黄粉虫抗菌肽TmAMP1m的原核表达及其与传统抗生素的联用研究  

Prokaryotic Expression of Antimicrobial Peptide TmAMP1m from Tenebrio molitor(Coleoptera:Tenebrionidae)and Its Combination with Conventional Antibiotics

作　　者：冯印 谢晓倩 颜鑫 周圆媛 仇伍霞 袁晓琴 毛新芳 刘忠渊 FENG Yin;XIE Xiaoqian;YAN Xin;ZHOU Yuanyuan;QIU Wuxia;YUAN Xiaoqin;MAO Xinfang;LIU Zhongyuan(College of Chemical Engineering,Sichuan University of Science&Engineering,Zigong 643000,China)

机构地区：[1]四川轻化工大学化学工程学院,自贡643000

出　　处：《中国生物工程杂志》2025年第2期34-43,共10页China Biotechnology

基　　金：国家自然科学基金(32260072)资助项目。

摘　　要：目的:原核表达黄粉虫(Tenebrio molitor)抗菌肽TmAMP1m,研究其单独使用及与传统抗生素联用时的抑菌活性,并探索其与传统抗生素联用产生协同效应的机制。方法:构建原核表达载体pET-30a-TmAMP1m,转化大肠杆菌Escherichia coli BL21异源表达,SDS-PAGE鉴定重组蛋白,对重组蛋白进行纯化、定量,测定其单独使用及联用的抑菌效果,并探索联用时产生协同效应的机制。结果:通过原核表达得到了目的蛋白TmAMP1m,经过蛋白质纯化后得到了浓度为57μg/mL的黄粉虫抗菌肽TmAMP1m,测定了TmAMP1m的抑菌谱,发现其对大肠杆菌、金黄色葡萄球菌、白念珠菌、铜绿假单胞菌、枯草芽孢杆菌、痤疮丙酸杆菌等常见菌有抑菌效果;测定了TmAMP1m对部分菌的最小抑菌浓度和最小杀菌浓度;测定了黄粉虫抗菌肽TmAMP1m与传统抗生素氨苄青霉素、庆大霉素、环丙沙星、头孢吡肟、米诺环素、多黏菌素B等的联用效果,发现其与部分抗生素联用时对部分菌有协同作用;联用时,抑菌组合对细菌细胞膜有明显的破坏作用,导致细胞内容物外流;对呼吸链脱氢酶有明显抑制效果。结论:TmAMP1m与抗生素联用有很好的抑菌效果,在对细菌细胞膜的破坏、细菌呼吸链的影响方面均有加强。Objective:To express Tenebrio molitor antimicrobial peptide TmAMP1m in prokaryotic cells,to study its antibacterial activity when used alone or in combination with traditional antibiotics,and to explore the mechanism of synergistic effect between Tenebrio molitor and conventional antibiotics.Methods:The prokaryotic expression vector pET-30a-TmAMP1m was constructed and transformed into Escherichia coli BL21 for heterologous expression.The recombinant protein was identified by SDS-PAGE,the recombinant protein was purified and quantified,the bacteriostatic effect of its single and combined use was determined,and the mechanism of synergistic effect was explored.Results:The target protein TmAMP1m was obtained by prokaryotic expression,and the antimicrobial peptide TmAMP1m with a concentration of 57μg/mL was obtained after protein purification.The antibacterial spectrum of TmAMP1m was determined,and it was found that it has bacteriostatic effect on Escherichia coli,Staphylococcus aureus,Candida albicans,Pseudomonas aeruginosa,Bacillus subtilis,Propionibacterium acne and other common bacteria,and the minimum inhibitory concentration and minimum bactericidal concentration of TmAMP1m on some bacteria were determined.The combined effect of antimicrobial peptide TmAMP1m with traditional antibiotics such as ampicillin,gentamicin,ciprofloxacin,cefepime,minocycline and polymyxin B was tested,and it was found that it has synergistic effect against some bacteria when combined with some antibiotics.The bacteriostatic combination had an obvious destructive effect on the bacterial cell membrane,resulting in leakage of cell contents and an obvious inhibitory effect on the respiratory chain dehydrogenase.Conclusion:The combination of TmAMP1m and antibiotics has a good bacteriostatic effect,is not easy to produce drug resistance,and strengthens the destruction of bacterial cell membrane and the effect of bacterial respiratory chain.

关 键 词：黄粉虫 抗菌肽 原核表达 联合使用 抑菌机制 

分 类 号：Q815[生物学—生物工程]