摘要
目的合成并筛选出具有良好体外抗菌活性的抗菌肽(AMP)。方法设计AMP序列并合成具有4条相似肽链的混合样品(肽库),将其与细菌细胞膜色谱固定相混合,通过高效液相色谱仪(HPLC)检测各混合物的峰面积变化率,以筛选目标AMP。通过琼脂平板法检测目标AMP的最小抑菌浓度(MIC)和抗细菌耐药性。通过四甲基偶氮唑盐比色法(MTT法)检测目标AMP的细胞相容性。结果HPLC检测结果显示,AMP RIKAHFKF分别与金黄色葡萄球菌和大肠杆菌细胞膜色谱固定相混合后溶液的峰面积变化率均较空白对照显著升高(F=15720.00,t_(LSD)=161.40、112.70,P<0.05)。MIC检测结果显示,AMP RIKAHFKF对金黄色葡萄球菌、大肠杆菌的MIC分别为0.060、0.100 g/L。耐药性检测结果显示,金黄色葡萄球菌、大肠杆菌与AMP RIKAHFKF共孵育10代后,其生长仍可被完全抑制。MTT法检测结果显示,与0.1 g/L相比,0.2和0.4 g/L AMP RIKAHFKF处理后L929细胞存活率无显著变化(P>0.05),而0.6、0.8、1.0 g/L AMP RIKAHFKF处理后L929细胞存活率显著降低(F=7.95,t_(LSD)=3.00~18.55,P<0.05)。结论AMP RIKAHFKF对金黄色葡萄球菌和大肠杆菌具有有效的生长抑制和抗细菌耐药性作用,并且具有较好的细胞相容性。
Objective To synthesize and screen for an antimicrobial peptide(AMP)with strong in vitro antibacterial activity.Methods The AMP sequence was designed,and a mixture(peptide library)consisting of four similar peptide chains was synthesized.The mixture was incubated with a bacterial cell membrane chromatographic stationary phase,and changes in peak area were detected using high-performance liquid chromatography(HPLC)to screen for the target AMP.The minimum inhibitory concentration(MIC)and antimicrobial resistance of the target AMP were determined using the agar plate method.The cytocompatibility of the target AMP was assessed by the tetrazolium salt colorimetric assay(MTT method).Results HPLC results showed that the peak area change rates of the solution after mixing AMP RIKAHFKF with Staphylococcus aureus and Escherichia coli cell membrane chromatographic stationary phases were significantly higher than those of the blank control(F=15720.00,t SLD=161.40,112.70,P<0.05).MIC testing showed that the MIC values of AMP RIKAHFKF against Staphylococcus aureus and Esc-herichia coli were 0.060 and 0.100 g/L,respectively.Resistance testing demonstrated that after10 generations of co-incubation with AMP RIKAHFKF,the growth of Staphylococcus aureus and Escherichia coli was completely inhibited.MTT assay results that compared to treatment with 0.1 g/L AMP RIKAHFKF,there was no significant change in the survival rate of L929 cells treated with 0.2 or 0.4 g/L AMP RIKAHFKF(P>0.05),whereas treatment with 0.6,0.8,or 1.0 g/L AMP RIKAHFKF significantly reduced cell viability(F=7.95,t_(LSD)=3.00-18.55,P<0.05).Conclusion AMP RIKAHFKF exhibits effective inhibitory activity against Staphylococcus aureus and Escherichia coli,including antibiotic-resistant strains,and demonstrates good cytocompatibility.
作者
李鉴人
徐子源
陈映欣
寇兆君
马学真
LI Jianren;XU Ziyuan;CHEN Yingxin;KOU Zhaojun;MA Xuezhen(Qingdao Medical College of Qingdao University,Qingdao 266042,China)
出处
《精准医学杂志》
2025年第6期486-491,共6页
Journal of Precision Medicine
关键词
抗菌肽类
色谱法
固态合成技术
抗药性
细菌
微生物敏感性试验
Antimicrobial peptides
Chromatographic method
Solid state synthesis technology
Drug resistance,bacteria
Microbial sensitivity test
