Cloning, expression, and analysis of the group 2 allergen from Dermatophagoides farinae from China

Abstract

To obtain the recombinant group 2 allergen product of Dermatophagoides farinae (Der f 2), the Der f 2 gene was synthesized by RT-PCR. The full-length cDNA comprised 441 nucleotides and was 99.3% identical to the reference sequence (GenBank AB195580). The cDNA was bound to vector pET28a to construct plasmid pET28a(+)-Der f 2, which was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE showed a specific band of about 14kDa in the hole cell lysate. As estimated by chromatography, about 3.86 g of the recombinant product as obtained, which conjugated with serum IgE from asthmatic children. The protein had a signal peptide of 17 amino acids. Its secondary structure comprised an alpha helix (19.86%), an extended strand (30.82%), and a random coil (49.32%). The subcellular localization of this allergen was predicted to be at mitochondria. Furthermore, its function was shown to be associated with an MD-2-related lipid-recognition (ML) domain. The results of this study provide a solid foundation for large-scale production of the allergen for clinical diagnosis and treatment of allergic disorders.

cDNA Cloning and Characterization of the House Dust Mite Allergen Sui p 2

“…1). The presence of the signal peptide is consistent with the amino acid sequences of other group 2 allergens (Cui et al, 2010;Horáčková et al, 2010) thus indicating that the Sui p 2 cDNA genes described in this study are the full-length sequences of the Sp group 2 allergen. Being an extracellular protein, the signal peptide present in Sui p 2 may play a major role towards the transport of the protein to the outside of the cell.…”

Experimental immunology Effects of the recombinant allergen rDer f 2 on neuro-endocrino-immune network in asthmatic mice

“…Animals were housed under sterile conditions in the Laboratory Animal Center of the Shanghai Second Military Medical University. The crude extracts of Dermatophagoides farinae and prepared recombinant protein of the group 2 allergen from D. farinae (Der f 2) was provided by Dr. Yubao Cui [ 6 ]. Mouse corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) radioimmunoassay kits were produced by DSL (USA).…”

“…C57BL/6 mice ( n = 28) were divided into four groups by the digit randomization method, n = 7 in each group: normal healthy control group (A); asthma control group (B); crude extract-treated asthma group (C); and rDer f 2-treated asthma group (D). To induce asthma in groups B, C, and D, animals were given a crude extract of D. farinae (50 ml) on day 1 and day 17 via intraperitoneal injection [ 6 ]. On day 15, 16, 17, and 18, the mice in groups B, C, and D were anesthetized with 0.1 ml 0.5% pentobarbital sodium via intraperitoneal injection, and then stimulated with a crude extract of D. farinae [ 6 ] 50 μl/per injection/day.…”

“…To induce asthma in groups B, C, and D, animals were given a crude extract of D. farinae (50 ml) on day 1 and day 17 via intraperitoneal injection [ 6 ]. On day 15, 16, 17, and 18, the mice in groups B, C, and D were anesthetized with 0.1 ml 0.5% pentobarbital sodium via intraperitoneal injection, and then stimulated with a crude extract of D. farinae [ 6 ] 50 μl/per injection/day. From day 31 to day 33, animals in group C and D were give a crude extract of D. farinae and rDer f 2 via intraperitoneal injection while animals in group A and B were given saline.…”