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Identification of gliadin-binding peptides by phage display

机译：通过噬菌体展示鉴定麦醇溶蛋白结合肽

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Background Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products. Results Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro . By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin. Conclusions We believe that several of the isolated and characterised gliadin-binding peptides described here could provide valuable tools for researchers in the field of CD by facilitating studies on localisation and uptake of various gliadin peptides in the small intestine. In future work, the potential of these peptides to detoxify gluten will be investigated.
机译：背景乳糜泻（CD）是一种小肠常见且复杂的疾病，由对小麦面筋和相关大麦和黑麦等可食用谷物的不耐受性引起。源自不完全的麦醇溶蛋白消化的肽激活固有层浸润的T细胞释放促炎细胞因子，进而引起小肠壁的深刻组织重塑。除了不食用含麸质的产品外，没有治愈CD的方法。结果通过针对固定化的麦醇溶蛋白的反复淘选富集了来自随机低聚物展示文库的噬菌体。将来自最后淘选轮的噬菌体进行铺板，挑选单个噬菌斑，与宿主细菌一起孵育，扩增至种群大小从10 11 至10 12 并纯化。从1000个纯化的噬菌体群体中分离DNA，并对覆盖36 bp寡核苷酸插入片段的区域进行测序，从中翻译出所展示的肽。总共鉴定出150多种不同的肽编码序列，其中许多是在各种实验条件下反复分离的。首先在库中测试各自表达一种肽的扩增的噬菌体群体，然后在ELISA分析中逐一测试其抑制人抗麦醇溶蛋白抗体结合能力。这些实验表明，测试的几种不同的表达肽的噬菌体均抑制麦醇溶蛋白和抗麦醇溶蛋白抗体之间的相互作用。最后，选择了四种不同的肽编码序列进行进一步分析，并在体外合成了相应的12-mer肽。通过ELISA测定法证明了几种肽抑制麦醇溶蛋白分子和血清抗麦醇溶蛋白抗体之间的相互作用。此外，ELISA竞争实验以及斑点印迹和蛋白质印迹表明，不同的肽与麦醇溶蛋白的不同分子部位相互作用。结论我们认为，本文所述的几种分离和表征的麦醇溶蛋白结合肽可以通过促进对小肠中各种麦醇溶蛋白肽的定位和摄取的研究，为CD领域的研究人员提供有价值的工具。在未来的工作中，将研究这些肽对面筋排毒的潜力。

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