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斑马鱼新型肽聚糖结合蛋白KRT8的模式识别活性和多肽抑菌活性鉴定
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Abstract:
目的：研究角蛋白KRT8在斑马鱼中的免疫相关功能，并在该蛋白的基础上筛选抗菌肽。方法：以斑马鱼KRT8为研究对象，通过蛋白免疫印记和酶联免疫吸附实验，研究该蛋白是否具有与肽聚糖、脂多糖以及革兰氏阴性与阳性菌结合的能力；通过脂多糖诱导斑马鱼发生炎症，探究krt8在炎症反应中的作用；通过斑马鱼体内细菌感染实验，进一步研究KRT8对炎症的作用；在该蛋白的基础上筛选抗菌肽，然后验证筛选出候选肽的抑菌能力。结果：本研究发现，KRT8可以与肽聚糖、脂多糖、革兰氏阴性和革兰氏阳性细菌结合。在脂多糖诱导的斑马鱼炎症模型中，krt8的表达水平发生变化，这说明krt8可能参与炎症反应。进一步研究发现，KRT8可缓解细菌诱导的炎症反应。从KRT8中筛选出的抗菌肽mP90-103具有直接杀菌作用，且无溶血活性。结论：KRT8可作为模式识别受体参与斑马鱼炎症免疫调控；其衍生肽mP90-103具有优良的抗菌活性和生物安全性。本研究为解释多种动物体内角蛋白的免疫功能提供了新见解。
Objective: To investigate the immune-related functions of keratin KRT8 in zebrafish, and screen antimicrobial peptides derived from KRT8. Methods: Taking zebrafish KRT8 as the research object, Western blot and ELISA were performed to detect the binding capacity of KRT8 to peptidoglycan (PGN), lipopolysaccharide (LPS), Gram-negative bacteria and Gram-positive bacteria. An LPS-induced zebrafish inflammatory model was constructed to explore the role of krt8 in inflammatory responses. In vivo bacterial infection assays in zebrafish were conducted to further clarify the regulatory effect of KRT8 on inflammation. Moreover, candidate antimicrobial peptides were screened based on the KRT8 sequence, and their antibacterial activities were verified subsequently. Results: KRT8 was capable of binding to PGN, LPS, Gram-negative and Gram-positive bacteria. In the LPS-induced inflammatory model, the expression of krt8 was significantly altered, suggesting that krt8 is involved in the inflammatory response. Further in vivo experiments confirmed that KRT8 could alleviate bacteria-triggered inflammatory reaction. The derived peptide mP90-103 screened from KRT8 exerted direct bactericidal activity with no hemolytic toxicity. Conclusion: KRT8 acts as a pattern recognition receptor and participates in the regulation of inflammatory immunity in zebrafish. Its derivative peptide mP90-103 possesses favorable antibacterial activity and high biosafety. This study provides novel insights into the immunological functions of keratins among diverse animal species.
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