设计多肽清除幽门螺杆菌在胃中定植

It has been found that Helicobacter pylori (Hp) gamma glutamyl peptidase and vacuolar toxin could induce immune tolerance, and the defensins which induced synthesis by Hp were degraded in the stomach, these factors resulted in failure for spontaneous elimination of Hp in the host. This study is going to obtain polypeptides with stability and anti Hp activity in the stomach based on partial amino acid sequences of LL-37 design. The mechanisms of polypeptides against Hp activity will be identified by microbiological and biochemical methods in stomach. Previous reports have showed that Hp up-regulated transcription factor Foxp3 promoted development of regulatory T cells then induced immune tolerance. It also reported that polypeptides could inhibit the activity of functional proteins. In this study, the immune tolerance induce by Hp is released by polypeptides inhibiting the activity of Foxp3. Firstly, the software of protein-peptide docking will be used to predict polypeptides which can specifically bind to Foxp3 and then be chemically synthesized. It is further screened and confirmed by the technology of protein heat transfer.The Foxp3 binding to specific sequence of DNA is inhibited by polypeptide using electrophoretic mobility shift assay (EMSA ) technique. The inhibitory effects of peptides on the interaction between Foxp3 and AML1 will be verified using mammalian double hybridization and co-immunoprecipitation techniques. The effect of Foxp3 nuclear localization on polypeptide will be detected in cells using confocal microscope technique. A method of flow cytometry analysis is used to confirm that the polypeptide really can down-regulate the development of regulatory T cells. In this study, a new technology and method is provided to solve the problem of clearing colonization of Hp in the stomach.

研究发现幽门螺杆菌（Hp）γ-谷氨酰转肽酶和空泡毒素诱发免疫耐受，Hp诱导合成的防御素在胃中被破坏，导致宿主自发清除Hp失败。本研究基于LL-37的部分氨基酸序列片段设计获得在胃中具有稳定性和抗Hp活性的多肽，利用微生物学和生化手段明确多肽在胃中的抗Hp机制。前期报道Hp诱发免疫耐受是因上调转录因子Foxp3促进调节T细胞发育。另有报道多肽可抑制功能蛋白活性。本研究利用多肽抑制Foxp3活性解除Hp诱导免疫耐受。首先采用蛋白-肽对接软件预测设计的多肽能与Foxp3结合并化学合成，进一步利用蛋白热转移技术加以筛选和证实。采用EMSA技术验证多肽抑制Foxp3与特异DNA结合；通过哺乳动物双杂交和免疫共沉淀验证多肽抑制Foxp3和AML1相互作用；共聚焦显微技术检测多肽对Foxp3在细胞核定位影响。利用流式细胞分析证实多肽确能下调调节T细胞发育。本研究为清除胃中Hp定植难题解决提供新技术方法。

本研究以人肠毒素LL-37在胃液中稳定存在的9个氨基酸片段（KRIVQRIKD）为基础，设计了28个含有12个氨基酸残基的短肽，并在肽的C端添加了3个氨基酸。所设计的多肽在低pH值下不被胃蛋白酶消化和降解。基于在线工具平台https://web.expasy. org/peptide_cutter/.对多肽进行了预测。然后，从28个短肽中筛选出最强的抗菌肽SAMP-12aa（KRIVQRIKDVIR）。进一步的研究发现SAMP-12aa在模拟胃液中培养后仍具有抗H.pylori活性。SAMP-12aa的MIC和MBC分别为8μg/mL和32μg/mL。SAMP-12aa具有良好的杀菌动力学。SAMP-12aa具有细胞选择性，能穿透和破坏细菌细胞膜，在较高浓度（128μg/mL）下对人体细胞几乎没有毒性。SAMP-12aa对耐药H.pylori活性具有很强的抑制作用，且不诱导H.pylori耐药的产生。其中6个多肽还具有很好抑制和消除幽门螺杆菌生物膜功能作用。控制幽门螺杆菌对抗生素耐药性的产生。我们进一步发现SAMP-12aa可以抑制转录因子Foxp3的活性。SAMP-12aa抑制了CD4+Foxp3CD25-T细胞向CD4+Foxp3CD25+TregT细胞的分化和发育。TregT是诱导幽门螺杆菌感染免疫耐受的重要因子。因此，SAMP-12aa可减轻H.pylori感染诱导的免疫耐受。综上所述，我们的研究结果表明SAMP-12aa可以作为一种新的抗幽门螺杆菌和抗药性幽门螺杆菌感染的药物。同时，SAMP-12aa可减轻H.pylori诱导的免疫耐受。