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The Dermatophagoides farinae group 22 allergen: cloning and expression in Escherichia coli
Author(s) -
Cui Yubao,
Cai Hongxing,
Zhou Ying,
Wang Nan,
Yu Lili,
Yang Li,
Zhang Chengbo
Publication year - 2015
Publication title -
international forum of allergy and rhinology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.503
H-Index - 46
eISSN - 2042-6984
pISSN - 2042-6976
DOI - 10.1002/alr.21571
Subject(s) - microbiology and biotechnology , complementary dna , recombinant dna , fusion protein , escherichia coli , biology , biochemistry , gene
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.