RESUMO
Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87 percent identity in amino acid sequence with Eur m 1 but only 80 percent with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96 percent), an extended strand (17.13 percent), a ß turn (5.61 percent), and a random coil (43.30 percent). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.
Assuntos
Animais , Alérgenos/imunologia , Antígenos de Dermatophagoides/genética , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Ácaros/imunologia , Sequência de Aminoácidos , Antígenos de Dermatophagoides/isolamento & purificação , Western Blotting , DNA Complementar/química , Poeira , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
To evaluate the feasibility of improving external quality assessment (EQA), we set up an experimental EQA survey that included 465 participants in China. During the period of this survey, we checked the quality of the EQA samples, divided the participants into different groups, each laboratory's result was assessed by calculation standard deviation index (SDI). The reference values were determined to evaluate the accuracy for peer groups. The data showed that the stability of the EQA samples was acceptable. Except for WBC count of the Abbott group, the mean, median and reference values for each parameter were very close. We found that the main reason affecting the performance of the participants was not using the reagents. calibrator and QC material recommended by manufacturer. From this survey, we obtain a good reference for future improvement.