新型杂合肽DY-CA的构建及抑菌活性评价  

Construction and antibacterial activity evaluation of a new hybrid peptide DY-CA

作　　者：邵学超 赵潇颖 王丹 马嘉辉 赵远 宋顺佳 贾慧建 孙丽媛 赵云冬 SHAO Xue-chao;ZHAO Xiao-ying;WANG Dang;MA Jia-hui;ZHAO Yuan;SONG Shun-jia;JIA Hui-jian;SUN Li-yuan;ZHAO Yun-dong(School of medical technology,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学医学技术学院,吉林132013

出　　处：《药物分析杂志》2022年第12期2082-2091,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：吉林省科技发展计划项目(20190301014NY,20190304115YY);吉林省教育厅“十三五”科学技术项目(JJKH20190657KJ);北华大学研究生创新项目(2021025);北华大学大学生创新创业训练计划项目(202210201074)。

摘　　要：目的:构建高活性、低毒性的新型杂合肽,对其抗肺炎克雷伯菌活性进行评价,探讨其在防治肺炎克雷伯菌引起的呼吸道感染的潜在应用价值。方法:通过生物信息学软件分析亲本肽dyskinbow-1ST的理化性质,预测杂合肽活性区域,确定与天蚕素Cecropin A的构建策略;应用生物信息学软件分预测杂合肽的理化性质及抑菌活性;通过固相法合成杂合肽DY-CA,用杂合肽对肺炎克雷伯菌的最小抑菌浓度(MIC)试验,杂合肽对肺炎克雷伯菌的时间生长曲线,羊血红细胞溶血试验,分析DY-CA的抑菌活性及生物相容性;通过结晶紫染色定量法观察生物膜的改变,研究杂合肽对肺炎克雷伯菌生物膜的抑制与清除效率。结果:经在线软件预测分析,杂合肽DY-CA为稳定的带正电荷疏水性抗菌肽,试验测得杂合肽的MIC为64μg·mL^(-1);对肺炎克雷伯菌的抑菌作用具有时间和浓度依赖性,杂合肽在3 h后表现出明显的抑制作用,2MIC、1MIC的杂合肽A基本上没有变化,肺炎克雷伯菌的生长完全被抑制;在有效浓度下,羊血红细胞溶血率为4.23%(<5%)为阴性;0.5MIC(t_(0.5MIC)=11.10,P<0.05)、1MIC(t_(1MIC)=17.96,P<0.05)、2MIC(t_(2MIC)=14.18,P<0.05)的杂合肽DY-CA均可抑制肺炎克雷伯菌生物膜的形成;0.5MIC(t_(0.5MIC)=11.10,P<0.05)的杂合肽DY-CA对肺炎克雷伯菌成熟生物膜清除效果不明显,1MIC(t_(1MIC)=17.96,P<0.05,)、2MIC(t_(2MIC)=14.18,P<0.05)的杂合肽DY-CA可显著破坏肺炎克雷伯菌成熟生物膜。结论:本试验设计构建并筛选了具有高活性、低毒性的新型杂合肽DY-CA,对肺炎克雷伯菌具有高效、快速的抑菌作用,有望成为防治肺炎克雷伯菌引起的呼吸道疾病的新型药物。Objective: To construct a new hybrid peptide with high activity and low toxicity, to evaluate its anti Klebsiella pneumoniae activity, and explore its potential application value in the prevention and treatment ofrespiratory tract infection caused by Klebsiella pneumoniae. Methods: The physical and chemical properties of parental peptide dyskinbow-1ST were analyzed by bioinformatics software. Its active region was predicted and the construction strategy of cecropina was determined. Bioinformatics software was used to predict the physical and chemical properties of hybrid peptides and their antibacterial activity. The heterozygous peptide DY-CA was synthesized by solid-phase method. The minimum inhibitory concentration(MIC) of heterozygous peptide against Klebsiella pneumoniae was studied. The time growth curve of heterozygous peptide against Klebsiella pneumoniae was plotted. The hemolysis test of sheep blood red blood cells was performed. The antibacterial activity and biocompatibility of DY-CA were analyzed. The changes of biofilm were observed by crystal violet staining to study the inhibition and clearance efficiency of heterozygous peptides on Klebsiella pneumoniae biofilm. Results: The hybrid peptide DY-CA was predicted to be a stable hydrophobic antibacterial peptide with positive charge by online software, and the MIC of the hybrid peptide was 64 μg·mL^(-1). The inhibitory effect on Klebsiella pneumoniae was time and concentration dependent. The heterozygous peptides showed obvious inhibitory effect after 3 h. The A of 2 MIC and 1MIC heterozygous peptides basically did not change and the growth of Klebsiella pneumoniae was completely inhibited. At the effective concentration, the hemolysis rate of sheep red blood cells was 4.23%(<5%), which was negative;0.5 MIC(t_(0.5MIC)=11.10, P<0.05), _(1MIC)(t_(1MIC)=17.96, P<0.05), 2 MIC(t_(2MIC)=14.18, P<0.05) DY-CA could inhibit the formation of Klebsiella pneumoniae biofilm;0.5 MIC(t_(0.5MIC)=11.10, P<0.05) DY-CA had no obvious effect on the clearance of mature

关 键 词：dyskinbow-1ST 天蚕素 杂合肽 生物信息学 抑菌活性 生物膜 

分 类 号：R917[医药卫生—药物分析学]