CN102741274A - 抗微生物肽 - Google Patents
抗微生物肽 Download PDFInfo
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- CN102741274A CN102741274A CN2010800584760A CN201080058476A CN102741274A CN 102741274 A CN102741274 A CN 102741274A CN 2010800584760 A CN2010800584760 A CN 2010800584760A CN 201080058476 A CN201080058476 A CN 201080058476A CN 102741274 A CN102741274 A CN 102741274A
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Abstract
本发明涉及具有抗微生物活性的新肽,以及含有该新肽的组合物。
Description
本发明涉及新的抗微生物肽、这些抗微生物肽的衍生物以及含有上述这些肽的组合物。
目前,几个抗微生物肽或衍生物正在进行进一步的临床开发,主要是以表面给药的药物的形式(Andrès,E.,Dimarcq,J.-L.Cationic anti-microbialpeptides:from innate immunity study to drug development.La revue demedicine interne(2004),25,629-635)。然而,这些分子中仅少数被建议用于于产养殖,例如分离自南美白对虾(Penaeus vannamei)的对虾抗菌肽(penaeidins)。参见EP1000153。
其原因可以通过这些抗微生物肽(AMP)中的某些在盐存在时被抑制这一事实来解释,然而本发明的肽并非如此,因为其分离自深海蠕虫:庞贝城蠕虫(Alvinella pompejana)或庞贝虫(Pompeii worm)。
该虫是科学界公认的最耐热的生物之一。实际上,它能忍耐从10℃至80℃的温度,这提示来自于该虫的分子例如本发明所述的肽,是热稳定的,并因此是药理学上感兴趣的。
通过大量使用抗生素而使水产养殖的产量增加已经成为可能。尽管存在法律约束,但预防性地使用抗生素很普遍,特别是在重要时期(例如,生命的早期、变态期、动物迁移期),在动物生长期也使用。大量使用抗生素会促进抗生素抗性株系的突生进化,从而使动物养殖遭到破坏。多重抗生素抗性(MAR)株系出现在80年代中期。最近三十年几乎没有发现任何新的抗生素家族,并且只提供极少的针对抗性株系的新产品。
可选择的解决方案之一将是使用AMP。这些由动植物天然产生的分子具有广谱的和持久的抗革兰氏阴性和阳性细菌、抗真菌以及抗包膜病毒的活性。此外,这些AMP不是免疫原性的,它们对细菌菌膜的作用不会有助于抗生素抗性株系的突生进化。开发于90年代的第一个AMP是爪蟾抗菌肽(Magainin)的衍生物:来自于Magainin Pharmaceuticals公司的培昔加南(Pexiganan)。在III期研究中,培昔加南被包含在霜剂中,其显示出与治疗糖尿病期间超感染的皮肤溃疡的口服抗生素氧氟沙星相同的治疗效果。目前开发的在不久的将来可能会用于临床的其它AMP中,应该提及的是Intrabiotics Pharmaceuticals公司开发的Iseganan、Micro logix Biotech公司开发的MBI肽和Periondotix公司开发的Histatin的衍生肽(Andres等,之前引用的)。但上文所述的对虾抗菌肽与本发明分离自庞贝城蠕虫的要求保护的肽相比似乎具有更大的分子量。
通过用本发明的肽替换一部分使用中的抗生素将减少水产养殖中抗生素的使用。由于AMP的无害性,它们的使用将有利于树立良好的品牌形象,也有利于被处理的动物、顾客和环境的健康,还不必担心促进抗生素抗性株系的突生进化。
因此,本发明的一个目的是提供具有广谱抗微生素活性的新的AMP。
本发明涉及包含或由SEQ ID NO:1组成的新肽,条件是所述肽不是由序列SEQ ID NO:2至5组成的那些。
术语“肽”的含义是指连续的氨基酸链。如果其含有至少50个氨基酸则该连续的氨基酸链可被称为蛋白质。该连续的氨基酸链可以是天然来源的或人工的(来自化学合成)。
术语“连续的氨基酸链”的含义是氨基酸的接触或连接贯穿于不间断序列。
因为SEQ ID NO:1来自于庞贝城蠕虫,因此称之为Alvinellacine。Alvinellacine是具有抗微生物活性的最小序列。该肽含有参与形成胱氨酸桥的4个半胱氨酸。
词语“微生物”的含义是指用显微镜才能看见的生物,如细菌、真菌和病毒。
本发明还涉及由SEQ ID NO:6组成的肽,或者SEQ ID NO:1的衍生物:
-通过在其C端和/或N端修饰,和/或
-通过在其肽链中置换和/或缺失和/或添加一个或几个氨基酸,和/或
-通过修饰其肽链中的至少一个-CO-NH-肽键,特别是通过引入反(retro)或反-倒(retro-inverso)型键,和/或
-通过用非蛋白形成性氨基酸置换其肽链中的至少一个氨基酸,
特别是那些具有80%以上,优选85%以上,优选90%以上,更优选95%以上同源性的肽或肽的片段。
根据本发明,衍生自SEQ ID NO:1的肽与SEQ ID NO:1的第1至22位的氨基酸具有至少80%的同一性,并具有抗微生物、抗病毒和/或杀真菌活性。SEQ ID NO:6对应于Alvinellacine的前肽。
术语“前肽”的含义是指包含N端信号肽的Alvinellacine的前体蛋白。该前肽在庞贝虫中经历翻译后修饰而变成SEQ ID NO:1所示的活性肽。
术语“前体蛋白”的含义是指不具有抗微生物活性的无活性的蛋白。
术语“抗微生物活性”的含义是指:针对细菌、真菌和病毒的作用,导致它们的群体的减少和/或导致微生物生长的抑制。
术语“信号肽”的含义是指SEQ ID NO:6所示的氨基酸链的片段,其是指导所述前体蛋白在庞贝城蠕虫中转运的短序列。SEQ ID NO:6中的信号肽是从第1位至第19位的氨基酸残基。
术语“在庞贝虫中的翻译后修饰”的含义是指发生在庞贝城蠕虫中的删除过程,相当于切除SEQ ID NO:6的N端部分的186个连续的氨基酸从而得到SEQID NO:1。
术语“其C端和/或N端的修饰”的含义是指替代位于SEQ ID NO:1所示的肽的第一位或最后一位的氨基酸中的基团,例如-COOH或-NH2,无论如何其目的是替代,例如包被该肽至载体。
术语“引入反型键”的含义是指引入反型酰胺键,其为SEQ ID NO:1中的-NH-CO-肽键。
术语“引入反-倒型键”的含义是指引入反-倒型酰胺键,其是-NH-CO-肽键并偶联有氨基酸的反式绝时构型。
“非蛋白形成性氨基酸”,必须理解为这些氨基酸未在蛋白质中发现(例如肉碱、L-刀豆氨酸或L-DOPA),或者在标准遗传密码中不被编码的氨基酸(例如羟基脯氨酸和硒代蛋氨酸)。
在有利的实施方式中,要求保护的肽与SEQ ID No:1的肽具有至少80%的同一性。因此,要求保护的肽可具有这样的氨基酸序列而无限制:其中在所述序列内的任何位置置换或缺失一个或多个氨基酸,或者其中向所述序列添加一个或多个蛋白形成性氨基酸,即天然氨基酸,特别是含有SEQ ID NO:1中参与形成胱氨酸桥的四个半胱氨酸的肽。
本发明还涉及上述所定义的肽,其特征在于所述肽是从庞贝城蠕虫中分离和纯化的。
“从庞贝城蠕虫中分离和纯化”的含义是指所述肽首先从庞贝城蠕虫中提取,然后再从其他有机碎片分离,例如有针对性地使用商品化的总蛋白质提取试剂盒,随后进行亲和层析,或者使用本领域技术人员通常使用的其它公知的技术。
本发明还涉及编码上述所定义的肽的分离的核酸分子,特别是SEQ ID NO:7的核酸分子,条件是所述核酸分子不是由SEQ ID NO:8至25组成的核酸分子。
SEQ ID NO:7对应于编码上述SEQ ID NO:6的前肽的核酸分子。
SEQ ID NO:8至25对应于编码SEQ ID NO:2至5的氨基酸链的分离的核酸分子。
词语“核酸分子”的含义是指编码要求保护的肽的单链或双链DNA,或者单链或双链RNA,包括DNA/RNA杂合分子。
本发明还涉及包含可操作地连接至表达载体的上述核酸分子的重组核酸构建体。
词语“可操作地连接”的含义是指上述核酸分子以共价连接的方式连接至表达载体,允许核糖体翻译要求保护的核酸分子,或允许RNA聚合酶产生编码要求保护的肽的mRNA。
术语“表达载体”的含义是指由核酸组成的分子,特别是包含启动子区和任选的增强子区的质粒。表达载体可以包含其它基因,例如抗生素抗性基因,以选择含有编码要求保护的肽的核酸分子的宿主细胞。使用上述表达载体的重组制备技术对本领域技术人员来说是公知的。
本发明还涉及包含上述重组核酸构建体的宿主细胞。
术语“宿主细胞”的含义是指任何活的细胞,特别是真核细胞,如真菌细胞(例如,黑曲霉(Aspergillus niger)),但也可以是细菌细胞,或者任何包含转录和/或翻译装置的非活体介质,任选是来自细胞的细胞器,其允许扩增要求保护的核酸构建体和/或产生要求保护的肽。
本发明进一步涉及上述所定义的肽,其用作药物,特别是作为抗微生物、抗病毒或杀真菌剂的用途。
本发明的另一个目的是提供包含至少一种上述所定义的肽的组合物。
本发明的另一个目的是提供上述定义的组合物,其用作针对革兰氏阴性和革兰氏阳性细菌的抗细菌剂的用途。
革兰氏阴性或革兰氏阳性细菌的列表可以在DSMZ目录中找到:(http://www.dsmz.de/microorganisms/bacteria_catalogue.php)。
本发明的另一个目的是提供上述定义的组合物在多种领域的用途,如人和动物卫生保健、农业和水产养殖业,以避免农场中传染病的发展。
术语“水产养殖”的含义是水生生物的养殖,特别是鱼类的养殖、藻类养殖和集成多营养养殖(IMTA)。
术语“水生生物”的含义是指任何淡水或咸水生物,包括但不限于下列生物:
-鱼类,例如鲤鱼、鲑鱼以及其它的鲤科及鲑科;
-软体动物类,例如牡蛎、蛤、鸟蛤、蚶、扇贝和贻贝;
-甲壳类,例如蟹、龙虾、虾和对虾;
-水生植物,例如藻类和浮游植物。
对本领域技术人员来说将要求保护的肽用于其它技术领域是显而易见的,例如用以减少不需要的微生物群的疫苗和物理方法。
本发明进一步涉及药物组合物,其包含与药学上可接受的载体组合的至少一种要求保护的肽。
“药学上可接受的载体”的含义是指由本领域技术人员选择的任何药物载体。
在这方面,所述肽能与任何药学上可接受的赋形剂、以任何药学上可接受的形式联合使用,例如小胶囊、硬胶囊,其它胶囊、皂和洗剂。
在另一个优选的实施方式中,要求保护的药物组合物进一步包含至少一种选自下组中的抗微生物剂:
-四环素类,如土霉素或金霉素;
-喹诺酮类,如奥索利酸、氟甲喹或沙氟沙星;
-磺酰胺,任选地用三甲氧苄二氨嘧啶或欧美德普(ormethoprim)增强效力;
-硝基呋喃类药,如呋喃唑酮;
-大环内酯类,如红霉素或螺旋霉素(E710);
-氟苯尼考;
-氯霉素。
本发明的另一个目的是提供上述药物组合物用于动物和/或人抗生素疗法中的用途。
最佳的给药途径(例如口服途径、表面途径、沐浴治疗)应由本领域技术人员确定,正如最佳的肽使用量和治疗持续时间。
本发明还涉及包含上述定义的组合物的膳食组合物,特别是还包含一种或多种营养成分的食品补充剂。
本发明的另一个目的是提供上述定义的组合物,其中所述肽用胶囊封装,特别是纳米胶囊封装。胶囊封装能有几方面的好处,特别是允许在一段时间内运送要求保护的肽。胶囊化和纳米胶囊化技术对本领域技术人员来说是公知的。
本发明的另一个目的是提供一种消毒剂,其包含含有至少一种要求保护的肽的组合物。
术语“消毒剂”的含义是:提供抗微生物、抗病毒和/或杀真菌活性并用于此目的化合物的混合物。
应当指出,在其它可能的应用中要求保护的肽可用于,例如生长培养基中、特别是用于昆虫和真核细胞的培养基中,以防止这些培养基被微生物污染。
下述实施例1至3和图1至3对本发明进行了举例说明。
图1显示了庞贝城蠕虫提取物的色谱图,如在实验部分所描述,其通过在Sep-Pak20g柱(WatersTM)预先纯化,用C18 250x4.1mm柱(218TP54 VydacTM)反向色谱(RP-HPLC)纯化获得。两条较粗的线显示的是具有抗微生物特性的级份。
x轴从左至右:洗脱时间(分钟);
y轴(左侧):255nm的光密度(O.D.);
y轴(右侧):洗脱液中ACN的百分率。
图2显示了Alvinellacine在脂质体中形成孔的能力,脂质体在此用于模拟细菌的膜,
x轴从左至右:时间(秒);
y轴(左侧):荧光(任意单位)。
箭头指示添加阿拉霉素(Alamethicin)和Alvinellacine的时刻。
阴性对照显示了背景噪声,对应于在未透化处理脂质体的情况下所测定的荧光。
图3显示了在通过有目的地使用Alvinellacine和两个其它的AMP(来源于猪的杀菌肽(Cecropin)和来源于两栖类的爪蟾抗菌肽)之后革兰氏阳性细菌(巨大芽孢杆菌(Bacillus megaterium))和革兰氏阴性细菌(大肠杆菌(Escherichiacoli))的通透化情况。实验在pH 7.4进行。对于巨大芽孢杆菌,在加入每一种AMP后10分钟测定细菌的通透性,对于大肠杆菌,在加入每一种AMP后120分钟测定。
x轴从左至右:Alvinellacine、杀菌肽(Cecropin)和爪蟾抗菌肽的浓度(nM);
y轴(左侧):通透化细菌(%)。
黑色符号代表大肠杆菌,白色符号代表巨大芽孢杆菌。
实施例1:来源于庞贝城蠕虫的内源Alvinellacine的提取、纯化和测序
在东太平洋海隆(East Pacific Rise)3,000米深处收集20条完整的、发育完全且性成熟的庞贝虫。将成虫碾碎,匀浆产生的混合物用1M HCl酸化至pH 3。然后,在10,000g离心30分钟。由此在离心沉淀中浓缩蛋白质并除去。
含有肽的上清用甲醇预填充的Sep-Pak20g(WatersTM)柱预纯化,然后用0.05%三氟乙酸(TFA)溶液预酸化的HPLC级水清洗柱子。用0.05%三氟乙酸(TFA)溶液预酸化的2%至60%的乙腈(ACN)梯度进行洗脱。
之后用C18250x4.1mm柱(218TP54 VydacTM)通过反相色谱(RP-HPLC)纯化以60%ACN洗脱剂获得的的级份。在该纯化中使用2%至62%的ACN梯度。随后对每个收集个级份的光密度进行检测,以获得色谱图(图1)。色谱图中的每个峰对应于一级份,该级份在干燥后重悬于纯水中,然后测试其抗微生物活性。
然后利用C18 250x2.1mm柱(218TP52VydacTM)通过连续RP-HPLC纯化具有活性的级份。通过质谱估计活性肽的纯度和分子量。
通过Edman降解法(对本领域技术人员而言公知的技术)对纯化的肽进行测序。结果显示对应于SEQ ID NO:1的活性肽的一个获得的序列,该序列是完全未知的序列。根据来源称该肽为Alvinellacine。使用经典的反向遗传学技术获得了编码该肽的前体蛋白的核酸分子。SEQ ID NO:6的前体蛋白包含序列中第1位至第19位氨基酸的信号肽。进一步从其推导出编码前肽的互补DNA,其由SEQ ID NO:7组成。
实施例2:抗微生物活性机制的评价
利用装有荧光试剂的脂质体确定抗细菌活性的机制。在此,含有双层磷脂的脂质体用于模拟细菌的膜。脂质体的透化处理将导致内部化合物的释放,因此与介质反应并产生可被光谱测定法检测的荧光。实验在设有阴性和阳性对照下进行。利用已知能在细菌的膜上形成孔的抗生素阿拉霉素作为阳性对照。阴性对照为脂质体溶液,目的是检测背景噪音(图2)。
结果显示,Alvinellacine与阿拉霉素同样地透化双层磷脂。
这种作用模式并不会有助于抗生素抗性株系的突生进化。
实施例3:抗细菌活性的评价及与其它AMP的比较
根据Hancock的方法(Hancock,R.E.W.September 19 1999,posting date.[Online.]Hancock Laboratory Methods.Department of Microbiology andImmunology,University of British Columbia,British Columbia,Canada.http://www.cmdr.ubc.ca/bobh/methods.htm[Antibiotics and AntimicrobialPeptides:MIC Determination by Microtitre Broth Dilution Method,last accessedSeptember 21st.])确定最小抑菌浓度(MIC)。利用先前描述的方法(Herbst,R.,Ott,C,Jacobs,T.,Marti,T.,Marciano-Cabral,F.,Leippe,M.Pore-formingpolypeptides of the pathogenic protozoon Naegleria fowleri.J.Biol.Chem.2002.277:22353-22360)检测细菌的膜的透性和孔形成活性。
在液体生长抑制分析中,纯化的Alvinellacine对于人病原体金黄色葡萄球菌(Staphylococcus aureus)(MIC 0.385-0.75μM)和蛤病原体溶藻弧菌(Vibrioalginolyticus)(MIC 0.006-0.012μM)具有非常好的活性。
为了研究Alvinellacine的作用模式,我们使用了荧光染料SYTOX-Green和革兰氏阳性细菌巨大芽孢杆菌或革兰氏阴性细菌大肠杆菌(图3)。试验结果证明Alvinellacine快速透化细菌的膜,并具有强的抗革兰氏阴性细菌的活性和程度弱些的抗革兰氏阳性细菌的活性。在检测期间,Alvinellacine的针对所测的革兰氏阴性细菌的活性比已知的抗微生物肽杀菌肽(Cecropin)P1和爪蟾抗菌肽II似乎高出一个数量级。其抗革兰氏阳性细菌的活性与这两个阳性对照相比较低。我们也通过使用最简膜系统检测了Alvinellacine的孔形成活性以进一步表征其作用模式。更准确地,我们监测了由偶氮植物凝血素(azolectin)组成的脂质体中诱导的膜电位,偶氮植物凝血素是来自大豆的粗磷脂混合物。其孔形成活性在终浓度达0.5nM时被检测到,而阳性对照阿拉霉素在0.1nM时产生信号。
Claims (16)
1.包含SEQ ID NO:1或由SEQ ID NO:1组成的肽或衍生自SEQ ID NO:1的肽,所述肽与SEQ ID NO:1的第1至22位氨基酸具有至少80%的同一性,并具有抗微生物、抗病毒和/或杀真菌活性,条件是所述肽不是由SEQ ID NO:2至5组成的肽。
2.权利要求1的肽,其由SEQ ID NO:6组成。
3.权利要求1或2的肽,其特征在于所述的肽分离并纯化自庞贝城蠕虫(Alvinella pompejana)。
4.编码权利要求1至3任意一项所述肽的分离的核酸分子,特别是SEQ IDNO:7所示的核酸分子,条件是所述核酸分子不是由SEQ ID NO:8至25组成的核酸分子。
5.重组核酸构建体,其包括与表达载体可操作性连接的权利要求4所述的核酸分子。
6.包含权利要求5所述的重组核酸构建体的宿主细胞。
7.权利要求1至3任意一项所述的肽,其用作药物,特别是用作抗微生物、抗病毒和/或杀真菌剂。
8.包含至少一种权利要求1至3所述肽的组合物。
9.权利要求8所述的组合物,其用作抗革兰氏阴性和/或革兰氏阳性细菌的抗细菌剂。
10.权利要求8或9所述的组合物,其用于水产养殖中。
11.药物组合物,其包含与药学上可接受的载体组合的至少一种权利要求1至3任一项所述的肽。
12.权利要求11所述的药物组合物,其进一步包含选自下列的至少一种另外的抗微生物剂:
-四环素类,如土霉素或金霉素;
-喹诺酮类,如奥索利酸、氟甲喹或沙氟沙星;
-磺酰胺,任选地用三甲氧苄二氨嘧啶或欧美德普(ormethoprim)增强效力;
-硝基呋喃类药,如呋喃唑酮;
-大环内酯类,如红霉素;
-氟苯尼考;
-氯霉素。
13.权利要求12所述的药物组合物,其用于动物和/或人体抗生素治疗。
14.膳食组合物,特别是进一步含有一种或多种营养成分的食品补充剂,其包含权利要求8或9所述的组合物。
15.权利要求8至14任意一项所述的组合物,其中所述肽是胶囊封装的,特别是纳米胶囊封装的。
16.包含权利要求8所述组合物的消毒剂。
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WO2022262385A1 (zh) * | 2022-01-24 | 2022-12-22 | 中国科学院南海海洋研究所 | 一种贝类来源的抗菌肽p-amp108及在制备治疗痤疮药物中的应用 |
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WO2007023163A1 (en) * | 2005-08-26 | 2007-03-01 | Novozymes A/S | Polypeptides having antimicrobial activity and polynucleotides encoding same |
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WO2007023163A1 (en) * | 2005-08-26 | 2007-03-01 | Novozymes A/S | Polypeptides having antimicrobial activity and polynucleotides encoding same |
WO2008066752A2 (en) * | 2006-11-22 | 2008-06-05 | Adnexus, A Bristol-Myers Squibb R & D Company | Targeted therapeutics based on engineered proteins for tyrosine kinases receptors, including igf-ir |
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CN106021999A (zh) * | 2016-05-17 | 2016-10-12 | 郑州轻工业学院 | 一种多功能抗微生物肽的最优多标记集成预测方法 |
CN106021999B (zh) * | 2016-05-17 | 2018-02-27 | 郑州轻工业学院 | 一种多功能抗微生物肽的最优多标记集成预测方法 |
WO2022262385A1 (zh) * | 2022-01-24 | 2022-12-22 | 中国科学院南海海洋研究所 | 一种贝类来源的抗菌肽p-amp108及在制备治疗痤疮药物中的应用 |
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