抗菌肽Lc-NKlysin-1a的抗菌稳定性及其抗菌机理

Antimicrobial Stability and Mechanism of Antimicrobial Peptide Lc-NKlysin-1a

Lc-NKlysin-1是从海洋鱼类——大黄鱼(Larimichthys crocea)中获得的抗菌肽,为进一步优化该抗菌肽和研究其结构与功能的关系,本文截取了其N末端的12个氨基酸残基,设计并合成了抗菌肽Lc-NKlysin-1a,同时,通过琼脂板打孔法,对其抗菌活性,不同温度、pH值、盐浓度等对其抗菌活性的影响,其溶血活性,最低抑菌质量浓度,其抗菌特征等进行了研究,并在扫描电镜下观察了其对细菌形态结构的影响.结果表明:抗菌肽LcNKlysin-1a对革兰氏阳性菌(金黄色葡萄球菌、枯草芽孢杆菌)和革兰氏阴性菌(大肠杆菌、副溶血弧菌、铜绿假单胞菌、抗链霉素大肠杆菌)均具有抗菌活性;但对金黄色葡萄球菌、大肠杆菌和抗链霉素大肠杆菌的抗菌活性最好,其最低抑菌质量浓度(MIC)在15.62531.25μg·mL^(-1)之间;相对于Lc-NKlysin-1,Lc-NKlysin-1a的抗菌活性明显增强;Lc-NKlysin-1a在15.62531.25μg·mL^(-1)时,其溶血率为3.80%7.37%,溶血活性较低;Lc-NKlysin-1a对温度、pH和盐等具有良好的耐受性,且稳定性高.此外,通过扫描电镜还观察到,抗菌肽Lc-NKlysin-1a作用于细菌后细菌的形态发生了明显改变,其细胞膜出现了褶皱和塌陷的现象,最终导致了内溶物外泻而死亡.改造后的抗菌肽其相对分子质量小,活性高,稳定性强,更易于被开发成抗菌肽类药物.

The antimicrobial peptide, Lc-NKlysin-1, was obtained from marine fish, the large yellow croaker （Larimiehthys crocea）. In the report, in order to study the relationship between structure and function of Lc- NKlysin-1, Lc-NKlysin-1a （12 N-terminal amino acid residues of Lc-NKlysin-1 ） was designed and synthesized. The effects of temperature, pH and salt concentration on antibacterial activity of Lc-NKlysin-1 a were determined by agar plate drilling method. The hemolytic activity of Lc-NKlysin-1 a was measured by hemoglobin releasing method. The minimum inhibitory concentration （MIC） of antimicrobial peptide was determined by two fold dilution method. The growth curve was drawn to analyze the antimicrobial characteristics of Lc-NKlysin-I a. Under scanning electron microscope, the effects of Lc-NKlysin-1a on the morphology and structure of bacteria was ob- served. The results indicated that Lc-NKlysin-1a has the antimicrobial activities against Gram-positive strains, Staphylococcus aureus , Bacillus subtilis , and Gram-negative strains, Escherichia coli , Pseudomonas aeruginosa , Vibrio and streptomycin-resistent E. coli. And the MIC of Lc-NKlysin-1 a against S. aureus, E. coli, and streptomycin-resistant E. coli were 15. 625-31.25 μg· mL-1. The antimicrobial activities of Lc-NKlysin-1 a were stronger than that of Lc-NKlysin-1. Lc-NKlysin-1 a had high stability against temperature, pH and salt concentration. After being treated with Lc-NKlysin-1a, the cell membrane was folded and collapsed, and leading to cell death. The higher antimicrobial activity, lower molecular mass and stabilities of Lc-NKlysin-1 a make it to be a promising candidate of novel antimicrobial agents or models for the development of novel antimicrobial peptides.