目的 鉴定美洲大蠊Periplaneta americanna中丰富的多肽类成分，寻找具有促创面修复活性多肽。方法 通过超滤技术从美洲大蠊中初步提取多肽成分，使用超高效液相色谱-高分辨质谱联合技术快速纯化鉴定多肽的氨基酸序列，使用PeptideRanker在线工具对鉴定多肽进行活性预测，应用Autodock 4.2分子对接软件初步预测鉴定多肽与创面修复相关靶蛋白结合作用强弱，最后通过体外实验验证筛选肽段的活性。结果 共鉴定出属于34个前体蛋白的449个多肽序列，选择相对丰度和评分较高的5条多肽，氨基酸序列分别为AGFAGDDAPR、DFIR、PGPIN、IGF、TLF，分子对接结果分析发现DFIR、PGPIN、IGF、TLF与胞外信号调节激酶/丝裂原活化蛋白激酶、细胞内非受体酪氨酸激酶/信号转导与转录激活子、磷脂酰肌醇-3-激酶/蛋白激酶B/一氧化氮合酶等相关信号通路上的胞外信号调节激酶2、丝裂原活化蛋白激酶、酪氨酸激酶、一氧化氮合酶等8个靶蛋白能够有效对接，体外实验结果表明在0.1～10 mmol/L浓度下，合成的DFIR、PGPIN、IGF、TLF对人脐静脉内皮HUVEC细胞和小鼠成纤维L929细胞2种细胞模型的增殖和迁移有不同程度的促进作用，说明DFIR、IGF、PGPIN及TLF具有潜在的促创面修复活性。结论 超高效液相色谱-高分辨质谱联合技术为美洲大蠊多肽类成分的分析鉴定提供有力的支持，多肽的分析鉴定同时为美洲大蠊促创面修复活性成分的探索提供了新的思路。

Objective To identify the abundant peptides in Periplaneta americana (PA) and search for peptides with the activity of promoting wound repair. Methods Peptide components were extracted preliminarily from PA by ultrafiltration technology, and the amino acid sequence of the peptides were quickly purified and identified using the combined technology of ultra-performance liquid chromatography and high-resolution mass spectrometry. PeptideRanker online tool was used to predict the activity of the peptides, and the Autodock 4.2 molecular docking software was used to predict and identify the binding effect of the peptides with the target proteins related to wound repair. Finally, the activity of the screened peptides was verified through experiments in vitro. Results A total of 449 peptide sequences belonging to 34 precursor proteins were identified, and five peptides with higher intensity and scores were selected, the amino acid sequences were AGFAGDDAPR, DFIR, PGPIN, IGF and TLF, respectively. Analysis of molecular docking results showed that DFIR, PGPIN, IGF and TLF were effectively docked with 8 target proteins including extracellular regulated protein kinases 2, mitogen-activated protein kinase, Janus kinase 2, and endothelial nitric oxide synthase in extracellular regulated protein kinases/mitogen-activated protein kinase, Janus kinase/signal transducer and activator of transcription, phosphatidylinositol 3 kinase/protein kinase B/endothelial nitric oxide synthase and other related signaling pathways. Meanwhile, the results showed that the Synthetic DFIR, PGPIN, IGF and TLF at the concentration of 0.1—10 mmol/L had different promotion effects on the proliferation and migration of human umbilical vein endothelial cells (HUVEC) and mouse fibroblasts (L929) cell models. It indicated that the four peptides of DFIR, IGF, PGPIN and TLF may have an impact on wound repair. Conclusion The combined technology of ultra-performance liquid chromatography and high-resolution mass spectrometry provided strong support for the analysis and identification of the peptide components of PA. The analysis and identification of peptides also provided new ideas for the exploration of the active ingredients of PA to promote wound repair.

R283.6

国家药品监督管理局中药材质量检测评价重点实验室开放课题:基于多肽组学的动物药美洲大蠊药材质量评价体系(2020NMPA- CDDC01)