单 位：
浙江大学饲料科学研究所动物分子营养学教育部重点实验室浙江省动物饲料与营养重点实验室
关键词：
猪;乳铁蛋白肽;抗菌活性;溶血性;细胞毒性
摘 要：
本研究采用化学合成的方法获得了猪源乳铁蛋白肽LFP-20及其改良肽,旨在通过研究该抗菌肽一级结构与抗菌活性的关系,获得抗菌活性更高的改良肽。微量肉汤稀释法测定的最小抑菌浓度(M IC)及溶血性分析结果表明,猪乳铁蛋白肽LFP-20及其改良肽LF2A、LF-1和LF-3对大肠杆菌、猪霍乱沙门氏菌、鼠伤寒沙门氏菌、金黄色葡萄球菌及表皮葡萄球菌均具有抗菌活性;LFP-20对5种试验菌株的M IC为64~128μg/mL,改良肽LF-1和LF-3的M IC降低了2~4倍;改良肽LF2A对5种试验菌株的抗菌活性没有提高,但溶血性降低;改良肽LF-1的抗菌活性有所提高,但在4、32、64、128和256μg/mL时的溶血性也显著提高(P<0.05)。WST-1和LDH法检测4种抗菌肽对人外周血单核细胞(PBMC)增殖的影响及细胞毒性结果表明,LF2A、LF-1和LF-3对PBMC增殖的影响具有剂量依赖性;与LFP-20相比,25~200μg/mL的LF-1显著提高了PBMC的增殖(P<0.05),但在400μg/mL浓度下却抑制了PBMC的增殖(P<0.05);200和400μg/mL LF-1使得PBMC的乳酸脱氢酶(LDH)释放百分率显著提高(P<0.05),在25~50μg/mL浓度下却对LDH释放具有降低作用(P<0.05)。对细胞膜去极化作用研究结果表明,4μg/mL的改良肽LF-3对大肠杆菌细胞膜的去极化作用明显增强,而抗菌活性较低的LFP-20和LF2A对细菌细胞膜的去极化作用低于LF-3。由此可知,改良肽LF-3的抗菌活性比猪乳铁蛋白肽LFP-20提高了2~4倍,且溶血作用和细胞毒性均没有显著增强;LF-3可通过对细胞膜的去极化作用破坏大肠杆菌细胞膜电势梯度平衡,推测对细胞膜的破坏作用是其发挥抗菌作用的途径之一。
译 名：
Molecular Improvement of Porcine Lactoferricin and Biological Activity of the Modified Peptides
作 者：
HAN Feifei AN Sha XIE Yonggang LIU Yifan WANG Yizhen (Institute of Feed Science,Key Laboratory for Molecular Animal Nutrition of Ministry of Education,Key Laboratory for Feed and Animal Nutrition of Zhejiang Province,Zhejiang University,Hangzhou 310029,China)
关键词：
porcine;lactoferricin;antimicrobial activity;hemolytic activity;cytotoxicity
摘 要：
In this study,porcine lactoferricin LFP-20 and its analogs were prepared by chemical synthesis with an aim to understand structure-function relationships of these peptides and thereby to obtain improved analogs.The minimum inhibitory concentration(MIC) measured by broth microdilution method and hemolytic analysis showed that the 20-residue porcine lactoferricin(LFP-20) and its analogs LF2A,LF-1,and LF-3 displayed the antimicrobial activity against Escherichia coli,Salmonella choleraesuis,Salmonella typhimurium,Staphylococcus aureus and Staphylococcus epidermidis.The minimum inhibitory concentrations of LFP-20 ranged from 64 to 128 μg/mL,and LF-1 and LF-3 were 2 to 4 times more effective than LFP-20.The studies demonstrated that the analog LF2A,replacing the 2-and 17-Cys of LFP-20 with Ala,did not show increased activities against bacteria,but exhibited decreased hemolytic activity.The analog LF-1,replacing the 9-and 18-Ile of LFP-20 with Trp,showed improved antimicrobial activity.But the hemolytic activity of LF-1 was also increased at 4,32,64,128,and 256 μg/mL(P<0.05).The cytotoxic potential of LFP-20 analogs was quantified by colorimetric WST-1 and LDH assays in peripheral blood mononuclear cell(PBMC).LF2A,LF-1 and LF-3 increased cell proliferation and viability in a dose dependent fashion.Compared with LFP-20,25 to 200 μg/mL LF-1 improved significantly cell proliferation(P<0.05),while 400 μg/mL LF-1 decreased cell proliferation(P<0.05).Both 200 and 400 μg/mL LF-1 induced an increase in lactate dehydrogenase(LDH) release from PBMC(P<0.05) whereas 25 to 50 μg/mL decreased the LDH release(P<0.05).Moreover,LF-3 exhibited obviously enhanced potential to depolarize the cytoplasmic membranes at relatively low concentrations(4 μg/mL).In contrast,LFP-20 and LF2A had more modest antibacterial activities,and a weaker ability to depolarize the cytoplasmic membrane.In conclusion,the antimicrobial activity of LF-3 is found to be 2 to 4 folds higher than that of LFP-20,which dose not couple with increased heomolysis and cytotoxicity to PBMCs.Moreover,LF-3 can disrupt the membrane potential by depolarizing the bacterial membrane,which is proposed to be one of the mechanisms of action of LF-3.
