CN114716512A - 一种青蟹广谱抗菌肽Scyampcin44-63及其应用 - Google Patents
一种青蟹广谱抗菌肽Scyampcin44-63及其应用 Download PDFInfo
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- CN114716512A CN114716512A CN202110722932.7A CN202110722932A CN114716512A CN 114716512 A CN114716512 A CN 114716512A CN 202110722932 A CN202110722932 A CN 202110722932A CN 114716512 A CN114716512 A CN 114716512A
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明公开了一种青蟹广谱抗菌肽Scyampcin44‑63及其应用，该抗菌肽首次发现于拟穴青蟹，其分子式为C113H188N30O24S2，其氨基酸序列如SEQ ID NO：01所示。本发明的青蟹广谱抗菌肽Scyampcin44‑63具有广谱抗微生物活性，对临床耐药性细菌和真菌具有很强的抗菌活性，对HEK‑293T和EPC细胞无细胞毒性，是一条水溶性好、抗菌活性高、抗菌谱广且安全、具有广阔应用前景的阳离子短肽。
Description
技术领域
本发明属于海洋分子生物学技术领域，具体涉及一种青蟹广谱抗菌肽Scyampcin44-63及其应用。
背景技术
由于抗生素的不当使用，导致临床出现了大量的多重耐药(MDR)、广泛耐药(XDR)和泛耐药(PDR)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。细菌耐药性问题在全球范围蔓延，每年有70万以上的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。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。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。
随着免疫功能缺陷人群的增多，侵袭性真菌感染呈上升趋势，每年由白色念珠菌、新型隐球菌、烟曲霉和荚膜组织胞浆菌等引起的侵袭性真菌感染导致的死亡人数高达150万人，严重威胁人类的健康。目前可使用的抗真菌药物较为有限，主要包括唑类(存在交叉耐药性问题)、多烯类(如两性霉素B，存在严重的肾毒性)、棘白菌素类(半衰期短)、氟胞嘧啶(存在恶心、呕吐、腹泻及骨髓抑制等不良反应)。抗真菌药物除了上述存在的问题，近年来真菌对抗真菌药物耐药问题也日益严峻，对唑类、多烯类、棘白菌素类和氟胞嘧啶的耐药问题层出不穷，最为典型的唑类耐药问题源于唑类药物广泛地在农业中应用。此外，侵袭性真菌感染的致死率>20％，新型抗真菌药物的开发迫在眉睫。
抗菌肽(Antimicrobial Peptide，AMP)又称为宿主防御肽，通常为两亲性的阳离子多肽，广泛存在于动物、植物、微生物等生物体中，具有广谱的抗细菌、抗真菌、抗病毒、抗寄生虫及抗肿瘤等活性。抗菌肽作为天然免疫的效应分子，相对于抗生素，抗菌肽抗菌机制多样、具有多个作用靶点、抗菌谱广、杀菌迅速、不易产生耐药性，此外，还具有促进伤口愈合、抗炎及免疫调控等功能，被认为是良好的天然抗生素替代品。已有天然多肽、人工合成或设计改造的多肽进入临床试验，用于真菌或细菌感染的治疗。鉴于其潜在的临床应用价值，开发和挖掘新型抗菌肽，寻找有效的抗生素替代品，以减少和改善临床耐药性问题变得尤为重要。
发明内容
本发明的目的在于提供一种青蟹广谱抗菌肽Scyampcin44-63。
本发明的另一目的在于提供上述青蟹广谱抗菌肽Scyampcin44-63的应用。
本发明的技术方案之一如下：
一种青蟹广谱抗菌肽Scyampcin44-63，其分子式为C113H188N30O24S2，其氨基酸序列如SEQ ID NO：01所示。
本发明的技术方案之二如下：
上述青蟹广谱抗菌肽Scyampcin44-63在制备抗细菌组合物中的应用。
在本发明的一个优选实施方案中，所述抗细菌组合物对金黄色葡萄球菌、屎肠球菌、单核细胞增生李斯特氏菌、蜡状芽孢杆菌、金黄色葡萄球菌、粪肠球菌、福氏志贺氏菌、鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌和阴沟肠杆菌具有抑制和杀灭作用。
本发明的技术方案之三如下：
一种抗细菌组合物，其有效成分包括上述青蟹广谱抗菌肽Scyampcin44-63。
在本发明的一个优选实施方案中，其有效成分为上述青蟹广谱抗菌肽Scyampcin44-63。
本发明的技术方案之四如下：
上述青蟹广谱抗菌肽Scyampcin44-63在制备抗真菌组合物中的应用。
在本发明的一个优选实施方案中，所述抗真菌组合物对酵母真菌和丝状真菌具有抑制和杀灭作用。
进一步优选的，所述酵母真菌为新型隐球菌或白色念珠菌，所述丝状真菌为腐皮镰孢或禾谷镰孢。
本发明的技术方案之五如下：
一种抗真菌组合物，其有效成分包括上述青蟹广谱抗菌肽Scyampcin44-63。
在本发明的一个优选实施方案中，其有效成分为上述青蟹广谱抗菌肽Scyampcin44-63。
本发明的有益效果是：
1.本发明的青蟹广谱抗菌肽Scyampcin44-63由20个氨基酸组成，C端进行酰胺化修饰，分子量为2428.03道尔顿，其中含6个赖氨酸、1个精氨酸和1个甘氨酸。HeliQuest预测该抗菌肽电荷数为+6，疏水性为0.211，是一条水溶性好、抗菌活性高、抗菌谱广且安全、具有广阔应用前景的阳离子短肽。
2.本发明的青蟹广谱抗菌肽Scyampcin44-63对临床耐药性细菌和真菌具有很强的抗菌活性，对HEK-293T和EPC细胞无细胞毒性。
附图说明
图1为本发明实施例1中青蟹广谱抗菌肽Scyampcin44-63对鲍曼不动杆菌CGMCC1.6769、金黄色葡萄球菌CGMCC1.2465的杀菌动力学曲线图。
图2为本发明实施例1中青蟹广谱抗菌肽Scyampcin44-63处理金黄色葡萄球菌CGMCC1.2465、大肠埃希菌CGMCC 1.2389、白色念珠菌CGMCC 2.2411引起的形态结构变化图。其中，A为金黄色葡萄球菌，B为金黄色葡萄球菌+6μM Scyampcin44-63，C为大肠埃希菌，D为大肠埃希菌+3μM Scyampcin44-63，E为白色念珠菌，F为白色念珠菌+6μM Scyampcin44-63。
图3为本发明实施例1中MTS法检测青蟹广谱抗菌肽Scyampcin44-63对HEK-293T细胞和EPC细胞细胞毒性实验结果图，其中，横坐标为Scyampcin44-63蛋白浓度(μM)，纵坐标为细胞存活率(％)。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
实施例1
本实施例中的青蟹广谱抗菌肽Scyampcin44-63的氨基酸序列为SEQ ID NO：01：GKKKKRNMMKTKEPGIIFFF-NH2，C端进行酰胺化修饰(-NH2)，即第20位的苯丙氨酸经-NH2修饰。
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％以上的青蟹广谱抗菌肽Scyampcin44-63，并提供多肽分子量、HPLC等检测信息，用HeliQuest预测其电荷和疏水性，其他理化参数均用ProtParam预测，抗菌肽Scyampcin44-63理化参数如表1所示。
表1抗菌肽Scyampcin44-63理化参数
一、Scyampcin44-63最小杀菌浓度(MBC：Minimum Bactericidal Concentration)的测定
1.涉及到的菌株有：新型隐球菌、白色念珠菌、腐皮镰孢、禾谷镰孢、金黄色葡萄球菌、屎肠球菌、粪肠球菌、单核细胞增生李斯特氏菌、蜡状芽孢杆菌、大肠埃希菌、鲍曼不动杆菌和铜绿假单胞菌购自中国科学院微生物研究所菌种保藏中心；临床耐药菌QZ19131、QZ18080、QZ19138、QZ20141、QZ18109、QZ18071、QZ18106、QZ18103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，将保种的细菌划线于营养肉汤(NB：Nutrientbroth)平板，37℃培养箱中静置培养；酵母真菌划线于YPD平板，丝状真菌划线于PDA平板，28℃培养箱中静置培养。
(2)待菌落生长至合适大小，细菌挑克隆至NB液体培养基中，37℃摇床，180rpm培养至对数期；酵母真菌挑克隆至YPD液体培养基，28℃摇床，230rpm培养至对数期。
(3)离心收集细菌或真菌，细菌用MH液体稀释，使得细菌终浓度约为5×105cfu/mL；酵母真菌用RPMI1640+0.165M MOPS+0.2％葡萄糖，真菌终浓度约为5×104cfu/mL；丝状真菌收集孢子后，计数，用培养基RPMI1640+0.165M MOPS调整孢子数至5×104cfu/mL。
(4)将已合成的Scyampcin44-63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稀释菌或孢子的培养基。
b阴性对照组：50μL无菌Milli-Q水和50μL菌或孢子悬液。
c待测实验组：50μL待测蛋白样品和50μL菌或孢子悬液。
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共孵的混合液点板至NB或YPD平板，于37℃或28℃培养，观察并记录MBC结果。
3.青蟹广谱抗菌肽Scyampcin44-63最小杀菌浓度(MBC)观察结果如表2所示。
表2抗菌肽Scyampcin44-63的抗菌活性
二、Scyampcin44-63杀菌动力学曲线
1.涉及到的菌株有：鲍曼不动杆菌CGMCC 1.6769、金黄色葡萄球菌CGMCC1.2465。
2.具体方法如下：
抗菌实验方法同最小杀菌浓度的测定方法一致，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.青蟹广谱抗菌肽Scyampcin44-63对鲍曼不动杆菌CGMCC 1.6769、金黄色葡萄球菌CGMCC1.2465杀菌动力学曲线如图1所示。
三、扫描电镜观察Scyampcin44-63处理后细菌或真菌的形态结构变化
1.涉及到的菌株有：金黄色葡萄球菌CGMCC1.2465、大肠埃希菌CGMCC 1.2389和白色念珠菌CGMCC 2.2411。
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个克隆至对应的营养肉汤液体培养基，摇至对数生长期，测OD，离心去上清，用MH培养基重悬菌体，调至OD为0.2，取500μL菌液+等体积抗菌肽，37℃共孵30min后(真菌培养时用YPD培养基，培养至对数生长期，测OD，离心去上清，用RPMI1640+0.165M MOPS+0.2％葡萄糖重悬菌体调至OD为0.5，取500μL菌液+等体积抗菌肽，37℃共孵60min)，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.青蟹广谱抗菌肽Scyampcin44-63对金黄色葡萄球菌CGMCC1.2465、大肠埃希菌CGMCC 1.2389和白色念珠菌CGMCC 2.2411形态结构变化如图2所示。
四、MTS法检测和评价Scyampcin44-63的细胞毒性
1.选取人肾上皮细胞(HEK-293T)和鲤鱼上皮细胞(EPC)细胞，对青蟹广谱抗菌肽Scyampcin44-63细胞毒性进行测定，如图3所示，横坐标为Scyampcin44-63蛋白浓度(μM)，纵坐标为细胞存活率(％)，
2.具体方法如下：
(1)收集生长状态良好的细胞，用DMEM+10％FBS稀释细胞浓度至105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Scyampcin44-63的培养基，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，酶标仪OD492nm读板，计算细胞存活率。
3.结果如图3，Scyampcin44-63对HEK-293T和EPC细胞无细胞毒性。
以上所述，仅为本发明的较佳实施例而已，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> 一种青蟹广谱抗菌肽Scyampcin44-63及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213> 拟穴青蟹(Scylla paramamosain)
<400> 1
Gly Lys Lys Lys Lys Arg Asn Met Met Lys Thr Lys Glu Pro Gly Ile
1 5 10 15
Ile Phe Phe Phe
20
Claims (10)
1.一种青蟹广谱抗菌肽Scyampcin44-63，其特征在于：其分子式为C113H188N30O24S2，其氨基酸序列如SEQ ID NO：01所示。
2.权利要求1所述的青蟹广谱抗菌肽Scyampcin44-63在制备抗细菌组合物中的应用。
3.如权利要求2所述的应用，其特征在于：所述抗细菌组合物对金黄色葡萄球菌、屎肠球菌、单核细胞增生李斯特氏菌、蜡状芽孢杆菌、金黄色葡萄球菌、粪肠球菌、福氏志贺氏菌、鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌和阴沟肠杆菌具有抑制和杀灭作用。
4.一种抗细菌组合物，其特征在于：其有效成分包括如权利要求1所述的青蟹广谱抗菌肽Scyampcin44-63。
5.如权利要求4所述的抗细菌组合物，其特征在于：其有效成分为如权利要求1所述的青蟹广谱抗菌肽Scyampcin44-63。
6.权利要求1所述的青蟹广谱抗菌肽Scyampcin44-63在制备抗真菌组合物中的应用。
7.如权利要求6所述的应用，其特征在于：所述抗真菌组合物对酵母真菌和丝状真菌具有抑制和杀灭作用。
8.如权利要求7所述的应用，其特征在于：所述酵母真菌为新型隐球菌或白色念珠菌，所述丝状真菌为腐皮镰孢或禾谷镰孢。
9.一种抗真菌组合物，其特征在于：其有效成分包括如权利要求1所述的青蟹广谱抗菌肽Scyampcin44-63。
10.如权利要求9所述的抗真菌组合物，其特征在于：其有效成分为如权利要求1所述的青蟹广谱抗菌肽Scyampcin44-63。
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| CN118047841B (zh) * | 2024-04-09 | 2024-06-11 | 中国农业科学院农业基因组研究所 | 一种发酵桑叶抗菌肽Squ8及其应用 |
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