title: The Dermatophagoides farinae group 22 allergen: cloning and expression in Escherichia coli.
abstract: BACKGROUND
  Dermatophagoides farinae (Hughes) (Acari: Pyroglyphidae) and other domestic mites produce allergens that affect people worldwide. Here, the complementary DNA (cDNA) coding for group 22 allergen of D. farinae (Der f 22) from China was cloned, sequenced, and expressed successfully.
  
  
  METHODS
  The cDNA encoding Der f 22 was synthesized by reverse transcription polymerase chain reaction (RT-PCR), then ligated to the pCold-TF for expression in Escherichia coli BL21 cells. The purified recombinant fusion protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western-blotting, and tandem matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF).
  
  
  RESULTS
  The full-length cDNA comprised 468 nucleotides and was 99.57% (466/468) identical with the reference sequence (GenBank: DQ643992). After the plasmid pCold-TF-Der f 22 was transformed into E. coli BL21 and expressed with the induction of IPTG, SDS-PAGE showed a specific band for the recombinant fusion protein. The recombinant fusion protein, which was purified by chromatography, bound with a His-tagged antibody by Western blotting. MALDI-TOF/TOF mass spectrometry revealed that the structure of the recombinant protein was identical to the predicted Der f 22 structure. The hydrophilic protein contains a signal peptide of 20 amino acids, and the mature Der f 22 consists of 135 amino acid residues with a molecular weight of 14.7 kDa and theoretical isoelectric points (pI) of 6.38. Its secondary structure comprises an alpha helix (38.5%), beta-sheet (45.9%), random coils (11.85%), and beta-turns (11.1%).
  
  
  CONCLUSIONS
  This work represents the first reported full-length sequence and successful cloning of Der f 22 from D. farinae in China; bioinformatics analysis can be used to further study the allergenicity and clinical utility of the recombinant Der f 22.
authors.affiliation: Department of Pathogenic Biology and Laboratory of Infection and Immunology, Xuzhou Medical College, Xuzhou, P.R. China.|Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, P.R. China.
authors.forename: Yu-bao
authors.initials: YB
authors.lastname: Cui
authors.affiliation: Department of Forensic Medicine, Xuzhou Medical College, Xuzhou, P.R. China.
authors.forename: Hong-xing
authors.initials: HX
authors.lastname: Cai
authors.affiliation: Department of Laboratory Medicine, Xuzhou Medical College, Xuzhou, 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, P.R. China.
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.affiliation: Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, P.R. China.
authors.forename: Li-li
authors.initials: LL
authors.lastname: Yu
authors.affiliation: Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, P.R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, P.R. China.
authors.forename: Cheng-bo
authors.initials: CB
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics; D040001:Dermatophagoides farinae / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins
keywords: Der f 22; Dermatophagoides farinae; domestic mite; molecular cloning; recombinant allergen