| The prevalence of allergic diseases has increased in recent years. House dust mite (HDM) is an important airborne allergen, which can induce allergic rhinitis (AR), bronchial asthma (BA) and other allergic diseases. Dust mite vaccine immunotherapy has been applicated widely in clinical practice as a disease-modified treatment. However, various factors such as allergen materials and extract technique make it difficult for allergen vaccines to be stardardized, which had an adverse impact on the efficacy and safety of immunotherapy.In our study, HDM specific IgE binding peptides were screened from a phage random peptide library, using positive and negative HDM IgE as specific bait respectively. Positive HDM IgE were selected from subjects with AR or/and BA, and negative HDM IgE from allergic subjects. HDM specific IgE binding peptides of B cell epitopes (both linear and conformational epitope) would be mapped by positive and negative screening simultaneously. The biological effects of these peptides were investigated in vivo. In order to observe the therapeutic effects on the allergic airway inflammation, HDM was used to induce AR&B A in mice, and the interventional effects of 1,2 and 3 soluble cyclic peptides by immunotherapy on mice's allergic airway damage were also observed. This study was expected to lay the foundation for the development of epitope vaccine to treat HDM induced allergic airway inflammation.?. Screening of dust mite IgE binding peptide1. The screening of phage peptide library:The positive and negative HDM IgE was used respectively as specific baits to screen phage peptide library. During bio-panning, non-binding and weakly binding peptide were removed by progressively decreasing of the concentration of specific bait, reducing of the interaction time of bait and library peptides, and strict washing conditions. At the same time, the phage clones with high affinity and great specificity were enriched after three rounds of bio-panning.2. Identifying the IgE binding phage clones:100 phage clones were selected randomly after three rounds of bio-panning. Subsequently, with ELISA technology 84 phage clones were found that identified as the HDM IgE binding phages.3. Sequence analysis of the effective phage DNA and Synthesis of the peptides:In order to deduce amino acid sequences of peptides, the DNA of 84 clones were sequenced. Excluding the same sequences and null clones,44 phage clones showing homology with allergen molecules of HDM were left. We compared primary and tertiary structures of the HDM molecules with the 44 phage clones by sequence alignment, the results showed that clone 41,44 and 9 showed highest similarity with HDM molecules (Der p1, Der p2, Der p5, Der p7), cycling peptides of these 3 clones were synthesized and designated as No.1,2 and 3 mimic peptides of target HDM, respectively.?. The effects of mimic peptides on HDM-induced AR&BA in miceTo examine the intervention effects of mimic peptide 1,2 and 3 on HDM-induced AR&BA in mice, a model of human allergic disease, we divided the mice into five groups named as control groups, AR&BA model groups and intervention groups of mimic peptide 1,2,3. Mice were transiently anaesthetized with anhydrous ether; 100?l HDM (Dermatophagoides pteronyssinus) extract was administered by intranasal instillation directly into the nares. At day 18 and day 36, control animals received sterile saline alone and the other mice were inoculated with 100?l HDM extract by intranasal instillation for three days. Before the second intranasal provocation, the intervention groups were administered by subcutaneous injection with mimic peptide 1,2 and 3 (500?g) were mixed in PBS (200?l) solution respectively; the other groups were injected with 200?l PBS simultaneously.After the last provocation, the behavioral scores and histological manifestations of the five groups were observed, the mRNA expressions of proinflammatory cytokines such as IL-25 and IL-33 in the nasal/lungs epithelium, and cytokines expression such as IL-4, IL-10, IL-25, IL-33, IFN-? in the nasal lavage fluid (NLF) and bronchoalveolar lavage fluid (BALF) were also investigated, the eosinophils in the nasal/lungs tissue and the sIgE and IgG1 of HDM in serum were assessed.The results showed that the intervention groups had no manifestation such as cyanotic lips, wheeze and tachypnea, the behaviors such as sneezing, nasal scratching and nasal discharge were significantly improved as compared with those of the model groups.The airway resistance of the intervention groups was significantly lower than model groups.The pathohistological examinations showed that the integrity of nasal epithelial cells were observed, no proliferation of columnar/goblet cells in the intervention groups. The infiltration of inflammatory cells in blood vessels and bronchia reduced obviously, and the situation of airway epithelium were also improved in the intervention groups.Compared with model groups, the mRNA expressions of IL-25 and IL-33 in the intervention groups were reduced, eosinophil counts were decreased, Th2 cytokines were significantly down-regulated and Thl cytokines were up-regulated. The intervention of mimic peptides led to remarkable reduction of HDM-sIgE and elevation of HDM-IgGl.In summary, HDM-IgE binding peptides obtained by phage display technique could obviously improve the symptoms of the mice's HDM-induced AR&BA, alleviate the allergic airway damage in vivo. This study would provide an experimental basis for the improvement of clinical SIT in the patients with AR and BA. |
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