猪乳铁蛋白肽的分子改良及改良肽的生物学活性研究

Molecular Improvement of Porcine Lactoferricin and Biological Activity of the Modified Peptides

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（MIC）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MIC为64~128 μg/mL，改良肽LF-1和LF-3的MIC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256 μg/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，25~200 μg/mL的LF-1显著提高了PBMC的增殖（P〈0.05），但在400 μg/mL浓度下却抑制了PBMC的增殖（P〈0.05）；200和400 μg/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25~50 μg/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，4 μg/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。

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（P0.05）.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（P0.05）,while 400 μg/mL LF-1 decreased cell proliferation（P0.05）.Both 200 and 400 μg/mL LF-1 induced an increase in lactate dehydrogenase（LDH） release from PBMC（P0.05） whereas 25 to 50 μg/mL decreased the LDH release（P0.05）.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.