The Dermatophagoides farinae group 22 allergen: cloning and expression in Escherichia coli

Int Forum Allergy Rhinol. 2015 Sep;5(9):794-800. doi: 10.1002/alr.21571. Epub 2015 Jun 11.

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%).

Conclusion: 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.

Keywords: Der f 22; Dermatophagoides farinae; domestic mite; molecular cloning; recombinant allergen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antigens, Dermatophagoides* / chemistry
  • Antigens, Dermatophagoides* / genetics
  • Antigens, Dermatophagoides* / metabolism
  • Arthropod Proteins / chemistry
  • Arthropod Proteins / genetics
  • Arthropod Proteins / metabolism
  • Base Sequence
  • Cloning, Molecular
  • Computational Biology
  • DNA, Complementary / genetics
  • Dermatophagoides farinae* / genetics
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Molecular Sequence Data
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA

Substances

  • Antigens, Dermatophagoides
  • Arthropod Proteins
  • DNA, Complementary
  • Recombinant Proteins