CN114516900A - 抗菌肽yhx-4及其应用 - Google Patents
抗菌肽yhx-4及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及一种抗菌肽YHX‑4及其应用。针对现有的抗菌肽不能满足临床治疗需求的上述问题，本发明提供一种抗菌肽YHX‑4及其应用。所述抗菌肽YHX‑4的氨基酸序列为：Trp‑Lys‑Leu‑Leu‑Ser‑Lys‑Leu‑Leu‑Gly‑Lys‑Leu‑Leu‑Lys‑NH2。本发明的抗菌肽对单核细胞增生李斯特氏菌、金黄色葡萄球菌、变异链球菌、大肠杆菌及沙门氏菌都有较好的抗菌活性。本发明的抗菌肽结构简单，合成难度小，安全性好。
Description
技术领域
本发明属于生物技术领域，具体涉及一种抗菌肽YHX-4及其应用。
背景技术
传统抗生素的广泛应用，在为人类抗击病原体提供有力武器的同时，也带来了严重的耐药性问题。根据中国细菌耐药性试验发布的数据，细菌耐药性逐年增加，一些细菌表现出多重耐药性和交叉耐药性，对治疗临床感染构成了严重的威胁。因此解决病原菌的耐药性问题已经刻不容缓。而开发新型抗菌药物是解决此问题的一个重要途径。其中抗菌肽凭借独特的穿膜机制以及快速的杀菌效率，不易产生耐药性，成为抗生素最有力的替代者。
抗菌肽(antimicrobial peptides，AMPs)，又称宿主防御肽，是一类小分子多肽，氨基酸总数一般在50以内。抗菌肽具有包括抗细菌、抗真菌、抗病毒、杀灭寄生虫、抑制肿瘤以及调节免疫系统的多种生物活性。同时，其独特的膜破坏机制和胞内杀菌机制不易产生耐药性，因而具有广泛的应用前景。
然而，尽管目前已被开发出的抗菌肽数量达到了3000余种，但目前只有少数的抗菌肽被用于临床治疗，如乳酸链球菌肽、短杆菌肽和多粘菌素等。当前，大部分抗菌肽还存在生产成本高、生物活性较低以及易引发溶血等问题，并不能满足临床治疗需求。因此，开发结构简单、生物活性高同时无明显毒副作用的肽是目前迫切需要解决的问题。
发明内容
针对现有的抗菌肽不能满足临床治疗需求的上述问题，本发明提供一种抗菌肽YHX-4及其应用，本发明的抗菌肽对革兰氏阳性菌(单核细胞增生李斯特氏菌、金黄色葡萄球菌、变异链球菌)、革兰氏阴性菌(大肠杆菌、沙门氏菌)都有较好的抗菌活性，并且结构简单，合成难度小，安全性好。
为实现上述目的，本发明提供一种抗菌肽YHX-4，所述抗菌肽YHX-4的氨基酸序列为：Trp-Lys-Leu-Leu-Ser-Lys-Leu-Leu-Gly-Lys-Leu-Leu-Lys-NH2，在序列表中的位置为SEQ ID NO.1。所述抗菌肽YHX-4包含13个氨基酸残基，分子量为1540.01Da，净电荷数为+4。
本发明的一个方面，提供所述抗菌肽YHX-4的抗菌用途。
进一步，所述抗菌用途为抗单核细胞增生李斯特氏菌、金黄色葡萄球菌、变异链球菌、大肠杆菌以及沙门氏菌。
进一步，所述抗菌肽YHX-4对单核细胞增生李斯特氏菌的最小抑菌浓度为4μg/mL。
进一步，所述抗菌肽YHX-4对变异链球菌的最小抑菌浓度为16μg/mL。
进一步，所述抗菌肽YHX-4对沙门氏菌的最小抑菌浓度为4μg/mL。
进一步，所述抗菌肽YHX-4对大肠杆菌的最小抑菌浓度为8μg/mL。
进一步，所述抗菌肽YHX-4对金黄色葡萄球菌的最小抑菌浓度为32μg/mL。
本发明还提供一种生物抗菌剂，其抗菌成分包含上述抗菌肽YHX-4。
本发明还提供一种动物饲料，其含有上述抗菌肽YHX-4。
本发明还提供一种防腐剂，其含有上述抗菌肽YHX-4。
优选地，所述防腐剂为用于食品的防腐剂或用于化妆品的防腐剂。
本发明还提供一种洗涤剂组合物，其含有上述抗菌肽YHX-4。
优选地，所述组合物为洗手液、香皂、沐浴液、洗发剂、牙膏、洗衣液或洗衣粉等。
本发明的有益效果在于：
(1)本发明具有广谱的抗菌活性，能够抑制革兰氏阳性菌和革兰氏阴性菌。革兰氏阳性菌例如单核细胞增生李斯特氏菌、金黄色葡萄球菌、变异链球菌等；革兰氏阴性菌例如大肠杆菌、沙门氏菌等。特别是对单核细胞增生李斯特氏菌、沙门氏菌和大肠杆菌具有非常低的最小抑菌浓度。
(2)本发明所述的抗菌肽，溶血活性较低，合成序列短，分子量小，化学合成难度小，能够更加特异性地杀死生物体内的病原菌同时很好的节约规模化生产成本。
附图说明
图1为本发明实施例1提供的抗菌肽YHX-4的二级结构预测示意图。
图2为本发明实施例2提供的抗菌肽YHX-4在333μg/mL浓度条件下对单核细胞增生李斯特氏菌的抑菌效果示意图。
图3为本发明实施例2提供的抗菌肽YHX-4在333μg/mL浓度条件下对大肠杆菌的抑菌效果示意图。
图4为本发明实施例2提供的抗菌肽YHX-4在333μg/mL浓度条件下对变异链球菌的抑菌效果示意图。
图5为本发明实施例2提供的抗菌肽YHX-4在333μg/mL浓度条件下对沙门氏菌的抑菌效果示意图。
图6为本发明实施例2提供的抗菌肽YHX-4在333μg/mL浓度条件下对金黄色葡萄球菌的抑菌效果示意图。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。
实施例1抗菌肽的设计
在抗菌肽从头设计及对构效关系理解的基础上，对从ADP3数据库中筛选得到的对革兰氏阳性菌和革兰氏阴性菌有抑菌作用的843条抗菌肽序列进行序列参数分析(包括序列长度、带电荷数、疏水氨基酸比例和氨基酸组成)，之后按照优势参数选取原则并结合理性设计思路，确定了新抗菌肽的各项序列参数，具体如下：
表1从头设计抗菌肽的序列参数
为降低合成成本，同时降低细胞毒性，序列长度选择频率第二位的13。选取带正电氨基酸Lys和极性不带电荷氨基酸Ser组成抗菌肽的极性面，选取疏水性氨基酸Leu和芳香族氨基酸Trp形成疏水面，来保证抗菌肽的两亲性结构。其中，Trp吲哚基存在的带负电π-电子云，在与阳离子氨基侧链形成阳离子-π键的同时又能与脂质双分子层表面的氨基胆碱相互作用，可以进一步提高抗菌肽对细菌细胞膜的吸附力和穿透力。带电荷数确定为+4，保证抗菌肽对细菌细胞膜的亲和力。
采用生物信息学预测及分析工具，对不同氨基酸排列顺序的肽序列分子量、平均疏水值、平均疏水力矩、不稳定性指数、脂肪族指数、半衰期、螺旋轮图、成为AMPs的可能性以及二级结构进行预测，选出最优的抗菌肽序列。所用生物信息学工具如下：
物化性质分析网站：Expasy ProtParam(https://web.expasy.org/protparam/)
螺旋轮图及疏水性分析网站：Heliquest(https://heliquest.ipmc.cnrs.fr/)
AMPs可能性预测网站：CAMPR3(http://www.camp.bicnirrh.res.in/)
二级结构预测网站：Pep-fold(https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/)；
Zhang Lab(https://zhanglab.ccmb.med.umich.edu/)
通过上述工具得到的最优抗菌肽序列及物化性质如表2所示，二级结构预测结果如图1所示。
表2抗菌肽YHX-4的序列及物化性质
实施例2抑菌活性
抗菌肽由强耀生物科技(上海)有限公司合成。
将金黄色葡萄球菌、大肠杆菌和沙门氏菌分别划线接种于LB固体培养基，将单核细胞增生李斯特氏菌和变异链球菌划线接种于BHI固体培养基，置于37℃恒温培养箱培养18h，挑取各个菌株的单菌落置于各自对应的液体培养基中，37℃恒温振荡培养12h。测量菌液的OD600值，并将其稀释至1×106CFU/mL。
①抑菌圈实验
配置LB和BHI半固体培养基(琼脂质量分数0.6％)，每皿20mL加入7μL菌液振荡混匀后倒入摆放好牛津杯的培养皿中，待培养基冷却凝固后拔除牛津杯完成打孔。每孔中加入160μL抗菌肽溶液(配置使用1mg抗菌肽溶于3mL超纯水)。置于37℃恒温培养箱培养24h后测量抑菌圈直径，测试结果如图2-6所示。
②最小抑菌浓度(MIC)的测定
向96孔板中加入培养至对数生长期的各菌株菌液(单核细胞增生李斯特氏菌、变异链球菌、金黄色葡萄球菌、沙门氏菌、大肠杆菌)50μL，同时在各孔中加入各浓度梯度抗菌肽溶液(512μg/mL、256μg/mL、128μg/mL、64μg/mL、32μg/mL、16μg/mL、8μg/mL、4μg/mL)50μL，并分别做阳性对照和阴性对照组。在37℃恒温培养箱培养8h后测量各孔的OD600值，取菌株生长被完全抑制的最低浓度作为抗菌肽的最小抑菌浓度(MIC)，测试结果如表3所示。
表3抗菌肽YHX-4的抑菌活性分析结果
实施例3溶血活性
取健康兔血液1mL加于肝素抗凝管中，在1000xg转速下离心10min后取沉淀，用PBS缓冲液冲洗3次，将红细胞用10mL PBS重悬。用PBS缓冲液将抗菌肽YHX-4的浓度调整至4-512μg/mL，以每孔50μL的剂量加入96孔板中，并加入等体积(50μL)的红细胞悬液混匀。以PBS缓冲液作为阴性对照，以0.1％Tritonx-100作为阴性对照，在37℃恒温培养1h后取出，1000xg离心10min，取出上清液用酶标仪在570nm处测OD值，测试结果如表4所示。
溶血率计算公式为：溶血率＝(AT-AO)/(AC-AO)×100％。
式中：AT为实验组的吸光值，AC为阳性对照组的吸光值，AO为阴性对照组的吸光值。
表4抗菌肽YHX-4的溶血活性(测定3次取平均值)
由表4可知，在最小抑菌浓度(MIC)条件下，抗菌肽YHX-4的溶血率均低于10％，说明抗菌肽YHX-4有较好的安全性，在制作生物抗菌剂、防腐剂和动物饲料等方面的应用前景较大，可进一步深入研究和开发利用。
以上所述的实施例仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案作出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 中国海洋大学
<120> 抗菌肽YHX-4及其应用
<141> 2022-01-26
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213> Artificial Sequence
<400> 1
Trp Lys Leu Leu Ser Lys Leu Leu Gly Lys Leu Leu Lys
1 5 10
Claims (10)
1.抗菌肽YHX-4，其特征在于，所述的抗菌肽YHX-4的氨基酸序列为：Trp-Lys-Leu-Leu-Ser-Lys-Leu-Leu-Gly-Lys-Leu-Leu-Lys-NH2。
2.根据权利要求1所述的抗菌肽YHX-4，其特征在于，包含13个氨基酸残基，分子量为1540.01Da，净电荷数为+4。
3.权利要求1所述的抗菌肽YHX-4在抑制单核细胞增生李斯特氏菌、变异链球菌、沙门氏菌、大肠杆菌以及金黄色葡萄球菌中的应用。
4.根据权利要求3所述的应用，其特征在于，所述抗菌肽YHX-4对单核细胞增生李斯特氏菌的最小抑菌浓度为4μg/mL；对变异链球菌的最小抑菌浓度为16μg/mL；对沙门氏菌的最小抑菌浓度为4μg/mL；对大肠杆菌的最小抑菌浓度为8μg/mL；对金黄色葡萄球菌的最小抑菌浓度为32μg/mL。
5.一种生物抗菌剂，其特征在于，其抗菌成分中包含权利要求1所述的抗菌肽YHX-4。
6.一种动物饲料，其特征在于，含有如权利要求1所述的抗菌肽YHX-4。
7.一种防腐剂，其特征在于，含有如权利要求1所述的抗菌肽YHX-4。
8.根据权利要求7所述的防腐剂，其特征在于，所述防腐剂为用于食品的防腐剂或者用于化妆品的防腐剂。
9.一种洗涤剂组合物，其特征在于，含有如权利要求1所述的抗菌肽YHX-4。
10.根据权利要求9所述的洗涤剂组合物，其特征在于，所述洗涤剂组合物为洗手液、香皂、沐浴液、洗发剂、牙膏、洗衣液或洗衣粉。
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