CN113121666A - 抗菌肽Scybaumancin105-127及其应用 - Google Patents
抗菌肽Scybaumancin105-127及其应用 Download PDFInfo
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- CN113121666A CN113121666A CN202110261150.8A CN202110261150A CN113121666A CN 113121666 A CN113121666 A CN 113121666A CN 202110261150 A CN202110261150 A CN 202110261150A CN 113121666 A CN113121666 A CN 113121666A
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- scybaumancin
- antibacterial peptide
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Abstract
本发明公开了抗菌肽Scybaumancin105‑127及其应用,该抗菌肽首次发现于拟穴青蟹(Scylla paramamosain),分子式为C106H188N36O28S3,其氨基酸序列如SEQ ID NO.1所示。本发明的抗菌肽Scybaumancin105‑127对鲍曼不动杆菌、福氏志贺氏菌、大肠埃希菌、铜绿假单胞菌、新型隐球菌、单核细胞李斯特氏菌、屎肠球菌及痤疮丙酸杆菌具有良好的抗菌活性,对正常细胞无细胞毒性。该抗菌肽Scybaumancin105‑127抗菌活性高、安全无毒,对临床耐药性细菌也具有较强的抗菌活性,具有广阔的应用前景。
Description
技术领域
本发明属于海洋分子生物学技术领域,具体涉及多肽及其应用。
背景技术
自1928年,弗莱明发现青霉素以来,抗生素的普及和应用挽救了无数的生命,推动了现代医疗的发展。然而,在持续性使用抗生素的过程中,抗生素的不当或过度使用,使得细菌耐药性基因被不断富集和传播扩散,导致临床中出现了大量的耐药性细菌,给临床治疗带来巨大挑战,甚至出现了无药可用的境地。细菌耐药性问题已在全球范围蔓延,2017年,世界卫生组织发表了首份抗生素耐药“重点病原体”清单,公布了12种对人类健康构成极大威胁的细菌种族目录,极为重要的1类重点清单包括碳青霉烯类药物耐药鲍曼不动杆菌、碳青霉烯类药物耐药铜绿假单胞菌、碳青霉烯类药物耐药/产超广谱β-内酰胺酶(ESBL)肠杆菌科细菌;万古霉素耐药屎肠球菌、甲氧西林耐药/万古霉素中介和耐药金黄色葡萄球菌位于十分重要的2类重点清单中。每年至少有70万人死于耐药性细菌感染,世界卫生组织预测,如不采取有效措施,这一数目在2050年将达到1000万。细菌对抗生素耐药的严峻形势,使得开发新型抗生素来替代传统抗生素变得尤为迫切。
抗菌肽(Antimicrobial Peptide,AMP)又称为宿主防御肽,广泛存在于动物、植物、微生物等生物体中,通常为具有两亲性的阳离子多肽,作为天然免疫的效应分子,具有广谱的抗菌、抗病毒、抗寄生虫、抗肿瘤活性;此外,还具有促进伤口愈合、抗炎及免疫调控等功能,相比于抗生素,抗菌肽抗菌机制多样、具有多个作用靶点、抗菌谱广、不易产生耐药性,被认为是良好的抗生素天然替代品。
自1981年,首个动物抗菌肽——天蚕素被分离得到后,大量抗菌肽被分离和鉴定,随着抗菌肽研究的开展,其功能被不断阐明,除了直接杀菌作用还具有免疫调控作用。同时,抗菌肽结构简单、化学合成方便快捷、杀菌迅速、不易产生耐药性等特点为其应用提供了更多的可能性。已有多种天然多肽、人工合成或设计改造的多肽进入临床试验,用于创面细菌性感染、痤疮、软组织感染、过敏性皮肤炎等疾病的治疗。鉴于其潜在的临床应用价值,开展抗菌肽的研究,获得活性高、安全性高的抗菌肽,以缓解临床耐药性问题,具有重要的意义。
发明内容
本发明的目的在于克服现有技术的不足之处,提供了抗菌肽Scybaumancin105-127及其应用。
本发明解决其技术问题所采用的技术方案之一是:
一种抗菌肽Scybaumancin105-127,分子式为C106H188N36O28S3,其氨基酸序列如SEQ IDNO.1所示,具体为:
Val-Val-Thr-Cys-Arg-Leu-Ala-His-Met-Ala-Leu-Lys-Ser-Ala-Lys-Ser-Ser-Arg-Ser-Leu-Leu-Cy s-His-NH2
本发明的拟穴青蟹抗菌肽Scybaumancin105-127由23个氨基酸组成,分子量为2511.06道尔顿,含2个赖氨酸、2个精氨酸和2个半胱氨酸残基,C端酰胺化修饰(-NH2)(即第23位的组氨酸His经-NH2修饰)。HeliQuest预测该抗菌肽电荷数为+4,疏水性为0.471。
所述抗菌肽Scybaumancin105-127可以以固体(如粉末等)、液体(如水溶液等)等多种形式应用。
本发明解决其技术问题所采用的技术方案之二是:
一种抗菌肽Scybaumancin105-127在制备抗菌剂中的应用。
本发明提供的一种新型抗菌肽Scybaumancin105-127,作为一种新型的抗菌活性物质,可以用于制备抗菌剂,作为抗菌剂中的有效成分,用于抑制或杀灭革兰氏阳性菌、革兰氏阴性菌或真菌。
本发明解决其技术问题所采用的技术方案之三是:
一种抗菌剂,所述抗菌剂包括氨基酸序列如SEQ ID NO.1所示的新型抗菌肽Scybaumancin105-127。
所述抗菌剂可抑制或杀灭革兰氏阳性菌、革兰氏阴性菌或真菌中的至少一种。
本发明所述的革兰氏阴性菌包括鲍曼不动杆菌、耐药鲍曼不动杆菌、福氏志贺氏菌、大肠埃希菌、耐药大肠埃希菌或铜绿假单胞菌中的至少一种;所述的真菌包括新型隐球菌;所述的革兰氏阳性菌包括单核细胞增生李斯特氏菌、屎肠球菌、耐药屎肠球菌或痤疮丙酸杆菌中的至少一种。
本发明所述的抗菌剂可以是药物形式的抗菌剂,即作为抗菌药物,也可以是非药物形式的抗菌剂,例如作为消毒产品,或作为纺织品、塑料等产品的添加剂等。
本发明解决其技术问题所采用的技术方案之四是:
一种抗菌肽Scybaumancin105-127在制备食品添加剂中的应用。
本发明提供的一种新型抗菌肽Scybaumancin105-127,安全性好,可以作为一种新型的食品添加剂,抑制和/或杀灭食源性污染微生物,尤其对食源性污染的单核细胞增生李斯特氏菌具有较强的杀菌活性。
本发明解决其技术问题所采用的技术方案之五是:
一种食品添加剂,所述食品添加剂包括氨基酸序列如SEQ ID NO.1所示的抗菌肽Scybaumancin105-127。所述食品添加剂尤其适合用于抑制和/或杀灭食源性污染的单核细胞增生李斯特氏菌。
本发明所涉及的设备、试剂、工艺、参数等,除有特别说明外,均为常规设备、试剂、工艺、参数等,不再作实施例。
本发明所列举的所有范围包括该范围内的所有点值。
本发明所述“大约”、“约”或“左右”等指的是所述范围或数值的±20%范围内。
本发明中,除有特别说明外,%均为质量百分比。
本技术方案与背景技术相比,它具有如下优点:
1.本发明的拟穴青蟹抗菌肽Scybaumancin105-127水溶性好、抗菌谱广、抗菌活性高且安全无毒,是一条具有广阔应用前景的阳离子短肽。
2.本发明的拟穴青蟹抗菌肽Scybaumancin105-127对单核细胞增生李斯特氏菌、屎肠球菌、福氏志贺氏菌、鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌、新型隐球菌及痤疮丙酸杆菌具有较强的抗菌活性,对HEK-293T、拟穴青蟹血细胞、EPC细胞无细胞毒性。
附图说明
图1为抗菌肽Scybaumancin105-127对鲍曼不动杆菌和新型隐球菌的杀菌动力学曲线,横坐标为时间(min),纵坐标为CFU(菌落形成单位)/mL。
图2为抗菌肽Scybaumancin105-127处理鲍曼不动杆菌非临床耐药菌株CGMCC1.6769和临床耐药菌株QZ18050引起的形态结构变化。其中,A为鲍曼不动杆菌非临床耐药菌株,B为鲍曼不动杆菌非临床耐药菌株+6μM Scybaumancin105-127,C为鲍曼不动杆菌临床耐药菌株,D为鲍曼不动杆菌临床耐药菌株+12μM Scybaumancin105-127。
图3为MTS法检测拟穴青蟹抗菌肽Scybaumancin105-127对HEK-293T细胞、拟穴青蟹血细胞(Hemocytes)和EPC细胞的细胞毒性实验;横坐标为Scybaumancin105-127蛋白浓度(μM),纵坐标为细胞存活率(cell viability)(%)。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
实施例1:拟穴青蟹抗菌肽Scybaumancin105-127
本实施例的一种拟穴青蟹抗菌肽Scybaumancin105-127的氨基酸序列如SEQ IDNO.1所示,具体为:
V-V-T-C-R-L-A-H-M-A-L-K-S-A-K-S-S-R-S-L-L-C-H-NH2,C端酰胺化修饰(-NH2)。
本实施例委托南京金斯瑞有限公司以固相合成方法合成获得纯度达95%以上的拟穴青蟹抗菌肽Scybaumancin105-127,并提供多肽分子量、HPLC等检测信息,用Helixquest预测其电荷和疏水性,其他理化参数均用ProtParam预测,拟穴青蟹抗菌肽Scybaumancin105-127理化参数如表1所示。
表1拟穴青蟹抗菌肽Scybaumancin105-127理化参数
实施例2:Scybaumancin105-127最小杀菌浓度(MBC:Minimum BactericidalConcentration)的测定
1、本实施例涉及到的菌株有:单核细胞增生李斯特氏菌(Listeriamonocytogenes)、屎肠球菌(Enterococcus faecium)、福氏志贺氏菌(Shigellaflexneri)、鲍曼不动杆菌(Acinetobacter baumannii)、大肠埃希菌(Escherichia coli)、铜绿假单胞菌(Pseudomonas aeruginosa)、新型隐球菌(Cryptococcus neoformans)购自中国科学院微生物研究所菌种保藏中心;痤疮丙酸杆菌(Propionibacterium acnes)购自广东省微生物菌种保藏中心;临床耐药菌QZ18080、QZ18081、QZ18050、QZ18055、QZ18109、QZ18110来源于福建医科大学附属第二医院检验科。
2、具体方法如下:
(1)活化菌种,将保种菌株划线于营养肉汤(NB:Nutrient broth)平板,37℃培养箱中静置培养(痤疮丙酸杆菌用脑心浸液(BHI:Brain heart immersion)+1%葡萄糖培养,并置于厌氧袋中培养;真菌划线于YPD平板,28℃培养箱中静置培养。)
(2)待菌落生长至合适大小,随机挑取3~5个单克隆于NB液体培养基中,37℃摇床,180rpm培养至对数期(痤疮丙酸杆菌挑克隆至BHI+1%葡萄糖培养,培养基液面上方加液体石蜡封顶;真菌挑克隆至YPD液体培养基,28℃摇床,230rpm培养至对数期)。
(3)4000g,离心3min收集细菌,最后用10mM磷酸钠缓冲液(NaPB,pH=7.4)与MH液体培养基混合液稀释细菌(NaPB:MH=3:2),使得菌体的最终浓度约为5×105cfu/mL(真菌抗菌条件为NaPB:YPD=3:2,菌体量约为5×104cfu/mL)。
(4)将已合成的Scybaumancin105-127粉末溶于无菌Milli-Q水,用0.22μm滤膜过滤,倍比稀释蛋白浓度至6μM、12μM、24μM、48μM、96μM、192μM;
(5)在96孔细胞培养板上,每种待测菌设置空白对照组、阴性对照组和待测实验组,每组设置三个平行:
a空白对照组:50μL待测蛋白样品和50μL稀释菌液的培养基
b阴性对照组:50μL无菌Milli-Q水和50μL菌悬液
c待测实验组:50μL待测蛋白样品和50μL菌悬液
将96孔细胞培养板置于37℃或28℃培养箱中,培养24h后吹打混匀取2μL点板至NB或YPD平板,于37℃或28℃培养,观察并记录MBC结果。
3、拟穴青蟹抗菌肽Scybaumancin105-127最小杀菌浓度(MBC)观察结果如表2所示。
表2拟穴青蟹抗菌肽Scybaumancin105-127的抗菌活性
注:MBC:最小杀菌浓度(μM),用a~b表示。a:平板可见菌落生长的最高蛋白浓度;b:平板未见菌落生长的最低蛋白浓度
实施例3:Scybaumancin105-127杀菌动力学曲线
1、涉及到的菌株有:鲍曼不动杆菌CGMCC 1.6769和新型隐球菌CGMCC 2.1563。
2、具体方法如下:
抗菌实验方法同最小杀菌浓度的测定一致,抗菌肽和细菌共孵一定时间后,不同时间点取10μL共孵的混合液至490μL PBS,充分混匀后取50μL稀释液涂NB平板,如果预实验涂板的菌落计数<50个克隆,则取25μL共孵的混合液直接涂板,37℃培养箱静置培养12h后,进行菌落计数(真菌共孵的混合液用PBS稀释10倍后,涂YPD平板,置于28℃培养箱静置培养48h后,进行菌落计数)。
3、拟穴青蟹抗菌肽Scybaumancin105-127对鲍曼不动杆菌和新型隐球菌的杀菌动力学曲线如图1所示。
实施例4:扫描电镜观察Scybaumancin105-127处理后鲍曼不动杆菌的形态结构变化
1、涉及到的菌株有:
鲍曼不动杆菌的非耐药菌株CGMCC 1.6769和临床耐药菌株QZ18050。
2、具体实验方法如下:
(1)扫描电镜样品制备
活化菌种,待克隆长至合适大小,随机挑取3~5个克隆至NB液体培养基,摇至对数生长期,测OD600,离心去上清,用MH培养基重悬菌体,调至OD为0.1,取500μL菌液+等体积抗菌肽,37℃共孵30min后,离心去上清,用PBS洗一次后收集菌体。
(2)固定、清洗、滴片:
用300μL 2.5%戊二醛重悬收集的菌体,4℃冰箱固定1.5h或更长时间。
固定后的菌体用PBS洗三次(20min/次)后,制成高浓度悬液,滴至已切割好的载玻片上,黏附30min,用滤纸吸干后进行乙醇梯度脱水。
(3)脱水:
30%乙醇5min;50%乙醇5min;70%乙醇10min,可4℃冰箱过夜。
以上操作在冰盒里进行。
80%乙醇10min;95%乙醇15min;100%乙醇15min×2次。
(4)临界点干燥后,10mA电流,喷金60s;扫描电镜观察并拍片记录。
3、拟穴青蟹抗菌肽Scybaumancin105-127对鲍曼不动杆菌的非耐药菌CGMCC 1.6769和临床耐药菌株QZ18050形态结构变化如图2所示。
实施例5:MTS法检测和评价Scybaumancin105-127的细胞毒性
1、选取人肾上皮细胞(HEK-293T)、拟穴青蟹血细胞(Hemocytes)、鲤鱼上皮细胞(EPC)细胞,横坐标为Scybaumancin105-127蛋白浓度(μM),纵坐标为细胞存活率(%),对拟穴青蟹抗菌肽Scybaumancin105-127细胞毒性进行测定。
2、具体方法如下:
(1)收集生长状态良好的细胞,用对应的细胞培养基(HEK-293T、EPC用DMEM+10%FBS;Hemocytes用L15+5%FBS+1.2%NaCl)稀释细胞浓度至103~104个/mL,在96孔细胞培养板中每孔加入上述细胞悬液100μL,HEK-293T置于37℃细胞培养箱,5%CO2;EPC和Hemocytes置于28℃细胞培养箱,5%CO2条件下静置培养。
(2)小心吸出50μL培养基,加入含有不同浓度Scybaumancin105-127的相应培养基,置于相应的细胞条件下静置培养24h。
(3)加入20μL MTS-PMS混合溶液,孵育4h后,使用酶标仪测得OD492值,评价Scybaumancin105-127的细胞毒性。
3、结果如图3。在浓度0~96μM范围内,Scybaumancin105-127对HEK-293T、拟穴青蟹血细胞、EPC细胞无细胞毒性。
以上所述,仅为本发明较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
序列表
<110> 厦门大学
<120> 抗菌肽Scybaumancin105-127及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> PRT
<213> 拟穴青蟹(Scylla paramamosain)
<220>
<221> AMIDATION
<222> (23)..(23)
<400> 1
Val Val Thr Cys Arg Leu Ala His Met Ala Leu Lys Ser Ala Lys Ser
1 5 10 15
Ser Arg Ser Leu Leu Cys His
20
Claims (10)
1.一种抗菌肽Scybaumancin105-127,其氨基酸序列如SEQ ID NO.1所示。
2.一种权利要求1所述的抗菌肽Scybaumancin105-127在制备抗菌剂中的应用。
3.一种抗菌剂,其特征在于:所述抗菌剂包括氨基酸序列如SEQ ID NO.1所示的抗菌肽Scybaumancin105-127。
4.根据权利要求3所述的抗菌剂,其特征在于:所述抗菌剂用于抑制和/或杀灭革兰氏阳性菌、革兰氏阴性菌或真菌中的至少一种。
5.根据权利要求4所述的抗菌剂,其特征在于:所述革兰氏阳性菌包括单核细胞增生李斯特氏菌、屎肠球菌、耐药屎肠球菌或痤疮丙酸杆菌中的至少一种。
6.根据权利要求4所述的抗菌剂,其特征在于:所述革兰氏阴性菌包括福氏志贺氏菌、鲍曼不动杆菌、耐药鲍曼不动杆菌、大肠埃希菌、耐药大肠埃希菌或铜绿假单胞菌中的至少一种。
7.根据权利要求4所述的抗菌剂,其特征在于:所述真菌包括新型隐球菌。
8.一种权利要求1所述的抗菌肽Scybaumancin105-127在制备食品添加剂中的应用。
9.一种食品添加剂,其特征在于:所述食品添加剂包括氨基酸序列如SEQ ID NO.1所示的抗菌肽Scybaumancin105-127。
10.根据权利要求9所述的食品添加剂,其特征在于:所述食品添加剂用于抑制和/或杀灭食源性污染的单核细胞增生李斯特氏菌。
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