牛血红蛋白β亚基片段衍生抗菌肽的分子设计及其抗菌活性分析

doi:10.16431/j.cnki.1671-7236.2022.09.033

摘要/Abstract

摘要： 【目的】试验旨在获得分子质量小、溶血性低、稳定性好的抗菌肽。【方法】以牛血红蛋白β亚基的氨基酸序列为基础，以抗菌肽的构效理论为指导，筛选出1条多肽（YKK-18），并以其为模板肽设计了3条长度为18个氨基酸残基的改造多肽（LJ-1、LJ-2和DLK-3），通过最小抑菌浓度的测定反映4条多肽的抗菌活性，采用琼脂糖扩散法分析有抗菌活性多肽的热稳定性、酸碱稳定性、盐离子稳定性和反复冻融稳定性，用分光光度法测定有抗菌活性多肽的溶血性。【结果】设计的4条多肽均为带正电荷的亲水性多肽，与APD3数据库中收录的抗菌肽氨基酸序列相似性均低于50%，二级结构中均有较高含量的α-螺旋，改造多肽的两亲性程度均高于模板肽，模板肽对测试菌株没有抗菌活性，而改造多肽对测试的大肠杆菌、沙门菌、绿脓假单胞菌、葡萄球菌、白色念珠菌等均有不同程度的抗菌活性，其中DLK-3和LJ-1的抗菌活性优于LJ-2，但对奇异变形杆菌均没有抗菌活性，在100 ℃高温、pH 4.0~10.0、生理浓度的盐离子（Na⁺、K⁺、Ca²⁺、Mg²⁺、Fe³⁺、Cu²⁺）和14次反复冻融等条件下仍有较好的抗菌活性，DLK-3的溶血作用呈剂量依赖性，在200 μg/mL时的溶血率已超过50%，而LJ-1和LJ-2的溶血率在高浓度（200 μg/mL）时仍低于5%。【结论】本研究结果为抗菌肽的分子设计和改造提供了参考，获得的抗菌肽LJ-1和LJ-2具有成为抗生素替代物的潜力。

关键词: 牛血红蛋白; 抗菌肽; 分子设计; 抗菌活性; 稳定性; 溶血作用

Abstract: 【Objective】 This research was aimed to obtain antimicrobial peptides with low molecular weight, low hemolysis and good stability.【Method】 One peptide (YKK-18) was selected from the amino acid sequence of bovine hemoglobin β-subunit with the guidance of the structure-activity theory of antimicrobial peptides, and three modified peptides (LJ-1, LJ-2 and DLK-3) with 18 amino acid residues were designed using YKK-18 as a template.The antimicrobial activities of four peptides were reflected by minimum inhibitory concentration (MIC), the thermal stability, acid-base stability, salt ion stability and repeated freeze-thaw stability of peptides with antimicrobial activities were analyzed by agarose diffusion method, and the hemolysis of peptides with antimicrobial activities was determined by spectrophotometry.【Result】 The four designed peptides were all positively charged hydrophilic peptides, and the similarity of amino acid sequence were less than 50% compared with antimicrobial peptides in APD3 database.Secondary structure of four designed peptides had high content of alpha helix.The amphiphilic degree of modified peptides were high than template peptide.The template peptide YKK-18 had no antimicrobial activity against all tested strains, while three modified peptides had different degrees of antimicrobial activity against Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus and Candida albicans. DLK-3 peptide and LJ-1 peptide had better antimicrobial activity than LJ-2. But four designed peptides had no antimicrobial activity against Proteus mirabilis.Under the conditions of high temperature of 100 ℃, pH 4.0 to 10.0, physiological concentration of salt ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Cu²⁺) and 14 times of repeated freezing and thawing, three modified peptides had good antimicrobial activity.The hemolysis of DLK-3 was dose-dependent, and at 200 μg/mL the hemolytic rate was over 50%.However, the hemolytic rates of LJ-1 and LJ-2 were still lower than 5% at high concentration (200 μg/mL).【Conclusion】 These findings would provide a reference for molecular design and modification of antimicrobial peptides, and the obtained antimicrobial peptides LJ-1 and LJ-2 would have the potential to be used as substitutes for antibiotics.

Key words: bovine hemoglobin; antimicrobial peptide; molecular design; antimicrobial activity; stability; hemolysis

中图分类号:

S859.79

邓丽, 李杰, 刘保国, 杭柏林. 牛血红蛋白β亚基片段衍生抗菌肽的分子设计及其抗菌活性分析[J]. 中国畜牧兽医, 2022, 49(9): 3589-3598.

DENG Li, LI Jie, LIU Baoguo, HANG Bolin. Molecular Design and Antimicrobial Activity Analysis of Antimicrobial Peptides Derived from Bovine Hemoglobin β-subunit[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3589-3598.

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