Cloning, expression, and analysis of a cDNA coding for the Dermatophagoides farinae group 21 (Der f 21) allergen

摘要

Domestic mite species like Dermatophagoides farinae induce allergies in people worldwide. Here, the cDNA coding for group 21 allergen of Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) from China was cloned, sequenced, and expressed in E. coli to aid in the development of diagnostic and treatment options for domestic mite hypersensitivity. First, the Der f 21 cDNA fragment was synthesized by RT-PCR; the confirmed full-length sequence comprised 411 nucleotides. The cDNA was ligated to the vector pCold-TF to construct an expression plasmid, pCold-TF-Der f 21. pCold-Tf-Der f 21 was transformed into E. coli BL21 cells, and its expression was induced by IPTG treatment. SDS-PAGE showed a specific band at the predicted molecular weight of Der f 21, demonstrating its successful expression. The recombinant fusion protein was obtained and its structure and molecular weight were confirmed by MALDI-TOF/TOF. Bioinformatics analysis revealed that the protein contained a signal peptide of 17 amino acids. The molecular weight of the mature Der f 21 allergen was approximately 14.16 kDa with a theoretical pI of 4.87. Its predicted secondary structure comprises a-helix (84.03%), extension chain (1.68%), and random coil (14.29%). The successful cloning of Der f 21 and a basic bioinformatics analysis of the protein provide a foundation for further study of this allergen in diagnosis and treatment of domestic mite hypersensitivity.