The Department of Pharmacy, Food and Drug School, Anhui Science and Technology University, Fengyang, China.
The Queensland Brain Institute, University of Queensland, Brisbane, Australia.
Appl Microbiol Biotechnol. 2018 Jun;102(11):4817-4827. doi: 10.1007/s00253-018-8982-9. Epub 2018 Apr 19.
Human neutrophil peptide 1 (HNP1) is a small (3.44 kDa) cationic peptide that is a distinct member of the defensin family. HNP1 plays a crucial role in controlling bacterial infections, particularly by antibiotic-resistant bacteria, through membrane perforation patterns. The structural characteristics of HNP1's three intramolecular disulfide bridges cause difficulty in its synthesis via chemical methods. In this study, bioactive recombinant HNP1 was produced using the Pichia pastoris (P. Pichia) expression system. HNP1 was fused with the polyhedrin of Bombyx mori and enhanced green fluorescent protein (EGFP) to prevent HNP1 toxicity in yeast host cells under direct expression. An enterokinase protease cleavage site (amino acid sequence DDDDK) was designed upstream of the HNP1 peptide to obtain the antibacterial peptide HNP1 with native structure after it was cleaved by the enterokinase. The fusion HNP1 protein (FHNP1) was successfully expressed and had a molecular mass of approximately 62.6 kDa, as determined using SDS-PAGE and Western blot. Then, the recovered FHNP1 was digested and purified; Tricine-SDS-PAGE results showed that HNP1 was successfully released from FHNP1. Functional analysis of induction against antibiotic-resistant Helicobacter pylori (H. pylori) showed that it was challenging for HNP1 to acquire resistance to the antibiotic-resistant H. pylori. Moreover, in vitro studies showed that HNP1 exerted a strong effect against antibiotic-resistant H. pylori activity. Furthermore, the animal model of H. pylori infection established in vivo showed that HNP1 significantly reduced the colonization of antibiotic-resistant H. pylori in the stomach. Our study indicated that this could be a new potential avenue for large-scale production of HNP1 for therapeutic application against the antibiotic-resistant H. pylori infection in humans.
人中性粒细胞肽 1（HNP1）是一种小（3.44 kDa）阳离子肽，是防御素家族的独特成员。HNP1 通过膜穿孔模式在控制细菌感染方面发挥着至关重要的作用，尤其是对抗生素耐药菌。HNP1 的三个分子内二硫键的结构特征使其难以通过化学方法合成。在这项研究中，使用巴斯德毕赤酵母（P. Pichia）表达系统生产具有生物活性的重组 HNP1。HNP1 与家蚕多角体蛋白和增强型绿色荧光蛋白（EGFP）融合，以防止 HNP1 在酵母宿主细胞中直接表达时的毒性。在 HNP1 肽的上游设计了肠激酶蛋白酶切割位点（氨基酸序列 DDDDK），以在被肠激酶切割后获得具有天然结构的抗菌肽 HNP1。融合 HNP1 蛋白（FHNP1）成功表达，分子量约为 62.6 kDa，通过 SDS-PAGE 和 Western blot 确定。然后，回收的 FHNP1 被消化和纯化；Tricine-SDS-PAGE 结果表明 HNP1 已成功从 FHNP1 中释放。针对抗生素耐药幽门螺杆菌（H. pylori）的诱导功能分析表明，HNP1 难以获得对抗生素耐药 H. pylori 的抗性。此外，体外研究表明 HNP1 对抗生素耐药 H. pylori 活性具有很强的作用。此外，体内建立的 H. pylori 感染动物模型表明，HNP1 可显著减少胃中抗生素耐药 H. pylori 的定植。我们的研究表明，这可能是大规模生产 HNP1 用于治疗人类抗生素耐药 H. pylori 感染的新途径。
