WO2004083438A1

WO2004083438A1 - A new peptide against enterococcus faecalis and the method of producing it

Info

Publication number: WO2004083438A1
Application number: PCT/CN2004/000048
Authority: WO
Inventors: Xiaoqing Qiu
Original assignee: Chengdu Photon Biotechnology Limited
Priority date: 2003-03-19
Filing date: 2004-01-14
Publication date: 2004-09-30
Also published as: CN1532282A

Abstract

The present invention provides a new recombination peptide against Enterococcus faecalis. The present invention also provides polynucleotide encoding the peptide which comprising the gene encoding the ion channel-forming colicin or the pore-forming domain thereof, and the gene encoding the pheromone from Enterococcus faecalis. The present peptide can provide improved bactericidal activity while avoiding to induce resistance of the bacterium.

Description

一种新型抗肠球菌多肽及其制备方法技术领域 Novel anti-enterococcal polypeptide and preparation method thereof

本发明涉及一种重组的抗肠球菌多肽，编码该多肽的核苷酸和氨基酸序列。本发明还涉及重组抗肠球菌多肽的制备方法。背景技术 The invention relates to a recombinant anti-enterococcus polypeptide, a nucleotide and amino acid sequence encoding the polypeptide. The invention also relates to a method for preparing a recombinant anti-Enterococcus polypeptide. Background technique

细菌感染是人类生命和健康的主要威胁，自磺胺和青霉素问世以来，人类陆续发明的抗菌素主要以通过抑制细菌胞壁合成、抑制或干扰细菌的核酸和蛋白质的代谢与合成途径来达到抗菌目的。然而这些抗菌途径容易诱使细菌发生突变而产生抗菌素耐药性。因此人们一直在致力于开发新型的抗菌素。在细菌胞膜上直接形成离子通道而致细菌死亡是比较有前途的抗菌素开发方向之一。自然界中，为数不少的细菌毒素就是以这种机制来杀死细菌的，其模式标本就是大肠杆菌 (Escherichia coli)分泌的一种毒素蛋白大肠菌素 (colicin), 1952 年 Jaco 发现其能特异地杀死其它株系的大肠杆菌和相关株系的某些杆菌，如 Shigella Sonnei ( Jacob et al, ) Sur labios nthese d'une colicine et son mode d' action, Annals of the Pasteur Institute. 83:295-315(1952))。 1978年 Finkelstem 等发现了可形成离子通道的大肠菌素可以在人工脂质双分子膜上形成电压依赖性离子通道，从而在根本上揭示了这一类细菌毒素的抗菌机制（ Scliein et al,. Colicin K acts by forming voltage dependent channels in phospholipid bilayer membranes. Nature 276:159-163(1973) )。 1996年 Qiu和 Finkelstein等揭示了大肠菌素 la在人工脂质双分子膜上形成的离子通道开放和关闭时的跨膜立体结构 ( Qiu et al., Major transmembrane movement associated with colicin la channel gating. J. Gen. Physiology. 107:313-328(1996) ), 为在分子水平上设计和制备新型的抗菌素奠定了理论基础。 Bacterial infection is a major threat to human life and health. Since the advent of sulfa and penicillin, humans have invented antibiotics mainly by inhibiting bacterial cell wall synthesis, inhibiting or interfering with bacterial nucleic acid and protein metabolism and synthesis pathways to achieve antibacterial purposes. However, these antibacterial pathways are prone to induce mutations in bacteria and develop antibiotic resistance. Therefore, people have been working on the development of new antibiotics. One of the more promising directions for the development of antibiotics is the formation of ionic channels directly on the bacterial cell membrane that cause bacterial death. In nature, many bacterial toxins use this mechanism to kill bacteria. The model specimen is a toxin protein colicin secreted by Escherichia coli. In 1952 Jaco discovered that it could be specific. E. coli and other strains of related strains, such as Shigella Sonnei (Jacob et al,) Sur labios nthese d'une colicine et son mode d 'action, Annals of the Pasteur Institute. 83: 295 -315 (1952)). In 1978, Finkelstem et al. Discovered that colistin, which can form ion channels, can form voltage-dependent ion channels on the artificial lipid bimolecular membrane, thereby fundamentally revealing the antibacterial mechanism of this type of bacterial toxins (Scliein et al ,. Colicin K acts by forming voltage dependent channels in phospholipid bilayer membranes. Nature 276: 159-163 (1973)). In 1996, Qiu and Finkelstein et al. Revealed the transmembrane three-dimensional structure of the ion channel formed by colicin la on the artificial lipid bilayer membrane when it is opened and closed (Qiu et al., Major transmembrane movement associated with colicin la channel gating. J Gen. Physiology. 107: 313-328 (1996)), laying a theoretical foundation for the design and preparation of new types of antibiotics at the molecular level.

近二十年，人们逐渐发现细菌也具有随外界环境变化而分泌的信号传导多肽，制备信号传导多肽的胞膜受体及胞内调节蛋白的基因往往偶联在一起，协调地控制着细菌的生长和***，但该方向的研究一直进展緩慢，比如金黄葡萄球菌的信号传导多肽 AgrD,—直到 199⁵年才最后搞清楚是一个八肽（Ji et al., Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc. Natl. Acad. Sci. USA 92:12055-12059(1995) )₀ 1999年发现金或抑制金黄色葡萄球菌的生长 ( Mayville et al" Structure activity analysis of synthetic autoinducing thiolactone peptides from staphylococcus aureus responsible for virulence. PNAS, 96:1218-1223(1999) )₀ In the past two decades, people have gradually discovered that bacteria also have signal transduction polypeptides that are secreted with changes in the external environment. Genes that make up the cell membrane receptors and intracellular regulatory proteins of signal transduction polypeptides are often coupled together to control bacterial growth and division, but research in this direction has been slow, such as Staphylococcus aureus signaling polypeptides AgrD, - until ¹⁹⁹⁵ before finally figuring out a octapeptide (Ji et al, Cell density control of staphylococcal virulence mediated by. an octapeptide pheromone. Proc. Natl. Acad. Sci. USA 92: 12055-12059 (1995)) ₀ Discovered or inhibited the growth of S. aureus in 1999 for virulence. PNAS, 96: 1218-1223 (1999)) ₀

如上所述，大肠菌素是一种理想的离子通道抗菌素，缺点是只能作用于大肠杆菌等格兰氏阴性杆菌，如果能利用致病菌特有的信号传导肽作为诱导物，将大肠菌素或其水性孔道结构或诱导至特定的细菌膜附近形成离子通道来杀伤该致病菌，应该是一种理想的抗菌素开发方向。发明内容 As mentioned above, colistin is an ideal ion channel antibiotic. The disadvantage is that it can only act on Gram-negative bacilli such as Escherichia coli. If the signal transduction peptide peculiar to pathogenic bacteria can be used as an inducer, colistin Or its aqueous pore structure or induced ion channels formed near specific bacterial membranes to kill the pathogenic bacteria should be an ideal direction for the development of antibiotics. Summary of the Invention

本发明的一个目的在于提供一种新型的重組抗菌多肽, 其含有大肠菌素和肠球菌信号传导多肽，从而能避免诱发细菌的耐药性, 提高杀菌能力。 It is an object of the present invention to provide a new type of recombinant antibacterial polypeptide, which contains colicin and enterococcus signaling polypeptide, so as to avoid inducing drug resistance of bacteria and improve bactericidal ability.

本发明的另一目的在于提供编码该重组抗肠球菌多肽的核苷酸序列。 Another object of the present invention is to provide a nucleotide sequence encoding the recombinant anti-coccus polypeptide.

本发明的再一目的在于提供一重组质粒, 其含有编码本发明重組抗肠球菌多肽的核苷酸序列。 Yet another object of the present invention is to provide a recombinant plasmid containing a nucleotide sequence encoding the recombinant anti-enterococcal polypeptide of the present invention.

本发明的再一目的在于提供一氨基酸序列，其编码本发明的重组抗肠球菌多肽。 Another object of the present invention is to provide an amino acid sequence which encodes the recombinant enterococcus polypeptide of the present invention.

本发明的再一目的在于提供含有本发明重组抗肠球菌多肽的药物组合物。本发明的再一目的在于提供本发明抗肠球菌多肽在制备抗菌药物中的应用。 Another object of the present invention is to provide a pharmaceutical composition containing the recombinant anti-enterococcal polypeptide of the present invention. Another object of the present invention is to provide an application of the anti-entococcal polypeptide of the present invention in the preparation of an antibacterial drug.

本发明的再一目的在于提供重组抗肠球菌多肽的制备方法。 Yet another object of the present invention is to provide a method for preparing a recombinant anti-Enterococcus polypeptide.

根据本发明的一方面, 将编码肠球菌信号传导多肽的基因与大肠菌素基因可操作地连接，从而获得表达重组抗肠球菌多肽的核苷酸序列。 According to one aspect of the present invention, a gene encoding an enterococcus signaling polypeptide and an colicin gene are operably linked to obtain a nucleotide sequence expressing a recombinant anti-entococcal polypeptide.

在本发明的一个优选实施方案中，采用编码肠球菌 ( Enterococcus faecalis ) 的信号传导多肽 cCFlO的基因 (SEQ ID NO. 1)作为抗菌多肽的信息素基因，其编码如 SEQ ID NO: 2所示的七肽；大肠菌素可选自能形成离子通道的大肠菌素 El、 Ia、 Ib、 A、 B和 N或其水性孔道结构域，在本发明的一个优选实施例中，将上述肠球菌信号传导多肽基因与大肠菌素 la ( SEQ ID NO. 3 )的羧基端连接而形成如 SEQ ID NO. 4的核苷酸序列。该核苷酸序列编码如 SEQ ID NO. 5 的氨基酸序列，该氨基酸序列所编码的多肽即为本发明的抗肠球菌多肽本发明中编码肠球菌信号传导多肽的核苷酸序列 SEQ ID NO. 1所编码氨基酸序列（SEQ ID NO: 2 )与 Genebank A30128号序列所表达的氨基酸序列相同。 In a preferred embodiment of the present invention, a gene encoding a signaling polypeptide cCFlO (SEQ ID NO. 1) of Enterococcus faecalis is used as a pheromone gene of an antibacterial polypeptide, and the encoding is shown in SEQ ID NO: 2 Heptapeptide; colicin may be selected from colistin El, Ia, Ib, A, B, and N or an aqueous channel domain thereof capable of forming an ion channel. In a preferred embodiment of the present invention, the aforementioned Enterococcus The signal transduction polypeptide gene is linked to the carboxy terminus of colicin la (SEQ ID NO. 3) to form a nucleotide sequence such as SEQ ID NO. 4. The nucleotide sequence is encoded as SEQ ID NO. 5 amino acid sequence, the polypeptide encoded by the amino acid sequence is the anti-enterococcal polypeptide of the present invention, the nucleotide sequence encoding the enterococcus signaling polypeptide of the present invention, the nucleotide sequence of SEQ ID NO. 1 encoded the amino acid sequence (SEQ ID NO: 2 ) Is the same as the amino acid sequence expressed by Genebank A30128 sequence.

在构建的重組抗肠球菌多肽中，可与靶细菌胞膜受体结合的信号传导多肽作为诱导物诱导大肠菌素通道结构域穿过细菌外膜与膜间隙到达靶细胞膜附近，然后大肠菌素水性孔道结构域在靶细菌胞膜上形成离子通道，使靶细菌胞内内容物泄漏而致靶细菌死亡，从而达到杀菌的目的。 In the constructed recombinant anti-enterococcal polypeptide, a signal transduction polypeptide capable of binding to the target bacterial cell membrane receptor was used as an inducer to induce the colicin channel domain to pass through the outer membrane of the bacteria and the membrane space to the vicinity of the target cell membrane, and then colicin The aqueous channel domain forms an ion channel on the target bacterial cell membrane, which causes the contents of the target bacterial cell to leak and cause the target bacterium to die, thereby achieving the purpose of sterilization.

根据本发明的另一方面，提供如图 1所示的包含本发明核苷酸的质粒载体，该质粒载体是将如上所述的编码肠球菌信号传导多肽的核苷酸序列经双链寡聚核苷酸点突变技术***大肠菌素 la基因的笫 626位氨基酸上而形成本发明的重組质粒。 According to another aspect of the present invention, there is provided a plasmid vector comprising the nucleotide of the present invention as shown in FIG. 1, which is a plasmid vector encoding a nucleotide sequence encoding an enterococcus signaling polypeptide as described above via double-stranded oligomerization. The nucleotide point mutation technology is inserted into amino acid 626 of the colicin la gene to form the recombinant plasmid of the present invention.

本发明中 , 构建质粒载体的原始质粒 pSELECT™- 1来自于 Promega公司，其中装载了大肠菌素 la和 immunity蛋白基因，针对肠球菌信号传导基因设计了一对引物，其序列分别如 SEQ ID NO.: 6和 SEQ ID NO: 7所示。利用双链寡聚核苷酸点突变技术，按照 Strategene公司药箱操作获得重组质粒，再将获得的重组质粒转染入大肠杆菌 TG1工程菌而获得宿主细胞。 ' In the present invention, the original plasmid pSELECT ™ -1 for constructing the plasmid vector is from Promega Company, which is loaded with colistin la and immunogenic protein genes, and a pair of primers are designed for enterococcus signaling genes, the sequences of which are as shown in SEQ ID NO .: 6 and SEQ ID NO: 7. Using double-stranded oligonucleotide point mutation technology, the recombinant plasmid was obtained according to the operation of Strategene's medicine cabinet, and the obtained recombinant plasmid was transfected into E. coli TG1 engineered bacteria to obtain host cells. '

根据本发明的再一方面，提供含有本发明重组抗肠球菌多肽的药物组合物，可以通过将本发明的多肽添加药学上可接受的载体或赋形剂或可选的其它成分而制成适于临床使用的药物组合物。 According to still another aspect of the present invention, a pharmaceutical composition containing the recombinant anti-enterococcal polypeptide of the present invention is provided, which can be prepared by adding the polypeptide of the present invention to a pharmaceutically acceptable carrier or excipient or optional other ingredients. Pharmaceutical composition for clinical use.

根据本发明的又一方面，提供本发明抗肠球菌多肽的制备方法，将编码肠球菌信号传导多肽的基因可操作地与大肠菌素基因连接获得编码重组抗肠球菌多肽的基因，将获得的基因导入到表达***中进行表达，分离表达的多肽而获得本发明的抗肠球菌多肽。 According to another aspect of the present invention, a method for preparing an anti-enterococcal polypeptide of the present invention is provided. A gene encoding an enterococcus signaling polypeptide is operably linked with an colistin gene to obtain a gene encoding a recombinant anti-entococcal polypeptide. The gene is introduced into an expression system for expression, and the expressed polypeptide is isolated to obtain the anti-entococcal polypeptide of the present invention.

本发明的抗肠球菌多肽相较于传统抗菌素的优点在于，其不会诱导细菌产生传统的耐药性。众所周知，细菌可以通过突变产生 β -内酰胺酶、减少摄入、改变药物作用位点等方式来改变其细胞壁结构，改变其蛋白廣和核酸的代谢等对传统抗菌素产生耐药性。但是细菌却较难以通过突变来改变其细胞膜的磷脂双分子层的结构，而这恰好是本发明的独到之处，本发明的重組多肽通过直接在靶细菌的月膜上形成离子通道而达到杀菌目的。附图的简要说明 The advantage of the anti-entococcal polypeptide of the present invention over traditional antibiotics is that it does not induce bacteria to develop traditional drug resistance. It is well known that bacteria can change their cell wall structure by changing mutations to produce β-lactamase, reduce intake, and change the site of drug action, and change their protein and nucleic acid metabolism to develop resistance to traditional antibiotics. However, it is more difficult for bacteria to change the structure of the phospholipid bilayer of their cell membrane by mutation, and this is exactly what is unique to the present invention. Ion channels are formed on the meninges of target bacteria to achieve sterilization. Brief description of the drawings

图 1 示出含有肠球菌信号传导多肽 cCFlO和大肠菌素 la的质粒 pCHCEF 的结构； Figure 1 shows the structure of a plasmid pCHCEF containing the enterococcus signaling polypeptide cCFlO and colicin la;

图 2示出抗肠球菌多肽的结构； Figure 2 shows the structure of an anti-enterococcal polypeptide;

图 3为本发明抗肠球菌多肽（Ph-EF )体外杀菌实验结果，其中左图为普通肠球菌，可见制备的 Ph-EF与青霉素一样，有效的抑制了肠球菌生长。右图为耐万古霉素肠球菌，万古霉素部分地抑制了肠球菌生长，制备的 Ph-EF抑菌效果比万古霉素至少强一倍。 FIG. 3 is the result of in vitro bactericidal experiment of anti-enterococcal polypeptide (Ph-EF) of the present invention, wherein the left is ordinary enterococci, and it can be seen that the prepared Ph-EF effectively inhibits the growth of enterococci as well as penicillin. The picture on the right shows vancomycin-resistant enterococci. Vancomycin partially inhibits the growth of enterococci. The prepared Ph-EF is at least twice as effective as vancomycin.

图 4为抗肠球菌多肽（Ph-EF )对耐万古霉素肠球菌 (ATCC 700802 VRE) 作用效果的透射电镜观察 (放大 15,000倍)。发明的具体实施方式 Figure 4 is a transmission electron microscope observation of the effect of anti-enterococcal polypeptide (Ph-EF) on vancomycin-resistant enterococci (ATCC 700802 VRE) (magnification 15,000 times). DETAILED DESCRIPTION OF THE INVENTION

下面结合附图，通过对本发明较佳实施例的描述许细说明本发明。 In the following, the present invention will be described in detail through the description of the preferred embodiments of the present invention with reference to the accompanying drawings.

【实施例 11 表达抗肠球菌多肽的质粒的构建和重组抗肠球菌多肽制备原始质粒为装载了大肠菌素 la和 immunity蛋白基因的 pSELECTTM一 1商用质粒 (质粒大小 8.3 kb， Promega公司） (UCSF, RGosh赠与）。经双链寡聚核苷酸点突变技术 (QuickChangeTM Kit, Strategeue公司）将编码肠球菌信号多肽 cCFlO基因***到大肠菌素 la基因的 1626位点上, 制备了抗肠球菌工程多肽的突变质粒 pCHCEF (如图 1 所示）。突变质粒转染入 E.coli TGI 工程菌 (AECOM, KJakes赠与)里制备多肽。 [Example 11 Construction of a plasmid expressing an anti-Enterococcus polypeptide and preparation of a recombinant anti-Enterococcus polypeptide The original plasmid was a pSELECTTM-1 commercial plasmid (plasmid size 8.3 kb, Promega) loaded with colicin la and immunogenic protein genes (UCSF) (UCSF , Gifted by RGosh). The double-stranded oligonucleotide point mutation technology (QuickChangeTM Kit, Strategeue) was used to insert the cCFlO gene encoding the Enterococcus signal peptide into the 1626 site of the colicin la gene, and a mutant plasmid pCHCEF was prepared against the enterococcus (As shown in Figure 1). The mutant plasmid was transfected into E.coli TGI engineering bacteria (AECOM, donated by KJakes) to prepare peptides.

突变程序按 Strategene QuikC ange Site-Directed Mutagenesis Kit (Catalog#200518) 药箱手册进行: Mutation procedures were performed according to the Strategene QuikC ange Site-Directed Mutagenesis Kit (Catalog # 200518) kit manual:

1. 准备点突变反应物： 1. Prepare point mutation reaction:

5 μ 1 10X buffer 5 μ 1 10X buffer

2 μ 1 (10ng)野生型大肠菌素质粒 2 μ 1 (10ng) wild type colicin plasmid

1.25 M l (125ng)设计的 5，-3，寡聚核苷酸引物（SEQ ID NO. 6) 1.25 M l (125ng) 5, -3, oligonucleotide primer (SEQ ID NO. 6)

1.25 l (125ng)设计的 3，-5，寡聚核苷酸引物（SEQ ID NO. ⁷) 1.25 l (125ng) designed 3, -5, oligonucleotide primers (SEQ ID NO. ⁷ )

1 μ 1 dNTP 00048 双蒸水 50 μΐ 1 μ 1 dNTP 00048 Double distilled water 50 μΐ

1 μ lpfu 1 μ lpfu

(除质粒、引物和双蒸水外，均为药箱所备试剂） (Except plasmids, primers and double distilled water, all reagents in the medicine box)

2. 进行 PCR扩增，扩增条件：变性 95°C, 35秒，退火 53°C,70秒，延伸 68°C, 17分共 20个循环； 2. Perform PCR amplification. Amplification conditions: Denaturation 95 ° C, 35 seconds, annealing 53 ° C, 70 seconds, extension 68 ° C, 17 minutes for 20 cycles;

3. 加入 Dpnl内切酶 1 μ 1消化母体 DNA链后 (37°C,1小时），取 1 μ 1反应物与 XLl-Blue感受态细胞 50μ1;水孵，行热冲击 42°C， 45秒，再置入冰中 2 分钟； 3. After adding Dpnl endonuclease 1 μ 1 to digest the mother DNA strand (37 ° C, 1 hour), take 1 μ 1 of the reaction and XLl-Blue competent cells 50 μ1; incubate in water and perform thermal shock 42 ° C, 45 Seconds, then put in ice for 2 minutes;

4. 加入 NZY培基 0.5ml后 220rpm, 37°C摇菌 1小时后，取 50-100μ1反应物铺板 (LB培基加 1%琼脂加 50 μ 1/ml氨苄青霉素， 37。C过夜) ； 4. After adding 0.5ml of NZY pegylate at 220 rpm and shaking at 37 ° C for 1 hour, plate 50-100 μ1 of the reaction (LB pegylate plus 1% agar plus 50 μ 1 / ml ampicillin, 37 ° C overnight);

5. 18小时后挑菌，循 Qiagene, Gibco等公司的各种商用提取质粒药箱提取质粒均可，测序确定突变成功。 5. After 18 hours, pick bacteria and use any of the commercial extraction plasmid kits from Qiagene, Gibco and other companies to extract plasmids. Sequencing confirms that the mutation is successful.

6. 将质粒 50ng与制备的 TG1工程菌感受肽细胞 50 μ 1冰孵 30分 42°C， 90秒热冲击，取 50-100 μΐ反应物加入 LB培基 0.5ml, 220rpm, 37°C摇菌 1 小时后铺板 (LB培基加 1%琼脂加 50 μ g/ml氨苄青霉素) 37V, 18小时后挑取菌落。 6. Incubate 50 ng of plasmid with the prepared TG1 engineered bacteria-sensing peptide cells in 50 μl for 30 minutes at 42 ° C and heat shock for 90 seconds. Take 50-100 μΐ of the reaction mixture and add 0.5ml of LB cultivar, 220rpm, 37 ° C. After 1 hour, the plate was plated (LB pegylated plus 1% agar plus 50 μg / ml ampicillin) at 37V, and the colonies were picked after 18 hours.

7. 大量增菌， 8-16升 FB培基， 250rpm, 37°C, 6-8小时；离心沉淀菌体， 4°C , 6000g, 20分钟，取 4°C、 50mM硼酸緩冲液 (2mM EDTA+ 2mM DTT) 50-80ητ1悬浮菌体,加 0.2Μ PMSF 250微升后超声破碎 (4 °C , 400W, 2分钟），高速离心破碎菌体（4°C, 75000g, 1.5小时），取上清加入硫酸链霉素 500万单位沉淀 DNA, 高速离心（⁴°C， 30000g, 10分钟）后装入分子量 OOO的透析袋于 4°C, 50mM硼酸緩冲液 4升透析过夜后，高速离心（4°C, 30000g, 10 分钟）后上清上样于 CM离子交换柱， 4°C0.3 M NaCl + 50mM硼酸緩冲液洗脱即可得到重组抗肠球菌多肽 Ph-EF, 其对应的氨基酸序列为 SEQ ID NO: 5。上述制备质粒中所设计的寡聚核苷酸序列 7. Large amount of bacteria, 8-16 liters of FB culture medium, 250rpm, 37 ° C, 6-8 hours; Centrifuge the bacteria, 4 ° C, 6000g, 20 minutes, take 4 ° C, 50mM boric acid buffer solution ( 2mM EDTA + 2mM DTT) 50-80ητ1 suspension bacteria, add 0.2M PMSF 250 microliters and ultrasonically crush (4 ° C, 400W, 2 minutes), high-speed centrifugal crush bacteria (4 ° C, 75000g, 1.5 hours), take Supernatant was added with 5 million units of streptomycin sulfate to precipitate DNA, centrifuged at high speed ( ⁴ ° C, 30,000 g, 10 minutes), and loaded into a dialysis bag with a molecular weight of 000 at 4 ° C, 4 liters of 50 mM boric acid buffer, and dialyzed overnight. After centrifugation (4 ° C, 30000g, 10 minutes), the supernatant was applied to a CM ion exchange column, and the recombinant anti-enterococcal peptide Ph-EF was obtained by eluting with 0.3 M NaCl + 50 mM boric acid buffer at 4 ° C. The corresponding amino acid sequence is SEQ ID NO: 5. Oligonucleotide sequence designed in the above preparation plasmid

5'-3'(SEQIDNO: 6) 5'-3 '(SEQIDNO: 6)

gcg aat aag ttc tgg ggt att CTG GTT ACC CTT GTG TTC GTG taa ata aaa tat aag aca ggc gcg aat aag ttc tgg ggt att CTG GTT ACC CTT GTG TTC GTG taa ata aaa tat aag aca ggc

3'-5'(SEQIDNO: 7) 3'-5 '(SEQIDNO: 7)

gcc tgt ctt ata ttt tat tta CAC GAA CAC AAG GGT AAC CAG aat acc cca gaa ctt att cgc 【实施例 2】重组抗肠球菌多肽对肠球菌的体外抑制作用 gcc tgt ctt ata ttt tat tta CAC GAA CAC AAG GGT AAC CAG aat acc cca gaa ctt att cgc [Example 2] In vitro inhibitory effect of recombinant anti-enterococcal polypeptide on enterococci

细菌为美国标准菌株， ATCC 29212(普通肠球菌)和 ATCC 700802(耐万古霉素肠球菌），如图 3所示，左图加入普通肠球菌菌液 5微升 (108 CFU/ml 级菌量）加入 1%胰蛋白胨， 1% NaCl， 0.5%酵母， 0.5%葡萄糖， 1%HK₂P0₄的培养液 10 ml中，共准备 4组，第一組加入 0.3 M NaCl + 50mM硼酸緩冲液 (量与实验组中加入的抗菌多肽液体量相同)作为对照，第二组加入 3 g/ml 青霉素 G钠，第三组加入 3 μ g/ml抗肠球菌多肽（保存液为 0.3 M NaCl + 50mM硼酸緩冲液)，笫四組待细菌生长三小时后加入 3 抗肠球菌多肽（保存液为 0.3 M NaCl + 50mM硼酸緩冲液)。 The bacteria are American standard strains, ATCC 29212 (common enterococcus) and ATCC 700802 (vancomycin-resistant enterococcus). As shown in Figure 3, 5 microliters of common enterococcus liquid (108 CFU / ml grade bacteria volume) is added to the left. ) Add 1% tryptone, 1% NaCl, 0.5% yeast, 0.5% glucose, 1% HK ₂ P0 ₄ in 10 ml of culture solution, prepare a total of 4 groups, the first group is added with 0.3 M NaCl + 50 mM borate buffer (The amount is the same as the amount of antibacterial peptide liquid added in the experimental group) As a control, the second group was added with 3 g / ml penicillin G sodium, and the third group was added with 3 μg / ml anti-enterococcal peptide (the storage solution was 0.3 M NaCl + 50mM borate buffer solution). In the fourth group, three anti-enterococcal peptides were added after the bacteria had grown for three hours (the storage solution was 0.3 M NaCl + 50mM borate buffer solution).

右图加入耐万古霉素肠球菌菌液 5微升 (108 CFU/ml 级菌量）加入 1%胰蛋白胨， l% NaCl, 0.5%酵母， 0.5%葡萄糖， 1%HK₂P0₄的培养液 10 ml中，共准备 4组，第一组加入 0.3 M NaCl + 50mM硼酸緩冲液 (量与实验组中加入的抗菌多肽液体量相同）作为对照 , 第二组待细菌生长两小时后 , 加入 10 μ g/ml 万古霉素，第三组待细菌生长两小时后加入 10 μ g/ml抗肠球菌多肽（保存液为 0.3 M NaCl + 50mM硼酸緩冲液)。 On the right, add 5 microliters of vancomycin-resistant enterococcus bacterial solution (108 CFU / ml), add 1% tryptone, 1% NaCl, 0.5% yeast, 0.5% glucose, 1% HK ₂ P0 ₄ In 10 ml, prepare 4 groups in total. The first group is added with 0.3 M NaCl + 50 mM boric acid buffer (the amount is the same as the amount of the antibacterial peptide liquid added in the experimental group) as a control. The second group is added after two hours of bacterial growth. 10 μg / ml vancomycin. In the third group, 10 μg / ml anti-enterococcal peptide was added after the bacteria had grown for two hours (preservation solution was 0.3 M NaCl + 50 mM borate buffer).

上迷各組液体置于 100ml三角烧瓶中， 200 ηι, 37°C生长,每小时采样 100 μ 1加入 96孔酶标板中经分光光度计 (Α 595 nm)比色测试细菌生长浊度，画出细菌生长曲线来比较抗菌多肽的抑菌效力。 The liquids of the above groups were placed in a 100ml Erlenmeyer flask, grown at 200 η, 37 ° C, and 100 μs were sampled every hour. 1 Added to a 96-well microtiter plate and tested for bacterial turbidity by spectrophotometer (Α 595 nm) colorimetry. Draw a bacterial growth curve to compare the antibacterial efficacy of the antimicrobial peptides.

由图 3的结果可见，制备的 Ph-EF与青霉素一样，有效地抑制了肠球菌生长。右图为耐万古霉素肠球菌，万古霉素部分地抑制了肠球菌生长，制备的 Ph-EF抑菌效果比万古霉素至少强一倍。 It can be seen from the results in FIG. 3 that the prepared Ph-EF effectively inhibits the growth of enterococci as well as penicillin. The picture on the right shows vancomycin-resistant enterococci. Vancomycin partially inhibits the growth of enterococci. The prepared Ph-EF has at least twice the antibacterial effect than vancomycin.

抗肠球菌多肽分子量为 70, 000, 是万古霉素分子量 1436的 49倍，如按同体积中相同药物分子数量比较，抗肠球菌多肽的杀菌效力是去甲万古霉素的数十乃至上百倍。 The molecular weight of anti-enterococcal peptide is 70,000, which is 49 times the molecular weight of vancomycin 1436. If compared with the number of the same drug molecules in the same volume, the bactericidal efficacy of anti-entococcal polypeptide is dozens or even hundreds of times of norvancomycin. .

【实施例 3】抗肠球菌多肽对耐万古霉素肠球菌 (ATCC 700⁸02)杀菌效果的透射电镜观察 (1%磷钨酸染色，放大 15,000倍)。 [Example 3] Transmission electron microscopy observation of bactericidal effect of anti-enterococcal polypeptide on vancomycin-resistant enterococcus (ATCC 700 ⁸ 02) (1% phosphotungstic acid staining, magnification 15,000 times).

抗肠球菌多肽对耐万古霉素肠球菌 (ATCC 700802 )杀菌效果的透射电镜观察 (1%磷钨酸染色，放大 15,000倍)如图 4所示。 Control, 对照组，细菌加入前述培养液中， 37°C 200ipm生长 2小时后仍呈现正常形态； Ph-SA, 细菌生长条件同对照组，加入抗金葡菌多肽 (10 g/ml) 生长两小时后，菌形态仍然正常； Vanco, 细菌生长条件同对照組，加入万古霉素 (20 g/ml)生长 2小时后，菌形态仍然正常； Ph-EF，细菌生长条件同对照组，加入抗肠球菌多肽 (10 g/ml)生长半小时后，菌皱缩，内容物泄漏，两小时后，上述病损更加明显。结论：抗肠球菌多肽可以有效地杀死耐万古霉素肠球菌 (ATCC 700802 )₀ Transmission electron microscopy (1% phosphotungstic acid staining, 15,000 times magnification) observation of the bactericidal effect of anti-enterococcal peptides on vancomycin-resistant enterococcus (ATCC 700802) is shown in FIG. 4. Control, the control group, the bacteria were added to the aforementioned culture solution, and the normal morphology was still displayed after 2 hours of growth at 200 ° m at 37 ° C; Ph-SA, the growth conditions of the bacteria were the same as those of the control group, and anti-staphylococcus aureus peptide (10 g / ml) was added to grow After hours, the morphology was still Normal; Vanco, bacterial growth conditions are the same as those in the control group, and the bacterial morphology is still normal after 2 hours of growth with vancomycin (20 g / ml); Ph-EF, bacterial growth conditions are the same as those in the control group, with anti-enterococcal peptide (10 g After growing for half an hour, the bacteria shrank and the contents leaked. After two hours, the above lesions became more pronounced. Conclusion: Anti-entococcal peptides can effectively kill vancomycin-resistant enterococci (ATCC 700802). ₀

【实施例 4】抗肠球菌多肽对耐万古霉素肠球菌 ATCC 700802 体内保护试验 [Example 4] In vivo protection test of anti-Enterococcus polypeptide against vancomycin-resistant Enterococcus ATCC 700802

小鼠 ⁵⁵只，体重 20-25gm, 分为 4组：对照组 (n=10)、青霉素组、万古霉素组、和抗肠球菌多肽（Ph-EF)组，各药物组又分为 3个剂量组，每组 5只小鼠。小鼠在腹腔注射致死剂量耐万古霉素肠球菌（ATCC 700802 , 菌量为 105CFU/ml)l小时后，分别由尾静脉注入图示剂量各种药物一次，每 12小时检查小鼠死亡情况，结果见表 1。 ⁵⁵ mice, weighing 20-25 gm, were divided into 4 groups: control group (n = 10), penicillin group, vancomycin group, and anti-enterococcal peptide (Ph-EF) group, and each drug group was divided into 3 groups. Dose groups of 5 mice each. Mice were injected intraperitoneally with a lethal dose of vancomycin-resistant enterococcus (ATCC 700802, the bacteria volume was 105CFU / ml) for one hour, and the illustrated doses of various drugs were injected into the tail vein, and the death of the mice was checked every 12 hours. The results are shown in Table 1.

Ph-EF对耐万古霉素肠球菌 ATCC 700802体内保护试验结果 Results of in vivo protection test of Ph-EF against vancomycin-resistant enterococcus ATCC 700802

药剂量途径 X 动感染后死亡分布死 Drug dose route X distribution of death after infection

、 ,

物给药量物 ττ Dosage ττ

数数 counting

(mg/kg) M20g鼠 (只） 24 48h 72h 96 120 144 16 八 (mg / kg) M20g rat (only) 24 48h 72h 96 120 144 16 Eight

重 h h h h 8h Weight h h h h 8h

Ph- 10 IV 0.5 5 0 0 0 0 0 0 0 0 Ph- 10 IV 0.5 5 0 0 0 0 0 0 0 0 0

EF 5 5 0 1 0 0 0 0 0 1 EF 5 5 0 1 0 0 0 0 0 1

2.5 5 0 2 0 0 0 0 0 2 青 10 IV χ θ.5 5 0 0 0 0 0 0 0 0 霉 5 5 0 1 0 0 0 0 0 1 素 2.5 5 1 0 0 0 0 0 0 1 万 10 IV 0.5 5 1 0 0 0 0 0 0 1 古 5 5 0 2 1 0 0 0 0 3 母 2.5 5 1 2 0 1 0 0 0 4 素 2.5 5 0 2 0 0 0 0 0 2 Green 10 IV χ θ. 5 5 0 0 0 0 0 0 0 0 Mold 5 5 0 1 0 0 0 0 0 1 Prime 2.5 5 1 0 0 0 0 0 1 10 10 IV 0.5 5 1 0 0 0 0 0 0 1 ancient 5 5 0 2 1 0 0 0 0 3 female 2.5 5 1 2 0 1 0 0 0 4 prime

对 IV X 0.5 10 3 3 1 0 3 0 0 10 照 0.9%NaQ For IV X 0.5 10 3 3 1 0 3 0 0 10 0.9% NaQ

組结果表明， Ph— EF在动物体内可有效地抗肠球菌感染，对动物有较强的保护作用。工业应用性 Group The results show that Ph-EF can effectively resist enterococcal infection in animals and has a strong protective effect on animals. Industrial applicability

本发明的抗菌多肽不会诱导细菌产生传统的耐药性，具有较强的抗菌能力，是新一代抗菌素，具有广阔的应用前景。以上对本发明的详细描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变和变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。 The antibacterial polypeptide of the present invention does not induce traditional drug resistance in bacteria, has strong antibacterial ability, is a new generation of antibiotics, and has broad application prospects. The above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and modifications according to the present invention, as long as they do not depart from the spirit of the present invention, all should fall within the scope of the appended claims of the present invention.

Claims

权利要求书 Claim

1、编码重组抗肠球菌多肽的核苷酸，其特征在于它含有 1. A nucleotide encoding a recombinant anti-coccus polypeptide, characterized in that it contains

编码可形成离子通道的大肠菌素或其水性孔道结构域的基因，以及编码肠球菌信号传导多肽的基因。 A gene encoding colicin or an aqueous channel domain thereof capable of forming an ion channel, and a gene encoding an enterococcus signaling polypeptide.

2、根据权利要求 1所述的核苷酸，其中所述编码肠球菌信号传导多肽的基因连接于所述编码可形成离子通道的大肠菌素或其水性孔道结构域的基因的 C末端。 2. The nucleotide according to claim 1, wherein the gene encoding the enterococcus signaling polypeptide is linked to the C-terminus of the gene encoding colicin or its aqueous channel domain that can form an ion channel.

3、根据权利要求 2所述的核苷酸，其中所述可形成离子通道的大肠菌素选自大肠菌素 El、 Ia、 Ib A、 B和N。 3. The nucleotide according to claim 2, wherein the colistin capable of forming an ion channel is selected from the group consisting of colistin El, Ia, Ib A, B, and N.

4、根据权利要求 3所述的核苷酸，其中所述可形成离子通道的大肠菌素为大肠菌素 Ia。 4. The nucleotide according to claim 3, wherein the colistin capable of forming an ion channel is colicin Ia.

5、根据权利要求 4所述的核苷酸,其中含有 SEQ ID NO. 4的核苷酸序列。 5. The nucleotide according to claim 4, which contains the nucleotide sequence of SEQ ID NO.

6、一种重组质粒，其特征在于其含有 6. A recombinant plasmid, characterized in that it contains

7、根据权利要求 6所述的重组质粒，其中含有 SEQ ID NO. 4的核苷酸序列。 The recombinant plasmid according to claim 6, which contains the nucleotide sequence of SEQ ID NO.

8、一种重組抗肠球菌多肽，其含有 8. A recombinant enterococcus polypeptide comprising:

可形成离子通道的大肠菌素或其水性孔道结构域，以及 Colistin or an aqueous channel domain thereof capable of forming an ion channel, and

肠球菌信号传导多肽。 Enterococcus signaling polypeptide.

9、根据权利要求 8所述的抗肠球菌多肽，其中包含 SEQ ID NO. 5的氨基酸序列。 The anti-enterococcal polypeptide according to claim 8, which comprises the amino acid sequence of SEQ ID NO.

10、含有权利要求 9所述的重组抗肠球菌多肽的药物组合物。 10. A pharmaceutical composition comprising the recombinant anti-enterococcal polypeptide according to claim 9.

11、权利要求 9所述抗肠球菌多肽在制备抗菌药物中的应用。 11. Use of the anti-enterococcal polypeptide according to claim 9 in the preparation of antibacterial drugs.

12、重組抗肠球菌多肽的制备方法，包括以下步骤： 12. A method for preparing a recombinant enterococcus polypeptide includes the following steps:

将编码肠球菌信号传导多肽的基因与编码可形成离子通道的大肠菌素或其水性孔道结构域的基因可操作地连接获得编码重组抗肠球菌多肽的基因；将获得的基因导入到表达***中进行表达； The gene encoding the enterococcus signaling polypeptide and the gene encoding the colistin or its aqueous channel domain that can form an ion channel are operably linked to obtain a gene encoding a recombinant anti-coccus polypeptide; the obtained gene is introduced into an expression system Express

分离表达的多肽而获得本发明的抗肠球菌多肽。 The expressed polypeptide is isolated to obtain the anti-entococcal polypeptide of the present invention.