[Cloning and sequence analysis of leptin receptor overlapping transcript-like 1 gene from Dermatophagoides farinae].

OBJECTIVE: To obtain the leptin receptor overlapping transcript-like 1 encoding gene (LepROTL1 gene) from Dermatophagoides farina, investigate the molecular characteristics of the gene and construct a prokaryotic expression vector to express this gene. METHODS: The LepROTL1 gene-encoding sequence fragments were captured based on the transcriptome sequencing results, and the full-length gene fragments were amplified from total RNA of D. farinae using a RT-PCR assay, and used to construct the expression plasmid pET28a(+)-LepROTL1, followed by sequencing. The plasmid was transformed into E. coli BL21 (DE3) T1R for the induction of IPTG expression. The expression product was characterized by SDS-PAGE and Western blotting. Bioinformatics analyses were performed to analyze the sequence and the molecular characteristics of its encoded protein. RESULTS: The amplification products of the RT-PCR assay showed a clear band on agarose gel electrophoresis, and sequencing analysis of the pET28a(+)-LepROTL1 plasmid showed 417 bp in length of the coding gene from the start codon ATG to the termination codon TAA. Following the plasmid transformation into E. coli and induction with IPTG, a specific band was seen on SDS-PAGE, indicating successful expression. Bioinformatics analysis showed that the LepROTL1 gene-encoded protein was composed of 134 amino acids, and had a relative molecular weight of 14 378.13 Da, a hydrophilicity index of 1.149, and certain hydrophobicity. The secondary structure was composed of alpha-helix (19 aa, 14.18%), extended strand (48 aa, 35.82%) and random coil (67 aa, 50.00%). The deduced amino acid sequence was used to obtain homologous genes by BLAST, and the phylogenetic tree showed that D. farinae was clustered with D. pteronyssinus. CONCLUSIONS: The full-length sequences and expression plasmid of the LepROTL1 gene are obtained, and the molecular features of the gene are demonstrated using bioinformatics analyses, which provide insights into further studies on the gene.

The draft genome, transcriptome, and microbiome of Dermatophagoides farinae reveal a broad spectrum of dust mite allergens.

BACKGROUND: A sequenced house dust mite (HDM) genome would advance our understanding of HDM allergens, a common cause of human allergies. OBJECTIVE: We sought to produce an annotated Dermatophagoides farinae draft genome and develop a combined genomic-transcriptomic-proteomic approach for elucidation of HDM allergens. METHODS: A D farinae draft genome and transcriptome were assembled with high-throughput sequencing, accommodating microbiome sequences. The allergen gene structures were validated by means of Sanger sequencing. The mite's microbiome composition was determined, and the predominant genus was validated immunohistochemically. The allergenicity of a ubiquinol-cytochrome c reductase binding protein homologue was evaluated with immunoblotting, immunosorbent assays, and skin prick tests. RESULTS: The full gene structures of 20 canonical allergens and 7 noncanonical allergen homologues were produced. A novel major allergen, ubiquinol-cytochrome c reductase binding protein-like protein, was found and designated Der f 24. All 40 sera samples from patients with mite allergy had IgE antibodies against rDer f 24. Of 10 patients tested, 5 had positive skin reactions. The predominant bacterial genus among 100 identified species was Enterobacter (63.4%). An intron was found in the 13.8-kDa D farinae bacteriolytic enzyme gene, indicating that it is of HDM origin. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed a phototransduction pathway in D farinae, as well as thiamine and amino acid synthesis pathways, which is suggestive of an endosymbiotic relationship between D farinae and its microbiome. CONCLUSION: An HDM genome draft produced from genomic, transcriptomic, and proteomic experiments revealed allergen genes and a diverse endosymbiotic microbiome, providing a tool for further identification and characterization of HDM allergens and development of diagnostics and immunotherapeutic vaccines.

Multiplex PCR for identifying common dust mites species (Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis).

INTRODUCTION: Dust mites are known to be an important source of inhalant allergens causing allergic rhinitis and asthma worldwide. The sizes of dust mite populations in patients' houses are useful to monitor the risk of allergen exposure. However, mite identification using the conventional microscopic technique requires specific expertise and is time consuming; therefore a molecular technique has been developed in order to solve these drawbacks. OBJECTIVE: To develop a multiplex PCR assay for identifying the three common dust mite species in Thailand, namely Dermatophagoides pteronyssinus (Dp), D. farinae (Df) and Blomia tropicalis (Bt), and to evaluate the efficacy of the technique. METHODS: Pairs of primers were designed and tested in either singleplex PCR or multiplex PCR. The multiplex PCR technique was also optimized in order to obtain specific products. The reaction mixture contained 5 pmole of individual primers, 10 mM dNTP, 5 units Taq DNA polymerase and genomic DNA (gDNA). The reaction was run for 25 cycles at 94 degrees C for 20 seconds, 58 degrees C for 20 seconds and 72 degrees C for 30 seconds. The PCR products were analyzed by 1.5% agarose gel electrophoresis with GelRed fluorescence dye. The optimized multiplex technique was also tested with 30 house dust samples and dust samples spiked with DNA from other insect and mite species. RESULTS: Three PCR products were obtained with the relevant gDNA templates as expected; 143 bp for DF, 221 bp for DP and 318 bp for BT, respectively. The detection limit of the tests was found to be as low 1 ng of gDNA, whereas mixed gDNA species confirmed the 100% specificity of this assay. The total duration from the preparation of the PCR reaction mixture until the analysis by agarose gel electrophoresis was approximately 2 hours. No amplified product was obtained from mites and insects of other species. CONCLUSION: The multiplex PCR was successfully developed for identifying 3 common dust mite species. This technique can be helpful, not only for non-acarologist personnel for dust mite identification, but also for patients who are allergic to dust mites.

The fauna and distribution of house dust mites in residential homes of Bandar Abbas District, Southern Iran.

This study was designed to determine the occurrence, distribution and abundance of house dust mites (HDM) in residential homes in Bandar Abbas (Hormozgan Province), because of numerous complaints of allergies in this oriental city. The study area was divided in five sampling zones based on population density and geographical distribution. In each sampling zone 10 houses were randomly selected. A total of 50 home dust samples were collected using a portable vacuum cleaner for 2 min from 1 m(2) of the surface of mattresses, carpets, sofas and furniture in residential houses. After collection, samples were immediately frozen. Mite species were identified and counted using standard methods and keys. Of the sampled houses 88% (44 houses) were contaminated with at least one HDM species. Three species were identified: Dermatophagoides pteronyssinus (63.1%), D. farinae (32.8%) and D. evansi (4.1%) (Pyroglyphidae). Our findings indicate a relationship between HDM density and moisture and temperature of residential places. The high contamination rate of residential houses (88%) and the favourable environmental conditions for these arthropods stress that they should be considered as important allergic causing agents.