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 (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.［Chinese Journal of Animal Nutrition，2011，23（2）：241-249］