Structures and Antigenic Epitopes of Dust Mite Allergens: Der p 2 and Der f 2: a Comparative Study / 现代医院

Objective To compare and analyze the primary and secondary structures and antigenic epitopes of the two allergens: Der p 2 and Der f 2. Methods The protein sequences of Der p 2 and Der f 2 were downloaded online. The primary and secondary structures of the dust mite allergens were compared and analyzed bioinformatically to determine the potential epitope and signal peptide sites. Results Both Der p 2 and Der f 2 contained 146 amino acids and 9 potential protein binding sites with a secondary structure that mainly contains [3 - sheets, and there might be signal peptides site at the 1st 17th segment of the N - terminus. B cell epitopes analysis revealed that both Der p 2 and Der f 2 have 9 potential linear B epitopes and 2 conformational B epitopes. NetMHCⅡserver prediction showed Der p 2 contains 6 high affinity sites, whereas Der f 2 0nly contains 5. Conclusion This study may lay the foundation for further research of the biochemical function of the 2 allergens and contribute to vaccine development for allergen - specific immunotherapy.

Influence of the Adjuvants and Genetic Background on the Asthma Model Using Recombinant Der f 2 in Mice

Der f 2 is the group 2 major allergen of a house dust mite (Dermatophagoides farinae) and its function has been recently suggested. To determine the optimal condition of sensitization to recombinant Der f 2 (rDer f 2) in murine model of asthma, we compared the effectiveness with different adjuvants in BALB/c and C57BL/6 mice. Mice from both strains sensitized with rDer f 2 by intraperitoneal injection or subcutaneous injection on days 1 and 14. The dosage was 20 microg. Freund's adjuvants with pertussis toxin (FP) or alum alone were used as adjuvants. On days 28, 29, and 30, mice were challenged intranasally with 0.1% rDer f 2. We evaluated airway hyperresponsivenss, eosinophil proportion in lung lavage, airway inflammation, and serum allergen specific antibody responses. Naive mice were used as controls. Airway hyperresponsiveness was increased in C57BL/6 with FP, and BALB/c with alum (PC200: 13.5+/-6.3, 13.2+/-6.7 vs. >50 mg/ml, p<0.05). The eosinophil proportion was increased in all groups; C57BL/6 with FP, BALB/c with FP, C57BL/6 with alum, BALB/c with alum (24.8+/-3.6, 20.3+/-10.3, 11.0+/-6.9, 5.7+/-2.8, vs. 0.0+/-0.0%, p<0.05). The serum allergen specific IgE levels were increased in C57BL/6 with FP or alum (OD: 0.8+/-1.4, 1.1+/-0.8, vs. 0.0+/-0.0). C57BL/6 mice were better responders to rDer f 2 and as for adjuvants, Freund's adjuvant with pertussis toxin was better.

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

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 percent 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. s estiated by chroatography, about 3.86 g of the recobinant 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 percent), an extended strand (30.82 percent), and a random coil (49.32 percent). 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 treatent of allergic disorders.

Com a finalidade de obter o produto recombinante do alergeno grupo 2 do Dermatophagoides farinae (Der f2), o gene Der f2 foi sintetizado por RT-PCR. O cDNA continha 441 nucleotídeos e era idêntico em 99,3 por cento à sequência de referência (GenBank AB195580). O cDNA foi ligado ao vetor pET28a para construir o plasmídeo pET28a(+)-Der f2, o qual foi introduzido por transformação em E. coli BL21 e induzido por IPTG. Em SDS-PAGE foi vista mia banda específica de 14 kDa no lisado celular. Conforme estimado por cromatografia, cerca de 3,86 mg do produto recombinante foi obtido, que reagia com IgE sérica de crianças asmáticas. A proteína continha um peptídeo sinal de 17 amino ácidos. Sua estrutura secundária consistia de uma alfa hélice (19,86 por cento), uma fita estendida (30,82 por cento), e uma sequência randômica (49,32 por cento). A localização subcelular desse alergeno foi predita ocorrer nas mitocôndrias. Sua função foi associada com o domínio de reconhecimento lipídico (ML) relacionado a MD-2. Os resultados desse estudo permitem a produção em larga escala do alergeno para o diagnóstico clínico e tratamento das doenças alérgicas.

Der f 2 activates phospholipase D in human T lymphocytes from Dermatophagoides farinae specific allergic individuals: Involvement of protein kinase C-alpha

The major house-dust mite allergen, Der f 2, stimulates the phospholipase D (PLD) in T lymphocytes from Dermatophagoides farinae specific allergic individuals. PLD activity increased more than two-fold in T cells from allergic patients compared with those cells from normal controls with maximal responses within 30 min after exposure of Der f 2. A well-known PLD activator PKC-alpha was found to be translocated to membrane from cytosol in Der f 2-treated T cells from Dermatophagoides farinae specific allergic individuals. Down-regulation of PKC-alpha with phorbol myristate acetate pretreatment for 24 h abolished Der f 2-induced PLD activation. Ro 320432, PKC inhibitor also reduced the effects of Der f 2-induced PLD activation suggesting that PKC-alpha acts as upstream activator of PLD in Der f 2-treated T cells. Taken together, the present data suggest that Der f 2 can stimulate PLD activity through the PKC-alpha activation in T cells from Dermatophagoides farinae allergic individuals

Study on the Localization of a Major Allergen Der f 2 by Fluorescence Immunohistochemical Method / 中国寄生虫学与寄生虫病杂志

Objective To localize Der f 2 in the body of Dermatophagoides farinae.Methods Live mites were embedded.Serial mite paraffin sections were made and checked under light microscope.The sections were incubated with anti-recombinant Der f 2 monoclonal antibody as the first antibody.They were then reacted with the fluorescent isothiocyanate conjugated goat anti-mouse IgG as the secondary antibody.The sections were examined with fluorescent light microscopy.Results Der f 2 reacting with immunofluorescent antibodies was found localized in the tissue and contents of the mid-gut of the mite.Conclusion The major allergen Der f 2 distributes in the gut and fecal pellets of Dermatophagoides farinae.