替代动物抗生素的多功能杂合肽分子设计、重组表达、生物学特性和机理研究

The goal of the project aims to provide solutions for problems hindered the healthy and sustainable development of animal husbandry, for example, 1) a variety of problems in animal disease-resistance and growth-promotion caused by used excessive antibiotics in the long term, 2) gram-negative (G-) pathogen infecting and releasing a large amount of endotoxin LPS induces inflammation, oxidative damage, and hypoimmunity, and 3) antibiotics cannot eliminate endotoxin and additionally release more endotoxin. In order to achieve the goal, the project will use database, of immune peptide and anti-inflammatory peptide sequences of various biological information, and a lot of literature with computational and bioinformatics tools to, firstly, design a new molecular of immunomodulatory and anti-inflammatory hybrid peptide (IAHP) and then chemical synthesize multiple hybrid peptides. Use screened high-activity IAHP to construct high efficient engineering bacterial or yeast. To study the biological characteristics and mechanism, the project team will heterologous express and purify the selected IAHP. Subsequently, to produce the anti-microbial, antiviral, immunomodulatory, and anti-inflammatory hybrid peptide (AMVIIHP), the team will re-hybrid the IAHP and the previous obtained antibacterial and antiviral hybrid peptide (AMVHP). Recombinant expression and purification of this hybrid peptide are performed to gain the new recombinant AMVIIHP. After that, through vitro tests and challenge experiments in broilers, the research will focus on studying the biological characteristics, mechanism of action, and antibiotic substitution effects of the recombinant ANVIIHP. The results of the research will provide a brand new, safety and environmental friendly strategy for anti-infection and substitution of antibiotics, and provide the new thoughts, methods, and technologies to study double or multiple effect hybrid peptides.

针对阻碍畜牧业健康持续发展的瓶颈问题：1)为动物抗病和促生长而长期使用抗生素引发的多种问题、2)G-病原菌感染释放大量内毒素LPS诱发炎症、氧化损伤和免疫力下降、3)抗生素无法消除内毒素而且促进内毒素释放等问题。本项目利用生物信息数据库中的免疫肽和抗炎肽序列及大量文献，借助计算机和生物信息工具，设计出新型免疫抗炎杂合肽(IAIHP)分子，化学合成多条IAIHP，筛选出高活性IAIHP，然后构建工程菌、表达和分离纯化，并研究其生物学特性和机理。随后将IAIHP与本实验室前期获得的抗菌抗病毒杂合肽进行二次杂合，形成抗菌抗病毒-免疫抗炎多功能杂合肽(AMVIIHP)，再重组表达、分离纯化，获得AMVIIHP。通过肉仔鸡攻毒和体外试验研究其生物学特性、作用机理和抗生素替代效果，研究成果将提供全新的、安全环保的抗感染和抗生素替代品策略，并为研究新型双效或多效杂合肽提供公用的新思路、新方法和新技术。

针对阻碍畜牧业健康持续发展的瓶颈问题:1)为动物抗病和促生长而长期使用抗生素引发的多种问题、2)G-病原菌感染释放大量内毒素LPS诱发炎症、氧化损伤和免疫力下降、3)抗生素无法消除内毒素而且促进内毒素释放等问题。本项目利用生物信息数据库中的免疫肽和抗炎 肽序列及大量文献，借助计算机和生物信息工具，设计出新型免疫抗炎杂合肽(IAIHP)分子， 化学合成多条IAIHP，通过体外试验筛选得到3条兼具免疫抗炎活性的杂合肽，并对其利用小鼠攻毒试验进行了抗炎、免疫体内活性验证，结果表明杂合肽能很好的缓解LPS诱导的小鼠肠道炎症，及CTX诱导的免疫抑制现象。然后构建工程菌、表达和分离纯化。随后将IAIHP与本实验室前期获得的抗菌抗病毒杂合肽进行二次杂合，形成抗菌抗病毒-免疫抗炎多功能杂合肽(AMVIIHP)，利用体外试验筛选获得1条兼具以上四种活性的杂合肽，并利用体外试验探究其抗菌、抗病毒机制，结果表明LPML可通过中和LPS，破坏细菌细胞膜，产生广谱抑菌效果，并能通过抑制RNA病毒复制，具有广谱抗RNA病毒活性。利用小鼠试验验证其体内抗菌、免疫、抗炎活性及机制。结果表明，杂合肽LPML可有效缓解LPS及大肠杆菌EHEC诱导的小鼠肠道炎症，并能增强CTX诱导的免疫抑制小鼠的免疫活性。对杂合图LPML进行了重组表达、分离纯化，获得AMVIIHP。研究成果将提供全新的 、安全环保的抗感染和抗生素替代品策略，并为研究新型双效或多效杂合肽提供公用的新思路 、新方法和新技术。