RESUMEN
Dermatophagoides farinae (Der f), one of the main species of house dust mites, produces more than 30 allergens. A recently identified allergen belonging to the alpha-tubulin protein family, Der f 33, has not been characterized in detail. In this study, we used bioinformatics tools to construct the secondary and tertiary structures and predict the B and T cell epitopes of Der f 33. First, protein attribution, protein patterns, and physicochemical properties were predicted. Then, a reasonable tertiary structure was constructed by homology modeling. In addition, six B cell epitopes (amino acid positions 34-45, 63-67, 103-108, 224-230, 308-316, and 365-377) and four T cell epitopes (positions 178-186, 241-249, 335-343, and 402-410) were predicted. These results established a theoretical basis for further studies and eventual epitope-based vaccine design against Der f 33.
Asunto(s)
Animales , Tubulina (Proteína)/química , Alérgenos/química , Epítopos de Linfocito T/química , Epítopos de Linfocito B/química , Dermatophagoides farinae/química , Antígenos Dermatofagoides/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/inmunología , Alérgenos/genética , Alérgenos/inmunología , Estructura Molecular , Estructura Terciaria de Proteína , Mapeo Epitopo , Epítopos de Linfocito T/genética , Epítopos de Linfocito B/genética , Biología Computacional , Análisis de Secuencia de Proteína , Dermatophagoides farinae/genética , Dermatophagoides farinae/inmunología , Antígenos Dermatofagoides/genética , Antígenos Dermatofagoides/inmunologíaRESUMEN
Crude extracts of house dust mites are used clinically for diagnosis and immunotherapy of allergic diseases, including bronchial asthma, perennial rhinitis, and atopic dermatitis. However, crude extracts are complexes with non-allergenic antigens and lack effective concentrations of important allergens, resulting in several side effects. Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) is one of the predominant sources of dust mite allergens, which has more than 30 groups of allergen. The cDNA coding for the group 5 allergen of D. farinae from China was cloned, sequenced and expressed. According to alignment using the VECTOR NTI 9.0 software, there were eight mismatched nucleotides in five cDNA clones resulting in seven incompatible amino acid residues, suggesting that the Der f 5 allergen might have sequence polymorphism. Bioinformatics analysis revealed that the matured Der f 5 allergen has a molecular mass of 13604.03 Da, a theoretical pI of 5.43 and is probably hydrophobic and cytoplasmic. Similarities in amino acid sequences between Der f 5 and allergens of other domestic mite species, viz. Der p 5, Blo t 5, Sui m 5, and Lep d 5, were 79, 48, 53, and 37%, respectively. Phylogenetic analysis indicated that Der f 5 and Der p 5 clustered together. Blo t 5 and Ale o 5 also clustered together, although Blomia tropicalis and Aleuroglyphus ovatus belong to different mite families, viz. Echimyopodidae and Acaridae, respectively.
Asunto(s)
Animales , Antígenos Dermatofagoides/genética , Proteínas de Artrópodos/genética , Dermatophagoides farinae/genética , Expresión Génica/genética , Secuencia de Aminoácidos , Antígenos Dermatofagoides/inmunología , Antígenos Dermatofagoides/metabolismo , Proteínas de Artrópodos/inmunología , Proteínas de Artrópodos/metabolismo , China , Clonación Molecular , Biología Computacional , ADN Complementario , Dermatophagoides farinae/inmunología , Dermatophagoides farinae/metabolismo , Escherichia coli/genética , Expresión Génica/inmunología , Datos de Secuencia Molecular , Filogenia , Plásmidos , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Animales , Preescolar , Humanos , Alérgenos/genética , Antígenos Dermatofagoides/genética , Dermatophagoides farinae/genética , Escherichia coli/genética , Secuencia de Aminoácidos , Alérgenos/química , Alérgenos/farmacología , Antígenos Dermatofagoides/química , Western Blotting , Clonación Molecular , ADN Complementario/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The present study aimed to characterize the group 2 allergens of the house dust mite Dermatophagoides farinae (Der f 2) from Hainan Island, a tropical region in Southeastern China. We cloned and sequenced cDNA coding for Der f 2 and found an additional region of 87 base pairs (bp) (from +77 to +163 bp) in our strain that was absent in the reference sequence (GenBank AB195580) used for primer design. However, the BLAST analysis identified the same sequence in strains reported from Reinbek, Germany, and Guangzhou, China. A phylogenetic tree was constructed using the Der f 2 nucleotide sequences from different regions or countries and showed that the Hainan sequence clustered with the strains from Reinbek and Guangzhou. Analysis of the translated amino acid sequence suggests that the encoded peptide is hydrophobic and extracellular with a cleavage site between the 17th and 18th amino acid residues and contains a strong trans-membrane helix from the 6th amino acid to the 24th amino acid, indicating a MD-2-related lipid recognition domain in this protein. Furthermore, the secondary structure of the pro-protein consists of 16.57% alpha helix, 32.57% extended strand and 50.86% random coil. In brief, we obtained a gene coding for Der f 2 and predicted the molecular characteristics of this protein using bioinformatics tools. Our analysis identified that this gene showed several significant differences to those reported previously.