*; D001483:Base Sequence; D002648:Child; D003001:Cloning, Molecular; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D007073:Immunoglobulin E
keywords: Dermatophagoides farinae; house dust mite; molecular cloning; recombinant allergen