CN110300760A - 新的内溶素 - Google Patents
新的内溶素 Download PDFInfo
- Publication number
- CN110300760A CN110300760A CN201780077502.6A CN201780077502A CN110300760A CN 110300760 A CN110300760 A CN 110300760A CN 201780077502 A CN201780077502 A CN 201780077502A CN 110300760 A CN110300760 A CN 110300760A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- seq
- present
- food
- endolysin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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Abstract
本发明涉及具有内溶素活性和根据SEQ ID No.1的氨基酸序列的多肽及其片段或衍生物。此外，本发明涉及编码所述多肽的核酸分子、包含所述核酸分子的载体和包含所述核酸分子或所述载体的宿主细胞。此外，本发明涉及所述多肽，其用作药物，特别是用于治疗或预防革兰氏阴性细菌感染、用作诊断手段、用作化妆用物质或用作消毒剂。本发明还涉及所述多肽用于处理或防止以下各项的细菌污染，特别是革兰氏阴性污染的用途：食品、食物加工设备、食物加工厂、与食品接触的表面、医疗装置、医院和医生诊室中的表面。此外，本发明涉及包含所述多肽的药物组合物。
Description
发明领域
本发明涉及具有内溶素活性和根据SEQ ID No.1的氨基酸序列的多肽及其片段或衍生物。此外，本发明涉及编码所述多肽的核酸分子、包含所述核酸分子的载体和包含所述核酸分子或所述载体的宿主细胞。此外，本发明涉及所述多肽，其用作药物，特别是用于治疗或预防革兰氏阴性细菌感染、用作诊断手段、用作化妆品物质或用作消毒剂(sanitizingagent)。本发明还涉及所述多肽用于处理或防止以下各项的细菌污染，特别是革兰氏阴性污染的用途：食品、食物加工设备、食物加工厂、与食品接触的表面、医疗装置、医院和医生诊室中的表面。此外，本发明涉及包含所述多肽的药物组合物。
发明背景
由细菌病原体引起的危及生命的人传染病现在部分由于对抗生素的抗性增加而重新出现。革兰氏阴性细菌对应于耐药性病原体的显著部分，并已成为导致由传染病引起的发病率和死亡率增加的主要公共健康威胁。已经提出了许多努力，但即使如此，多药物抗性革兰氏阴性细菌的比例在全世界仍在上升，并且已成为一个增长的问题。与革兰氏阳性细菌发生的情况相比，缺乏或甚至不存在特别针对多药物抗性革兰氏阴性细菌开发的新型活性抗生素，并因此，现在在临床样品和环境中分离的分类为泛耐药性隔离群(isolate)(即对所有可用抗生素都有抗性的细菌)的革兰氏阴性生物体的数目在增长。
革兰氏阴性细胞壁由外膜构成，所述外膜是大多数抗微生物剂的主要障碍。除了少数例外，外膜由以下组成：磷脂(内小叶)、脂多糖(LPS)(外小叶)和与肽聚糖共价结合并锚定到内小叶的蛋白质如孔蛋白(充当缓和通道(defusing channel))和脂蛋白的不对称膜。LPS是外膜的主要组分并且对于许多外部试剂为细菌提供渗透性屏障。LPS是极其多样的结构，其可以响应于当时的环境条件进行修改(Nikaido,2003)。三个不同的区域可以根据它们的化学结构、生物合成、遗传学和功能而区分：i)脂质A组分，ii)核心区域和iii)O-抗原，也称为O-特异性多糖或O-侧链。LPS可以呈现为粗糙(R-型，无O-抗原)、平滑(S-型，有O-抗原)或混合物(SR-型)。总体外膜完整性由两个主要稳定力维持，即LPS阳离子结合位点(离子相互作用)和脂质A酰基链基团的致密疏水堆积(疏水相互作用)，并且作用为有效的扩散屏障功能，作为针对抗微生物剂的天然屏障。因此，需要针对革兰氏阴性细菌有活性的新抗微生物剂。
用作“酶抗生素(enzybiotics)”——“酶”和“抗生素”的杂合术语——的内溶素完美地满足了此需求。内溶素是由所有双链DNA噬菌体编码的特化的肽聚糖降解酶(Young etal.,Trends Microbiol.2000Mar；8(3):120-128)。内溶素介导的作用是由其他蛋白质协调且调节的事件以在噬菌体复制周期结束时降解和损害细菌肽聚糖(PG)层。根据它们的活性和它们水解的键，内溶素可以分为不同的类别：i)糖苷酶，其在N-乙酰葡糖胺(GlcNAc)的还原末端或在N-乙酰胞壁酸(MurNAc)的还原末端切割聚糖组分；ii)N-乙酰基-β-D-胞壁质酶(通常称为溶菌酶或胞壁质酶)，其与裂解性转糖苷酶共享相同的聚糖靶标，但提供不同的终产物；iii)酰胺酶，其催化MurNAc和L-丙氨酸之间的关键酰胺键的水解，从而将聚糖链与茎肽分开；和iv)不同类别的内肽酶和羧肽酶，其攻击交联细胞壁的茎肽中的LD-和DD-键。
虽然内溶素的潜力久为人知，但它们的抗细菌用途却由于抗生素的成功和主宰地位而相形见绌。仅仅由于抗生素抗性细菌的令人惊恐的出现，科学界和商业参与者已经恢复了他们对内溶素作为替代疗法的兴趣。2001年，第一个内溶素研究揭示了在几秒钟内破坏A、C和E组链球菌，使其他14种共生链球菌未受伤害的巨大能力。内溶素在外源添加时以物种特异性方式快速切割PG的这种独特的能力促使研究人员分离出大量的成功靶向其他革兰氏阳性病原体的内溶素。然而，主要由于高度保护性的外膜的存在，攻击革兰氏阴性细胞的内溶素几乎没有引起注意，所述内溶素单独不能破坏所述高度保护性的外膜以便能够作用于PG。因此，靶向革兰氏阴性细胞(如医院和食源性病原体(例如沙门氏菌属(Salmonella)、大肠杆菌(Escherichia coli)、假单胞菌属种(Pseudomonas spp.)、不动杆菌属种(Acinetobacter spp.)))的基于内溶素的技术可能是当今内溶素疗法中最重要的挑战之一。
发明概述
本发明涉及从克氏柠檬酸杆菌(Citrobacter koseri)噬菌体CkP1中分离的具有肽聚糖降解活性的新型多肽，其具有针对革兰氏阴性细菌的抗微生物活性。
本发明的第一个目的提供具有内溶素活性的多肽，其包含根据SEQ ID No.1的氨基酸序列或其片段或衍生物。包含根据SEQ ID No.1的氨基酸序列的多肽优选由根据SEQID No.2的核苷酸序列编码。在优选的实施方案中，所述片段包含根据SEQ ID No.3的氨基酸序列，其优选由核苷酸序列SEQ ID No.4编码。在另一个优选实施方案中，所述衍生物在根据SEQ ID No.1和SEQ ID No.3的氨基酸序列中具有缺失、添加、插入和/或取代。在另一个优选实施方案中，根据本发明的所述多肽在N或C端处包含具有膜或LPS破坏活性的肽段，特别是阳离子或聚阳离子肽。所述多肽可以另外地包含标签，优选His6标签。
本发明的第二个目的提供编码根据本发明的多肽的分离的核酸分子。
本发明的第三个目的涉及包含根据本发明的核酸分子的载体。
本发明的第四个目的涉及包含根据本发明的核酸分子或根据本发明的载体的宿主细胞。
本发明的第五个目的涉及根据本发明的多肽，其用于通过手术或疗法治疗人或动物体的方法中和或用于在人或动物体上实施的诊断方法中。特别地，本发明涉及根据本发明的多肽，其用于治疗或预防受试者(例如人或动物)中的革兰氏阴性细菌感染的方法中。根据本发明的多肽也可以用作诊断物质。例如，细菌在与根据本发明的多肽离体接触时的裂解(例如，在从受试者的样品或从环境建立的细菌培养物中)表明存在革兰氏阴性细菌。
本发明的第六个目的涉及根据本发明的多肽作为食物、饲料中或化妆用中的抗微生物剂或者作为消毒剂的用途。另一个优选的实施方案涉及根据本发明的所述多肽用于处理或防止以下各项的革兰氏阴性细菌污染的用途：食品、食物加工设备、食物加工厂、与食品接触的(无生命的)表面、饲料、饲料加工设备、饲料加工厂、与饲料接触的(无生命的)表面、医疗装置、医院和医生诊室中的(无生命的)表面。
发明详述
附图说明
以下附图用于说明本发明。
图1.使用BLASTp和Pfam系统对野生型克氏柠檬酸杆菌内溶素CkP1gp131的生物信息学分析。DNA和氨基酸序列在它们出现在野生型噬菌体中时分别是A)SEQ ID No.2和B)SEQ ID No.1。溶菌酶样超家族的催化域(氨基酸1-163，SEQ ID No.3，加下划线)在图解(图1C)和序列(图1A和B；下划线)中显现。
图2.CkP1gp131针对几种革兰氏阴性细菌病原体的抗细菌活性。将指数生长的细胞(≈3x107CFU/ml)在37℃与重悬于20mM HEPES pH 7.0中的50μg/ml(2.4μM)的CkP1gp131一起温育2小时，或与HEPES一起温育2小时作为阴性对照。温育后，通过定量CFU的数量来评估抗细菌作用，并以对数单位表示为相对失活(＝log10(N0/Ni)，其中N0＝未经处理的细胞数并且Ni＝处理后的细胞数)。对于n＝3次重复，给出平均值±标准偏差。CK-克氏柠檬酸杆菌菌株(#1至11)；CF-弗氏柠檬酸杆菌(Citrobacterfreundii)菌株(#1至7)；*低于检测限(<10cfu/ml)。
图3.CkP1gp131的抗细菌活性：A)不同pH值下的活性。B)不同离子强度下的活性。测试了两种浓度的内溶素(5和50μg/ml)。活性以对数单位表示为相对失活(＝log10(N0/Ni)，其中N0＝未经处理的细胞数并且Ni＝处理后的细胞数)。对于n＝4次重复，给出平均值±标准偏差。*低于检测限(<10CFU/ml)。
图4.CkP1gp131的抗细菌活性：A)不同温度下的活性。测试了两种浓度的内溶素(5和50μg/ml)。对于n＝4次重复，给出平均值±标准偏差。B)30天时期内的活性(50μg/ml)。活性以对数单位表示为相对失活(＝log10(N0/Ni)，其中N0＝未经处理的细胞数并且Ni＝处理后的细胞数)。*低于检测限(<10CFU/ml)。
说明书
定义：
如果出现在本文，则以下术语应具有下列定义。
如本文所用，术语“多肽”同义地指术语“蛋白质”。如本文所用，术语多肽”是指通过肽键以特定序列连接的氨基酸残基的线性多聚体。蛋白质的氨基酸残基可以通过例如共价附接各种基团如碳水化合物和磷酸盐来修饰。其他物质可以更松散地与多肽链缔合，如血红素或脂质，从而得到缀合的蛋白质，其也包含于如本文所用的术语“多肽”中。已经阐明了多肽链折叠的各种方式，特别是关于alpha螺旋和beta折叠片的存在。如本文所用，术语“多肽”是指所有四种类别的蛋白质，即全alpha、全beta、alpha/beta和alpha加上beta。
如本文所用，术语“肽段”是指与蛋白质如内溶素连接的任何种类的肽。然而，本发明意义上的肽段不指His6标签、Strep标签、Avi标签、Myc标签、Gst标签、JS标签、半胱氨酸(cystein)标签、FLAG标签或本领域已知的其他标签、硫氧还蛋白或者麦芽糖结合蛋白(MBP)。与如本文所用的术语“肽段”形成对比，术语“标签”是指下述肽，其可以用于促进多肽的表达和/或亲和纯化，以将多肽固定化至表面或充当标志物或标记部分以检测多肽，例如通过不同ELISA测定形式中的抗体结合，只要使标签可用于上面列出的促进之一的功能不是由所述肽的正电荷引起的。然而，取决于各自的pH，His6标签也可以带正电荷，但由于其与固定化的二价阳离子结合而用作亲和纯化工具，并且不用作根据本发明的肽段。
如本文所用，术语“肽段”是指由约2至10个约100个氨基酸残基，更优选约4至约50个氨基酸残基，更优选约5至约30个氨基酸残基组成的短多肽序列，其中一个氨基酸残基的氨基通过肽键与另一个氨基酸残基的羧基连接。
如本文所用，术语“内溶素活性”是指水解细菌细胞壁的活性，特别是通过降解细菌肽聚糖如革兰氏阴性细菌的肽聚糖。
如本文所用，术语“野生型”或“wt”是指如SEQ ID No.1中所描述的内溶素CkP1gp131的氨基酸序列。编码野生型内溶素CkP1gp131的核酸序列描述于SEQ ID No.2中。
如本文所用，术语“缺失”优选是指与相应的参照序列相比，衍生序列中不存在1、2、3、4、5(或甚至多于5个)连续氨基酸或核酸残基。衍生物可以表现出一个、两个或更多个此类缺失。
如本文所用，术语“插入”优选是指与相应的参照序列相比，衍生序列中1、2、3、4、5(或甚至多于5个)连续氨基酸或核酸残基的另外的顺序内(intrasequential)存在。本发明的衍生物可以表现出一个、两个或更多个此类插入。
如本文所用，术语“添加”优选是指与相应的参照序列相比，衍生序列的N和/或C端处另外存在1、2、3、4、5(或甚至多于5个)连续氨基酸或核酸残基。
如本文所用，术语“取代”是指位于衍生序列的某个位置处的氨基酸或核酸残基的存在，其不同于参照序列中相应的位置处存在或不存在的氨基酸或核酸残基。本发明的衍生物可以表现出此类取代的1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20或更多个。如上所述，优选地此类取代是保守取代。
如本文所用，术语“细胞壁”是指形成革兰氏阴性细菌的外细胞外壳(enclosure)并因此保证其完整性的所有组分。特别地，如本文所用，术语“细胞壁”是指肽聚糖、具有脂多糖的革兰氏阴性细菌的外膜、细菌细胞膜，而且也指以例如荚膜、外蛋白质层或粘液沉积在肽聚糖上的另外的层。
本发明涉及自克氏柠檬酸杆菌噬菌体CkP1分离的新的内溶素，其可用于制备针对革兰氏阴性细菌的新型抗细菌剂。特别地，本发明涉及包含根据SEQ ID No.1的氨基酸序列的多肽或其片段或衍生物。包含根据SEQ ID No.1的氨基酸序列的多肽优选由根据SEQ IDNo.2的核苷酸序列编码。
具有根据SEQ ID No.1的氨基酸序列的内溶素CkP1gp131具有164个氨基酸的长度。酶活性域(EAD)(aa 1至163)遵从溶菌酶样超家族域基序，其具有根据SEQ ID No.3的氨基酸序列和根据SEQ ID No.4的核苷酸序列。
因此，SEQ ID No.1的优选片段是包含根据SEQ ID No.3的氨基酸序列的多肽。SEQID No.1的其他优选片段是缺少CkP1gp131的N端甲硫氨酸的多肽(例如参见SEQ ID No.5和SEQ ID No.6)。优选地，根据本发明的多肽的片段为至少80个、更优选地至少100个、甚至更优选地至少120个、甚至更优选地至少140个、最优选地至少160个氨基酸长。优选地，包含SEQ ID No.1的片段的根据本发明的多肽不包含SEQ ID No.1的序列。优选地，包含SEQ IDNo.1的片段的根据本发明的多肽基本上表现出wt CkP1gp131内溶素(SEQ ID No.1)的裂解活性。所述活性优选表示约10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或约200％的wt CkP1gp131内溶素的活性。
在一些实施方案中，本发明的多肽包含氨基酸序列，其是根据SEQ ID No.1的氨基酸的衍生物。此类衍生物可以是基本上包含根据SEQ ID No.1、SEQ ID No.3、SEQ ID No.5和/或SEQ ID No.6的氨基酸序列，但具有另外的修饰和/或改变的多肽。
在一个实施方案中，根据本发明的所述内溶素的衍生物的所述修饰和/或改变可以是突变，特别是缺失、插入、添加、取代或其任何组合。根据本发明的所述衍生物基本上表现出wt CkP1gp131内溶素(SEQ ID No.1)的裂解活性。所述活性优选表示约10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或约200％的wt CkP1gp131内溶素的活性。
包含wt CkP1gp131内溶素(SEQ ID No.1)的片段和衍生物的多肽的活性可以通过本领域技术人员熟知的测定法测量，如例如平板裂解测定法或液体裂解测定法，其例如描述于(Briers et al.,J.Biochem.Biophys 20Methods 70:531-533,(2007))中。
在本发明的优选的实施方案中，根据本发明的多肽在N端处或在C端处另外包含标签，例如His6标签、Strep标签、Avi标签、Myc标签、Gst标签、JS标签、半胱氨酸标签、FLAG标签或本领域已知的其他标签。在本发明的优选的实施方案中，所述标签在C端处与SEQ IDNo.1的序列或其片段和/或衍生物连接(例如，N-标签-SEQ ID No.1-C)。所述标签可以通过另外的氨基酸残基与SEQ ID No.1或其片段或衍生物连接。所述另外的氨基酸残基可以由至少1、2、3、4、5、6、7、8、9或10个另外的氨基酸残基组成。在本发明的优选的实施方案中，标签通过另外的氨基酸残基Leu-Glu或Lys-Gly与SEQ ID No.1、片段或衍生物连接。在优选的实施方案中，本发明涉及包含根据SEQ ID No.7的氨基酸序列的多肽。与具有根据SEQ IDNo.1的氨基酸序列的多肽相比，具有根据SEQ ID No.7的氨基酸序列的多肽包含分别与SEQID No.1的N端连接的另外的His6标签。包含根据SEQ ID No.7的氨基酸序列的多肽优选由根据SEQ ID No.8的核苷酸序列编码。
本发明进一步涉及编码根据本发明的多肽的分离的核酸分子。根据本发明特别优选的分离的核酸分子包含根据SEQ ID No.2、SEQ ID No.4和/或SEQ ID No.8的核酸序列。优选地，核酸分子包含与SEQ ID No.1异源的启动子。
本发明进一步涉及包含根据本发明的核酸分子的载体。所述载体可以提供根据本发明的所述多肽的组成型或诱导型表达。
本发明还涉及包含根据本发明的多肽、核酸和/或载体的宿主细胞。宿主细胞可以是表达所述多肽的遗传修饰的合适宿主细胞。所述宿主细胞可以是微生物如细菌或酵母，或动物细胞如例如哺乳动物细胞，特别是人细胞。根据本发明的宿主细胞不是多细胞生物体的一部分，而是分离的(例如，永生化细胞系等)。在本发明的一个实施方案中，宿主细胞是大肠杆菌细胞。可以仅仅由于生物技术原因(例如产率、溶解度、成本等)选择宿主，但也可从医学观点选择宿主，例如，非病理性细菌或酵母或人细胞。
本发明的另一方面涉及用于遗传转化合适的宿主细胞以获得根据本发明的多肽的表达的方法，其中通过将编码根据本发明的所述多肽的根据本发明的核酸引入到宿主细胞中对宿主细胞进行遗传修饰。根据本发明的多肽的翻译和表达可以通过本领域技术人员熟知的基因工程化方法获得。
本发明还涉及从根据本发明的宿主细胞(例如表达所述多肽的经遗传修饰的合适宿主细胞)获得根据本发明的多肽的方法，其中所述方法包括从所述宿主细胞分离根据本发明的多肽。
在进一步的方面，本发明涉及组合物，优选药物组合物，其包含根据本发明的多肽和/或用根据本发明的核酸分子或载体转化的宿主细胞，并且进一步包含稀释剂、赋形剂或载体，例如药学上可接受的稀释剂、赋形剂或载体。
在本发明的优选的实施方案中，组合物另外地包含透化革兰氏阴性细菌的外膜的试剂，例如金属螯合剂如例如EDTA、TRIS、乳酸、乳铁蛋白、多粘菌素、柠檬酸和/或如例如Vaara所述(Agents that increase the permeability of the outer membrane.VaaraM.Microbiol Rev.1992Sep；56(3):395-441)的其他物质。还优选的是包含上述透化剂的组合的组合物。特别优选的是包含以下各项的组合物：约10μM至约100mM EDTA，更优选地约50μM至约10mM EDTA，更优选地约0.5mM至约10mM EDTA，更30优选地约0.5mM至约2mM EDTA，更优选地约0.5mM至约1mM EDTA。还优选的是包含以下各项的组合物：约0.5mM至约2mM EDTA，更优选地约1mM EDTA并且另外地约10至约100mM TRIS。
本发明还涉及根据本发明的多肽和/或用包含编码根据本发明的多肽的核苷酸序列的核酸转化的宿主，其用于通过手术或疗法治疗人或动物体的方法以及在人或动物体上实施的诊断方法中。在进一步的方面，本发明涉及在用于治疗和/或预防与革兰氏阴性细菌相关的病症、疾病或状况的方法中，根据本发明的多肽和/或用包含核酸分子的载体转化的宿主的用途，所述核酸分子包含编码根据本发明的多肽的核苷酸序列。特别地，所述病症、疾病或状况可以由细菌组(group)、科、属或种的革兰氏阴性细菌引起，其包含人或动物病原性菌株，如肠杆菌科((埃希氏菌属(Escherichia)，特别是大肠杆菌、沙门氏菌属(Salmonella)、志贺氏菌属(Shigella)、柠檬酸杆菌属(Citrobacter)、爱德华氏菌属(Edwardsiella)、肠杆菌属(Enterobacter)、哈夫尼菌属(Hafnia)、克雷伯氏菌属(Klebsiella)，特别是肺炎克雷伯氏菌(K.pneumoniae)、摩根氏菌属(Morganella)、变形杆菌属(Proteus)、普罗维登斯菌属(Providencia)、沙雷氏菌属(Serratia)、耶尔森氏菌属(Yersinia))、假单胞菌科(Pseudomonadaceae)(假单胞菌属(Pseudomonas)、特别是铜绿假单胞菌(P.aeruginosa))、伯克霍尔德氏菌属(Burkholderia)、寡养单胞菌属(Stenotrophomonas)、希瓦氏菌属(Shewanella)、鞘氨醇单胞菌属(Sphingomonas)、丛毛单胞菌属(Comamonas)、奈瑟氏菌属(Neisseria)、莫拉氏菌属(Moraxella)、弧菌属(Vibrio)、气单胞菌属(Aeromonas)、布鲁氏菌属(Brucella)、弗朗西丝氏菌属(Francisella)、博德特氏菌属(Bordetella)、军团菌属(Legionella)、巴尔通氏体属(Bartonella)、考克斯氏体属(Coxiella)、嗜血菌属(Haemophilus)、巴斯德氏菌属(Pasteurella)、曼氏菌属(Mannheimia)、放线杆菌属(Actinobacillus)、加德纳氏菌属(Gardnerella)、螺旋体科(Spirochaetaceae)(密螺旋体属(Treponema)和疏螺旋体属(Borrelia))、钩端螺旋体科(Leptospiraceae)、弯曲杆菌属(Campylobacter)、螺杆菌属(Helicobacter)、螺菌属(Spirillum)、链杆菌属(Streptobacillus)、类杆菌科(Bacteroidaceae)(拟杆菌属(Bacteroides)、梭杆菌属(Fusobacterium)、普雷沃氏菌属(Prevotella)、卟啉单胞菌(Porphyromonas))、不动杆菌属(Acinetobacter)、特别是鲍氏不动杆菌(A.baumanii)。特别地，治疗和/或预防病症、疾病或状况可以由铜绿假单胞菌、恶臭假单胞菌(Pseudomonasputida)、假单胞菌伯克霍尔德氏菌(Burkholderia pseudomallei)、大肠杆菌和/或鼠伤寒沙门氏菌(Salmonella Typhimurium)引起。
在进一步的方面，本发明涉及在需要治疗和/或预防的受试者中治疗病症、疾病或状况的方法，所述方法包括向所述受试者施用有效量的根据本发明的多肽和/或有效量的用包含编码根据本发明的多肽的核苷酸序列的核酸转化的宿主，或有效量的根据本发明的组合物。受试者可以是例如人或动物。特别地，所述治疗的方法可以用于治疗和/或预防由革兰氏阴性细菌，特别是由如上面列出的革兰氏阴性细菌引起的皮肤、软组织、呼吸系统、肺、消化道、眼、耳、齿、鼻咽、口、骨、阴道、菌血症的伤口和/或心内膜炎的感染。
在根据本发明的治疗(或预防)的方法中使用的剂量和施用的途径取决于待治疗的特定疾病/感染的部位。施用的途径可以是例如口服、局部、鼻咽、肠胃外、静脉内、直肠或任何其他施用的途径。为了将根据本发明的多肽和/或有效量的用包含编码根据本发明的多肽的核苷酸序列的核酸转化的宿主，或根据本发明的组合物应用于感染的部位(或要被感染的危及部位)，可以使用保护活性化合物免受环境影响(如蛋白酶、氧化、免疫应答等)的制剂，直至其到达感染的部位。因此，制剂可以是胶囊、糖衣丸、丸剂、栓剂、可注射溶液或任何其他医学上合理的盖伦制剂。优选地，盖伦制剂可以包含合适的载体、稳定剂、调味剂、缓冲剂或其他合适的试剂。例如，对于局部施用，制剂可以是洗剂或膏药，对于鼻咽应用，制剂可以是盐水溶液，经由喷雾应用于鼻。
优选地，如果待治疗(或待预防)的感染由多抗性细菌菌株引起，特别是由针对一种或多种下列抗生素具有抗性的菌株引起：链霉素、四环素、头孢金素(cephalothin)、庆大霉素(gentamicin)、头孢噻肟(cefotaxime)、头孢菌素(cephalosporin)、头孢他啶(ceftazidime)或亚胺培南(imipenem)，则根据本发明的多肽用于医学治疗。此外，根据本发明的多肽可以通过与常规抗细菌剂(例如抗生素、羊毛硫抗生素(lantibiotics)、细菌素或内溶素等)组合施用而用于治疗的方法中。
本发明还涉及包含一个或多个隔室的药物包(pharmaceutical pack)，其中至少一个隔室包含一种或多种根据本发明的多肽，一种或多种用包含编码根据本发明的多肽的核苷酸序列的核酸转化的宿主和/或根据本发明的组合物。
在另一方面，本发明涉及制备药物组合物的方法，所述方法包括将一种或多种根据本发明的多肽和/或一种或多种用包含编码根据本发明的多肽的核苷酸序列的核酸转化的宿主与药学上可接受的稀释剂、赋形剂或载体混合。
在甚至进一步的方面，根据本发明的组合物是化妆用组合物(cosmeticcomposition)。几种细菌物种可以对患者的身体的环境暴露表面如皮肤造成刺激。为了预防此类刺激或者为了消除所述细菌病原体的轻微表现(minor manifestation)，可以采用特定的化妆用制备物，其包含足够量的根据本发明的多肽以降解已经存在或新沉降的病原性革兰氏阴性细菌。
在进一步的方面，本发明涉及根据本发明的多肽，其用作医药诊断、食物诊断、饲料诊断或环境诊断中的诊断手段，特别是作为用于诊断细菌感染或污染，特别是由革兰氏阴性细菌造成的细菌感染或污染的诊断手段。在此方面，根据本发明的多肽可以用作工具以特异性降解病原性细菌，特别是革兰氏阴性病原性细菌。通过根据本发明的多肽对细菌细胞的降解可以通过添加去污剂如Triton X-100或其他减弱细菌细胞包膜的添加剂如多粘菌素B来支持。需要特定细胞降解作为后续使用基于核酸的方法如PCR、核酸杂交或NASBA(基于核酸序列的扩增)，免疫方法如IMS、免疫荧光或ELISA技术，或其他依赖于细菌细胞的细胞组分的方法如使用对独特细菌组或种具有特异性的蛋白质的酶测定法(例如，用于肠杆菌的β-半乳糖苷酶，用于凝固酶阳性菌株的凝固酶)对细菌进行特异性检测的起始步骤。
在进一步的方面，本发明涉及根据本发明的多肽用于处理或防止以下各项的革兰氏阴性细菌污染的用途：食品、食物加工设备、食物加工厂、与食品接触的(无生命)表面(如货架和食物存放区)和在其中病原性、兼性病原性或其他不期望的细菌可以潜在侵染食物材料的所有其他情况下的用途。同样地，根据本发明的多肽可以用于处理或防止以下各项的革兰氏阴性细菌污染：饲料、饲料加工设备、饲料加工厂、与饲料接触的(无生命)表面(如货架和饲料存放区)和在其中病原性、兼性病原性或其他不期望的细菌可潜在侵染饲料原料的所有其他情况下使用。根据本发明的多肽还可以用于处理或防止以下各项的革兰氏阴性细菌污染：医疗装置以及医院和医生诊室中所有种类的(无生命)表面。
特别地，本发明的多肽可以预防性地用作消毒剂。所述消毒剂可以在手术之前或之后使用，或者例如在血液透析过程中使用。此外，早产儿和免疫受损者，或有假体装置需要的那些受试者，可以用根据本发明的多肽治疗。所述治疗可以是预防性或在急性感染过程中进行。在相同背景下，医源性感染，特别是由抗生素抗性菌株如铜绿假单胞菌(FQRP)，不动杆菌种和肠杆菌科如大肠杆菌、沙门氏菌属、志贺氏菌属、柠檬酸杆菌属、爱德华氏菌属、肠杆菌属、哈夫尼菌属、克雷伯氏菌属、摩根氏菌属、变形杆菌属、普罗维登斯菌属、沙雷氏菌属和耶尔森氏菌属种造成的感染可以预防性地或在急性期期间用本发明的多肽来治疗。因此，根据本发明的多肽也可以与其他可用于消毒溶液的成分如去污剂、tensids、溶剂、抗生素、羊毛硫抗生素或细菌素组合用作消毒剂。
以下实施例解释本发明，但不认为是对本公开或所附权利要求书的限制。除非另有说明，使用如例如由Sambrock et al.,1989,Molecular Cloning:A LaboratoryManual,2nd edition,Cold Spring Harbor Laboratory Press,Cold 15Spring Harbor,New York所述的分子生物学标准方法。
实施例1.克氏柠檬酸杆菌噬菌体CkP1的内溶素CkP1gp131的克隆、表达和纯化
CkP1gp131(164个氨基酸长，MW＝18609Da；SEQ ID No.1)是源自克氏柠檬酸杆菌噬菌体CkP1的球状内溶素，预测其具有溶菌酶样超家族催化域(从氨基酸1至163；SEQ IDNo.3)(见图1)。
噬菌体CkP1的经纯化的基因组DNA用作模板，用于使用以下PCR参数在具有KAPAHiFi聚合酶(Kapa Biosystems)的标准PCR反应中扩增编码内溶素CkP1gp131的开放阅读框ORF131：
开始：95℃,2min；随后是35个循环的[98℃(20sec)→60℃(15sec)→72℃(20sec)]，随后是72℃(5min)以及冷却至4℃以及储存。
在编码内溶素CkP1gp131的ORF131的标准PCR扩增期间使用的引物：
ORF131正向引物5’gggCATATGAACATTTTTAAAATGCTTCG 3’(SEQ ID No.9)
ORF131反向引物5’gggGGATCCTCATTTATATGCGTTCCATG 3’(SEQ ID No.10)
然后将获得的PCR片段连接在市售的pET28a表达载体(Novagen)中。获得的用于重组CkP1gp131的氨基酸和DNA序列分别列于SEQ ID No.7和SEQ ID No.8。
在用0.5mM IPTG(异丙基硫代半乳糖苷)在37℃诱导4小时的时期后，在指数生长的大肠杆菌BL21(λDE3)细胞中进行CkP1gp131的重组表达。然后，使用由pET28a表达载体编码的N端6xHis标签，将内溶素应用于堆积在HisTrapTM HP 1ml柱(GE Healthcare)中的Ni2 +-NTA树脂用于纯化。纯化方法包括四个步骤：
1.平衡步骤–10mL的洗涤缓冲液(25mM咪唑、0.5mM NaCl和20mM NaH2P04–NaOH于pH7.4的浓度)。
2.加样步骤–加样总可溶性表达的大肠杆菌提取物，所述提取物重悬于洗涤缓冲液(25mM咪唑、0.5mM NaCl和20mM NaH2P04–NaOH于pH 7.4的浓度)中。
3.洗涤步骤–10mL的洗涤缓冲液(25mM咪唑、0.5mM NaCl和20mM NaH2P04–NaOH于pH7.4的浓度)。
4.洗脱步骤–2mL的洗脱缓冲液(250mM咪唑、0.5mM NaCl和20mM NaH2P04–NaOH于pH7.4的浓度)。
通过标准变性SDS-PAGE凝胶将CkP1gp131的洗脱的蛋白质级分(MW＝20772Da)可视化(图2)，获得高于95％的纯度。将蛋白质在pH 7.0的10mM HEPES中透析(使用MaxiGeBAflex-tube Dialysis Kit-Gene Bio-Application L.T.D)，并通过BCATM蛋白质测定试剂盒测定浓度，其中牛血清白蛋白作为标准品(Thermo Scientific)。获得每升大肠杆菌表达培养物37.2mg的表达产率。
实施例2.克氏柠檬酸杆菌噬菌体CKP1内溶素(SEQ ID No.7)对一组(panel)革兰氏阴性病原性细菌的抗细菌作用
为了评价抗细菌作用，使用50μg/ml的重组CkP1gp131(SEQ ID No.7)对10mMHEPES(pH7.0)中的指数中期细胞进行所有抗细菌实验，并37℃温育2小时(图3)。
以对数单位的相对失活(＝log10(N0/Ni)，其中N0＝未经处理的细胞数(在阴性对照中)并且Ni＝温育后计数的经处理的细胞数)用于定量抗细菌活性。对于n＝4次重复，给出平均值±标准偏差。
使用上式，CkP1gp131对所有测试的革兰氏阴性细菌具有抗细菌活性，范围从1直至>5个对数(log)(达到<100cfu/ml的检测限)。
实施例3.克氏柠檬酸杆菌噬菌体CKP1内溶素(SEQ ID No.7)在各种pH条件下对克氏柠檬酸杆菌的抗细菌作用
将指数生长的克氏柠檬酸杆菌细胞(≈3x109CFU/ml)以100倍稀释到20mM的不同的缓冲系统中：柠檬酸钠(pH 6)；HEPES(pH 7-8)和硼酸(pH 9)。通过将50μL的细胞与50μL的重组内溶素(或Hepes缓冲液作为阴性对照)混合以使其具有5或50μg/ml的终浓度来开始反应。温育2小时后，通过定量CFU的数量来评估抗细菌作用，并以对数单位表示为相对失活(＝log10(N0/Ni)，其中N0＝未经处理的细胞数并且Ni＝处理后的细胞数)。
实施例4.克氏柠檬酸杆菌噬菌体CKP1内溶素(SEQ ID No.7)在各种离子强度条件下对克氏柠檬酸杆菌的抗细菌作用
将指数生长的克氏柠檬酸杆菌细胞(≈3x109CFU/ml)以100倍稀释到20mM的HEPESpH 7中，并且在增加量的NaCl(0-500mM)的情况下与5或50μg/ml的内溶素(终浓度)一起温育2小时。温育2小时后，通过定量CFU的数量来评估抗细菌作用，并以对数单位表示为相对失活(＝log10(N0/Ni)，其中N0＝未经处理的细胞数并且Ni＝处理后的细胞数)。
实施例5.克氏柠檬酸杆菌噬菌体CKP1内溶素(SEQ ID No.7)在各种温度条件下对克氏柠檬酸杆菌的抗细菌作用
使用20mM的HEPES缓冲液,pH 7.0；0mM NaCl将指数生长的克氏柠檬酸杆菌细胞与内溶素一起温育。温育2小时后，通过定量CFU的数量来评估抗细菌作用，并以对数单位表示为相对失活(＝log10(N0/Ni)，其中N0＝未经处理的细胞数并且Ni＝处理后的细胞数)。
实施例6.克氏柠檬酸杆菌噬菌体CKP1内溶素(SEQ ID No.7)随时间推移对克氏柠檬酸杆菌的抗细菌作用
将内溶素(SEQ ID No.7)在20mM Hepes pH 7.0缓冲液中在37℃下保持数天。a)在第0天、第5天、第10天、第20天和第30天，a)针对指数生长的克氏柠檬酸杆菌细胞(≈3x107CFU/ml)在37℃测试50μg/ml的内溶素且进行2小时，再次使用HEPES缓冲液作为阴性对照。温育后，通过定量CFU的数量来评估抗细菌作用，并以对数单位表示为相对失活(＝log10(N0/Ni)，其中N0＝未经处理的细胞数并且Ni＝处理后的细胞数)。
在第0天、第5天、第10天、第20天和第30天，在远紫外区和近紫外区(190至260nm)的圆二色光谱上分析相同的样品。内溶素表明是高度稳定的，因为没有观察到显著的二级结构变化。因此，CD热稳定性分析表明，在37℃下一个月后内溶素活性仍然存在。
考虑到本文公开的本发明的说明书和实践，本发明的其他实施方案对于本领域技术人员而言将是显而易见的。意图认为说明书和实施例仅是示例性的，本发明的真实范围和精神由所附权利要求书指示。
序列表
<110> 米尼奥大学
<120> 新的内溶素
<130> UDM-001 PCT
<150> EP16204770.8
<151> 2016-12-16
<160> 10
<170> PatentIn version 3.5
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atgaacattt ttaaaatgct tcgtatcgat gaaggatacg attctaaaat ctataaagat 60
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Claims (14)
1.多肽，其包含根据SEQ ID No.1的氨基酸序列或其片段或衍生物。
2.根据权利要求1的多肽，其中所述片段包含根据SEQ ID No.3的氨基酸序列。
3.根据权利要求1或2的多肽，其中所述衍生物在根据SEQ ID No.1、3、5、6和/或7的氨基酸序列中具有缺失、添加、插入和/或取代。
4.根据权利要求1至3的多肽，其中所述多肽在N或C端处与具有膜或LPS破坏活性的肽段融合。
5.根据前述权利要求中任一项的多肽，其另外地包含标签，优选His6标签。
6.根据前述权利要求中任一项的多肽，其中所述多肽具有内溶素活性。
7.分离的核酸分子，其编码根据权利要求1至6中任一项的多肽。
8.载体，其包含根据权利要求7的核酸分子。
9.宿主细胞，其包含根据权利要求7的核酸分子或根据权利要求8的载体。
10.根据权利要求1至6中任一项的多肽，其用于通过手术或疗法治疗人或动物体的方法以及在人或动物体上实施的诊断方法中。
11.根据权利要求1至6中任一项的多肽作为食物、饲料中或化妆品中的抗微生物剂或者作为消毒剂(disinfecting agent)的用途。
12.根据权利要求1至6中任一项的多肽，其用于治疗或预防革兰氏阴性细菌感染的方法中。
13.根据权利要求1至6中任一项的多肽用于处理或防止以下各项的革兰氏阴性细菌污染的用途：食品、食物加工设备、食物加工厂、与食品接触的表面、饲料、饲料加工设备、饲料加工厂、与饲料接触的表面、医疗装置、医院和医生诊室中的表面。
14.药物组合物，其包含根据权利要求1至6中任一项的多肽和药学上可接受的稀释剂或赋形剂。
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| CN102575240A (zh) * | 2009-08-24 | 2012-07-11 | 勒芬天主教大学，K.U.勒芬R&D | 新的细胞内溶素OBPgpLYS |
| CN103201381A (zh) * | 2010-11-03 | 2013-07-10 | 勒芬天主教大学，K.U.勒芬R&D | 新的细胞内溶素 |
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- 2017-12-18 EP EP17837873.3A patent/EP3555121B1/en active Active
- 2017-12-18 US US16/469,800 patent/US20190330609A1/en not_active Abandoned
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Patent Citations (4)
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| WO2010023207A2 (en) * | 2008-08-26 | 2010-03-04 | Katholieke Universiteit Leuven K.U. Leuven R&D | Antimicrobial agents |
| CN102197132A (zh) * | 2008-08-26 | 2011-09-21 | 勒芬天主教大学，K.U.勒芬R&D | 抗微生物剂 |
| CN102575240A (zh) * | 2009-08-24 | 2012-07-11 | 勒芬天主教大学，K.U.勒芬R&D | 新的细胞内溶素OBPgpLYS |
| CN103201381A (zh) * | 2010-11-03 | 2013-07-10 | 勒芬天主教大学，K.U.勒芬R&D | 新的细胞内溶素 |
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| EDWARDS,G.B.等: "《lysozyme [Citrobacter phage Moon]》", 《NCBI REFERENCE SEQUENCE: YP_009146566.1》 * |
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Also Published As
| Publication number | Publication date |
|---|---|
| CN110300760B (zh) | 2023-10-20 |
| EP3555121A1 (en) | 2019-10-23 |
| KR20190110094A (ko) | 2019-09-27 |
| CA3045387A1 (en) | 2018-06-21 |
| WO2018109229A1 (en) | 2018-06-21 |
| JP2020504602A (ja) | 2020-02-13 |
| US20190330609A1 (en) | 2019-10-31 |
| AU2017375050B2 (en) | 2021-07-22 |
| KR102503567B1 (ko) | 2023-02-24 |
| EA201991233A1 (ru) | 2020-01-13 |
| EP3555121B1 (en) | 2021-09-22 |
| JP7033596B2 (ja) | 2022-03-10 |
| AU2017375050A1 (en) | 2019-06-06 |
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