靶向抗菌肽的分子设计，表达及应用研究

The extensive use of broad-spectrum antibiotics in clinical practice has led to the rising prevalence of medically relevant multidrug-resistant pathogens worldwide. In addition, the application of broad-spectrum antimicrobials often results in killing pathogenic and beneficial microorganisms indiscriminately. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. Antimicrobial peptides (AMPs) were admired for their excellent antimicrobial activity and distinct action mechanism from antibiotics. Previously, we have designed and optimized α-helix and β-sheet antimicrobial peptides, and exploited the relationship between structure and function. A few optimized or ideal peptide sequences were obtained. In this study, we further design excellent antimicrobial peptides with specificity by tuning physicochemical parameters and synthesizing with pheromone, and evaluate the in vitro and in vivo antimicrobial activity, with the aim to obtain optimal peptide sequence of targeting action. Then, action mechanisms of these peptides will be further investigated and different protein expression system, such as Escherichia. coli, Pichia pastoris will be constructed, and the optimal peptide sequences will be expressed. The in vitro and iv vivo biological activity of the expressed antimicrobial peptides will be evaluated, and we believe that this work will further lay the foundation for the application of the "smart" antimicrobial peptides.

广谱抗生素的大量使用不仅导致了耐药菌在世界范围内频繁出现，而且其在清除病原菌的同时也杀灭了正常细菌，对机体微生态平衡造成了严重破坏，在临床上经常出现继发感染等负面后果。抗菌肽以其卓越的抗菌活性及与抗生素不同的作用机制为克服抗生素耐药性的出现而倍受青睐。本课题组已于前期对α-螺旋和β-折叠抗菌肽进行了结构改造，探讨了其结构和功能的关系，有了扎实的工作基础。为了进一步筛选出具有特异抗菌活性的抗菌肽并深入研究其作用机理最后实现量化生产，在原有基础上，通过序列优化及嵌合的方式进一步对抗菌肽进行靶向改造，通过体内、外生物学活性测定，筛选出2-3种理想型的抗菌肽，在确定了其作用机理及信号转导通路后通过大肠杆菌、毕赤酵母等系统建立表达体系，最终筛选出理想的专杀型抗菌肽分子进行发酵罐扩大试验，并进行动物体内实验，评价其体内生物学效价和治疗潜力，为生产和应用“智能型”抗菌肽奠定基础。

通过天然抗菌肽库筛选及结构优化设计出以F4为代表的短序列靶向革兰氏阴性菌抗菌肽；利用无乳链球菌的信息素为靶向段，以阳离子细胞穿透肽作为穿透段，设计具有特异性抗无乳链球菌及胞内杀菌能力的多重功能抗菌肽L1、L2；利用具有识别效果的靶向结构域和具有抗菌活性的杀菌结构域设计了靶向金黄色葡萄球菌抗菌肽S2，且不易产生耐药性；以WKKIWKDPGIKKWIK多肽序列为模板，通过截断、延长、增减电荷数和改变两亲性等参数，将特异性识别结合大肠杆菌的噬菌体展示肽与优化模板杂合，设计出靶向大肠杆菌抗菌肽KIGQK；通过氨基酸替换的方式降低杀菌区域中正电荷数量，将广谱抗菌肽C4与粪肠球菌信息素cCF10连接构建特异性靶向粪肠球菌抗菌肽；利用枯草芽孢杆菌表达系统和毕赤酵母表达系统实现了抗菌肽T9W的重组表达。