Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

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.

Techniques for covering soft tissue defects resulting from plantar ulcers in leprosy: Part I--General considerations and summary of results.

Recurrent plantar ulceration is a common and serious complication occurring consequent to impairment of the tibial nerve in leprosy patients. In spite of many therapies and long therapeutic course, it is extremely difficult to abolish this complication in many cases because of extensive skin and soft tissue cushion loss due to repeated infection. Since the early 70's we have been using microscopic surgical techniques to reconstruct the ulcerated area using eight types of the flaps. In this series of papers we review our experience (76 patients). Post-operatively, the flaps survived in all cases, the long-term results have proved satisfactory, and recurrent ulceration occurred in only three patients.