WO2018086516A1 - 一种抑制耐药性细菌的抗菌肽及其应用 - Google Patents

一种抑制耐药性细菌的抗菌肽及其应用 Download PDF

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WO2018086516A1
WO2018086516A1 PCT/CN2017/109799 CN2017109799W WO2018086516A1 WO 2018086516 A1 WO2018086516 A1 WO 2018086516A1 CN 2017109799 W CN2017109799 W CN 2017109799W WO 2018086516 A1 WO2018086516 A1 WO 2018086516A1
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drug
resistant
lys
bacteria
arg
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French (fr)
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罗小芳
覃佐东
何福林
欧阳晓平
李治章
李常健
彭倩芮
陈哲生
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湖南科技学院
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Priority to US16/407,132 priority Critical patent/US10603350B2/en
Priority to US16/790,666 priority patent/US10918692B2/en
Priority to US16/790,669 priority patent/US10898541B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to antibacterial peptides, particularly antibacterial peptides capable of inhibiting and killing bacteria, and in particular to an antibacterial peptide capable of inhibiting and killing multi-drug resistant bacteria.
  • MDR multidrug resistance
  • the technical problem to be solved by the present invention is to provide an antimicrobial peptide capable of inhibiting and killing multi-drug resistant bacteria and an application thereof.
  • the present invention provides an antimicrobial peptide capable of inhibiting and killing a multi-drug resistant bacterium, which is one of the following:
  • XH-12C Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile-Trp-Arg; as described in SEQ ID NO: 3;
  • XH-12B Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile-Phe; as described in SEQ ID NO: 2;
  • XH-12A Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile; ie as described in SEQ ID NO: 1.
  • the invention also discloses the use of the above antibacterial peptide in the preparation of a medicament for inhibiting and/or killing fungi, Gram-positive bacteria, Gram-negative bacteria, and drug-resistant bacteria.
  • the fungus is Candida albicans
  • the Gram-positive bacteria are Staphylococcus aureus, Listeria,
  • the Gram-negative bacteria is Escherichia coli
  • the drug-resistant bacteria are drug-resistant golden yellow grape ball, drug-resistant Acinetobacter baumannii, drug-resistant Klebsiella pneumoniae, drug-resistant Enterobacter sakazakii, drug-resistant Salmonella typhimurium, drug-resistant Streptococcus agalactiae , drug-resistant Enterococcus faecalis, resistant R. anatipestifer.
  • the invention also provides the use of the above antibacterial peptide in the preparation of an anticancer drug.
  • the cancer is H460, KB-3-1 or drug-resistant cancer MX20, KB-C2, KB-CV60.
  • the invention also provides the use of the above antimicrobial peptide in the preparation of a medical carrier.
  • the medicament includes at least one of XH-12C, XH-12B, and XH-12A.
  • the drug is mixed with at least one pharmaceutically acceptable carrier or additive.
  • the application field thereof is any of the following: human medicine (antibacterial, anticancer, burn treatment, etc.), veterinary medicine, medical carrier, food additive, feed additive, daily chemical product.
  • the preparation method of the above antibacterial peptide is prepared by the BOC method in the conventional solid phase chemical synthesis method.
  • the N-terminal Boc protecting group must be removed with TFA prior to HF cleavage.
  • the method of manually cutting the N-terminal Boc group was to wash the reaction with a TFA/DCM ratio of 1:1 at room temperature for 15 min.
  • Trp-containing polypeptide resin placed in a round bottom flask at a volume ratio of 1:10 piperidine/DMF, and cooled in an ice bath; added polypeptide resin (1 g/10 mL) at 0 ° C The reaction was stirred for 2 h; washed twice with DMF (5 volumes), DCM twice, MeOH twice.
  • the Cys-containing polypeptide was HF/anisole/DMS/p-thiophenol (10:1:1:0.2), and Cys-free HF/DMS/anisole (10:1:1).
  • the use of the antibacterial peptide of the present invention may be, for example, application in the preparation of a medicament for treating Gram-negative bacteria infected with Staphylococcus aureus, Listeria as Gram-negative bacteria or Escherichia coli, in particular In the preparation of a medicament for treating or preventing veterinary or human use, for killing and inhibiting Staphylococcus aureus, Listeria, and Escherichia coli;
  • the medicament may comprise a composition of one or more of the above-described antimicrobial peptides defined in the present invention.
  • the medicament may further comprise one or more pharmaceutically acceptable carriers or additives, such as an active enzyme for promoting enzymatic hydrolysis, or an active agent for enhancing dispersibility;
  • the dosage and use conditions of the antimicrobial peptide of the present invention in the above applications can be determined according to methods known in the art.
  • the present invention has been experimentally shown to inhibit and kill Gram-positive bacteria represented by Staphylococcus aureus and Listeria, or Gram-negative bacteria represented by Escherichia coli, and fungi represented by Candida albicans. Significant effect; the invention It has a significant effect on a variety of drug-resistant bacteria, cancer cells, and drug-resistant cancer cells.
  • the antibacterial peptide obtained by the invention has broad-spectrum antibacterial activity, has low hemolytic activity and anti-cancer activity, and has a good therapeutic effect on drug-resistant bacteria and drug-resistant cancer cells, and has certain therapeutic effects. Application prospects in the fields of medicine, food and daily chemicals.
  • Figure 1 is a diagram showing the effect of the antibacterial peptide XH-12A on killing Staphylococcus aureus (A), Listeria (B), and Escherichia coli (C);
  • FIG. 2 is an effect diagram of the antibacterial peptide XH-12B killing Staphylococcus aureus (A), Listeria (B), and Escherichia coli (C);
  • Fig. 3 is a diagram showing the effects of the antibacterial peptide XH-12C against Staphylococcus aureus (A), Listeria (B), and Escherichia coli (C).
  • Example 1 Evaluation of minimum inhibitory concentration (MIC) using a micro-broth dilution method.
  • the drug concentration range should include resistance, mediation, and sensitive cut-off values.
  • NMCLS recommends the use of Mueller-Hinton (MH) broth, Ph 7.2 ⁇ 7.4. Aerobic bacteria and facultative anaerobic bacteria grow well in this medium.
  • MH Mueller-Hinton
  • 3 to 5 colonies of similar morphology to be examined were picked up by inoculating loops, inoculated in 4-5 mL of hydrolyzed casein (MH) broth, and incubated at 35 ° C for 2-6 h.
  • the logarithmic growth phase of the bacteria after the enrichment was corrected to 0.5 McMurray standard with physiological saline or MH broth, and contained about 1 to 2 ⁇ 10 8 CFU/mL.
  • MIC board Preparation of MIC board: Aseptic operation, add different concentrations of antibiotic solution to the sterilized 96-well polystyrene plate, and add the solution to the first to eleventh holes, 10 ⁇ L per well, the 12th hole. No drug was added as a growth control, sealed after freeze-drying, and stored at -20 ° C or less.
  • the suspension prepared by the growth method or the direct bacterial suspension method having a concentration equivalent to 0.5 McPherson standard is diluted with 1:1000 in MH broth, 100 ⁇ L is added to each well, and sealed at 35 ° C for ordinary air. In the incubator, incubate for 16 to 20 hours to judge the result. At this time, the drug concentrations of the first to the eleventh holes were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 ⁇ g/mL, respectively.
  • Escherich ATCC 25922, Monocute proliferation ATCC 19115, Staphylococcus aureus ATCC29213 were used in this experiment.
  • MBC Minimum bactericidal concentration
  • Double dilution to determine the minimum bactericidal concentration the medicinal concentration is halved (double dilution), such as MBC in a row/column of 96-well plates, and 10 ⁇ L of different concentrations of drug solution are added to each well to make the first
  • the final concentration of the solution was 128 ⁇ g/mL
  • the second well was 64 ⁇ g/mL
  • the third 32 ⁇ g/mL ..., and so on.
  • the concentration is MBC. The results are shown in Figures 1-3.
  • Escherich ATCC 25922, Monocute proliferation ATCC 19115, Staphylococcus aureus ATCC29213 were used in this experiment.
  • Example 3 Determination of inhibitory activity against multi-drug resistant bacteria and minimum inhibitory concentration (MIC)
  • the minimum dilution factor was used to detect the minimum inhibitory concentration (MIC).
  • the tested strains were multi-drug resistant Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus and multi-drug resistant sakisaki Enterobacter, Salmonella typhimurium, Streptococcus agalactiae, Enterococcus faecalis, R. anatipestifer. Each strain was inoculated on MH solid medium and cultured in an inverted incubator at 37 °C. After the colony grows, the single colony is picked up by the inoculating loop and transferred to the MH liquid medium.
  • the incubator is shaken to a logarithmic growth phase at 37 ° C, and the OD 600 is detected by an ultraviolet spectrophotometer according to 1 OD 600 ⁇ 1 ⁇ . 10 9 cfu/ml, the concentration of the bacterial solution was calculated, and it was diluted to 2 ⁇ 10 5 cfu/ml using MH medium. 90 ⁇ L of bacterial solution and 10 ⁇ L of the polypeptide solution were added to each well, mixed uniformly, and then cultured at 37 ° C for 18 hours, and OD 600 was measured using a microplate reader. The minimum sample concentration that inhibits bacterial growth is the minimum inhibitory concentration (MIC) of the sample. The experiment was repeated 3 times and averaged. The results obtained are as described in Tables 2 and 3 below.
  • Enterobacter sakazakii Salmonella typhimurium Streptococcus agalactiae Enterococcus faecalis Riemerella anatipestifer XH-12C 300 ⁇ g/ml 300 ⁇ g/ml 50 ⁇ g/ml 200 ⁇ g/ml 300 ⁇ g/ml XH-12B 300 ⁇ g/ml 300 ⁇ g/ml 50 ⁇ g/ml 50 ⁇ g/ml 300 ⁇ g/ml XH-12A 250 ⁇ g/ml 300 ⁇ g/ml 50 ⁇ g/ml 50 ⁇ g/ml 300 ⁇ g/ml
  • Example 4 Determination of inhibitory activity against fungi and minimum inhibitory concentration (MIC)
  • the experimental results show that the above antibacterial peptides have inhibitory activities against Candida albicans, especially the antimicrobial peptides XH-12C and XH-12A.
  • Example 5 Inhibitory activity against cancer resistant cancer cells and determination of semi-inhibitory concentration (IC 50 )
  • the toxicity of antimicrobial peptides to cancer cells was analyzed by MTT assay.
  • the cells were first suspended in a culture dish with trypsin-digested moisture, and 180 ⁇ L of the cell solution was seeded in a 96-well plate to a final concentration of 5 ⁇ 10 3 cells/well, and incubated for 24 hours. Then add 20 ⁇ L of different concentrations of peptide, use topotecan, paclitaxel, vincristine and doxorubicin as positive controls, continue incubation for 68 hours, add MTT reagent (4mg/mL), incubate for 4 hours, then discard The supernatant was dissolved in 100 ⁇ L of DMSO to dissolve the crystals. Cell viability was measured at a wavelength of 570 nm. IC 50 values calculated from the survival curves. The experiment was repeated 3 times and averaged. The results obtained are as described in Table 5.
  • MX20 is a H460 cell resistant strain overexpressing BCRP protein induced by Mitoxantrone
  • KB-C2 is a drug-resistant strain of K-3-1 cells overexpressing P-gp protein induced by Colchicine;
  • KB-CV60 is a KB-3-1 cell-resistant strain overexpressing MRP1 protein induced by Cepharanthine and Vincritine.
  • Hemolysis rate (%) (D test - D negative ) / (D positive - D negative ) ⁇ 100%.
  • the standard hemolysis rate is less than 5%, which is a negative reaction, no hemolysis; the hemolysis rate is more than 5% is a positive reaction, and there is hemolysis.
  • the results obtained are as described in Table 6.
  • Hemolysis rate (%) (D -D-negative sample) / (D -D-negative-positive) ⁇ 100%.
  • the standard hemolysis rate is less than 5%, which is a negative reaction, no hemolysis; the hemolysis rate is more than 5% is a positive reaction, and there is hemolysis.
  • the results obtained are as described in Table 7.

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Abstract

本发明提供了一类能抑制和杀灭多种耐药性细菌的抗菌肽，包括XH-12C、XH-12B、XH-12A。本发明还提供了上述抗菌肽在制备抑制和/或杀灭真菌、革兰氏阳性菌、革兰氏阴性菌、耐药性细菌药物、抗癌药物中的应用以及在医用载体中的应用。

Description

[根据细则37.2由ISA制定的发明名称]　一种抑制耐药性细菌的抗菌肽及其应用 技术领域
本发明涉及抗菌肽，特别是能抑制和杀灭细菌的抗菌肽，具体是一种能抑制和杀灭多种耐药性细菌的抗菌肽。
背景技术
抗生素滥用导致病原微生物产生高水平抗药性和多药物抗性(multidrug resistance，MDR)已经成为世界性的难题。近几年来，具有MDR的“超级病菌”在世界各地相继爆发，严重威胁着人类的生命和健康安全。大量研究表明，致病菌通过基因的水平转移等机制可获得MDR，并抵抗多种临床一线使用的抗生素。为了应对来自“超级耐药细菌”日益严峻的挑战，人们除了对现有抗生素进行修饰改良和发掘新型抗生素外，更寄希望于挖掘抗生素的替代物以缓解“超级耐药细菌”所造成的世界卫生安全危机。
发明内容
本发明要解决的技术问题是提供一种能抑制和杀灭多种耐药性细菌的抗菌肽及其应用。
为了解决上述技术问题，本发明提供一种能抑制和杀灭多种耐药性细菌的抗菌肽，其为以下任一：
XH-12C：Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile-Trp-Arg；即如SEQ ID NO:3所述；
XH-12B：Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile-Phe；即如SEQ ID NO:2所述；
XH-12A：Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile；即如SEQ ID NO:1所述。
本发明还同时公开了上述抗菌肽在制备抑制和/或杀灭真菌、革兰氏阳性菌、革兰氏阴性菌、耐药性细菌药物中的应用。
作为上述应用的改进：
所述真菌为白色念珠菌；
所述革兰氏阳性菌为金黄色葡萄球菌、李斯特菌，
所述革兰氏阴性菌为大肠杆菌；
所述耐药性细菌为耐药金黄色葡萄球、耐药鲍曼不动杆菌、耐药肺炎克雷伯菌、耐药阪崎氏肠杆菌、耐药鸡伤寒沙门氏菌、耐药无乳链球菌、耐药粪肠球菌、耐药鸭疫里默氏杆菌。
本发明还同时提供了上述抗菌肽在制备抗癌药物中的应用。
作为本发明的上述应用的改进：所述癌为H460、KB-3-1或耐药癌MX20、KB-C2、KB-CV60。
本发明还同时提供了上述抗菌肽在制备医用载体中的应用。
作为本发明抗菌肽上述的应用的改进，所述药物包括XH-12C、XH-12B、XH-12A中的至少一个。所述药物中混合有至少一种的医药上可接受的载体或添加剂。
作为本发明抗菌肽上述的应用的改进，其应用领域为以下任一：人用药(抗菌、抗癌、烧伤处理等)、兽用药、医用载体、食品添加剂、饲料添加剂、日化用品。
上述抗菌肽的制备方法，采用现有的固相化学合成法中的BOC法制备。
具体包括：
一、树脂合成
1)Peptide acid Merrifield Resin and PAM Resin
2)Peptidecarboxamide MBHA Resin
二、肽链合成
1)氨基酸的偶联；
2)N-端Boc基团的切除；
在HF切割以前须将N-端Boc保护基团用TFA除去。手工切割N-端Boc基团方法是用TFA/DCM比为1：1的溶液在室温条件下洗涤反应15min。
三、切割
1)切割前的树脂准备：所有树脂在切割前必须完全的洗净和干燥。
特殊情况处理：
A)His的Dnp保护基团的切割：用最小体积的DMF溶胀树脂；用20mol的苯硫酚处理1-2h；将树脂转移到黏结玻璃漏斗中，在用HF液体或TFMSA处理前以DMF，甲醇和冷乙醚频繁冲洗。
B)含Trp的多肽树脂的脱甲酰基：以体积比为1：10哌啶/DMF液放入圆底烧瓶中，并在冰浴中冷却；加入多肽树脂(1g/10mL)，在0℃下搅拌反应2h；用DMF(5倍体积)洗两次，DCM两次，MeOH两次；用HF切割前高真空干燥上述所得树脂至少4h。
2)HF切割
标准HF切割法(0.2mmol)
A)将多肽树脂，聚四氟乙烯管和净化剂混合物加入反应容器中。含Cys的多肽用HF/苯甲醚/DMS/p-苯甲硫酚(10：1：1：0.2)，而不含Cys的用HF/DMS/苯甲醚(10：1：1)。
B)将盖子旋紧，切割前在冰的纯甲醇中冷却至少5min。
C)在参照生产商的说明下蒸馏10mL的HF到瓶中。因为含Arg(Tos)的多肽切割会持续2h以上。
D)反应最后，通过氮气流蒸发HF和DMS。
E)用TFA从树脂上吸取出切除下的多肽。
F)负压下过滤移走树脂，用TFA洗树脂两次。过滤筛选，加入8-10倍的冷乙醚。有时需要蒸发大多数TFA以得到粗品的沉淀物。
3)切割后期处理：
A)沉淀，在真空下在Hirsch漏斗中用硬滤纸过滤沉淀的肽。用冷醚冲洗沉淀物，在合适的缓冲溶液中溶解，然后冻干。
B)离心，加入少量体积的叔丁甲醚在残留物中，研磨，直到得到悬浮物，将悬浮物转移到一个干净的离心管中，密闭离心，自动离心机在这过程中是必须的。然后将醚从管中小心的倒出，并用醚重复洗涤，在合适的缓冲溶液里溶解残留物，然后冻干。
C)水溶性肽，沉淀后，加水到残留物中，然后把混合物转移到分液漏斗中，可以加入少量乙醇助溶。
D)充分摇晃堵塞的漏斗，分散混合物，静止分离，分离下层液(水)。
E)加少量水到漏斗中，重复上述的摇晃—静止—分离三次，移去上层液，然后放置于干净的烧瓶中，将混合液转移到分液漏斗中。
F)加少量的新配二乙基醚，重复上述的摇晃—静止—分离二到三次，每次都把醚层移去，把水层放到分液漏斗中，收集水层到干净的烧瓶，冻干。
本发明的抗菌肽的用途，例如可以是：在制备治疗以金黄色葡萄球菌、李斯特菌为革兰氏阴性菌或以大肠杆菌为代表的革兰氏阴性菌感染药物中的应用，特别是在制备治疗或预防兽用或人用的药物中的应用，用以杀灭和抑制金黄色葡萄球菌、李斯特菌、大肠杆菌；
所述的药物可以包含上述本发明所定义的抗菌肽中的一种或多种的组合物；
所述的药物还可以包含有一种或一种以上医药上可接受的载体或添加剂，如为了实现促进酶解的活性酶，或者是增强分散能力的活性剂；
本发明的抗菌肽在上述应用中的使用剂量和使用条件，可以根据本领域的已知方法来确定。
本发明经实验表明，用于抑制和杀灭以金黄色葡萄球菌、李斯特菌为代表的革兰氏阳性菌或以大肠杆菌为代表的革兰氏阴性菌、以白色念珠菌为代表的真菌有显著的效果；本发明 对多种耐药性细菌、癌细胞、耐药癌细胞有显著的效果。
综上所述，本发明所得的抗菌肽具有广谱的抗菌活性，且具有低溶血性、抗癌细胞活性，特别是对于耐药菌和耐药癌细胞具有很好的治疗作用，具有一定的医药、食品和日化领域的应用前景。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为抗菌肽XH-12A杀灭金黄色葡萄球菌(A)、李斯特菌(B)、大肠杆菌(C)的效果图；
图2为抗菌肽XH-12B杀灭金黄色葡萄球菌(A)、李斯特菌(B)、大肠杆菌(C)的效果图；
图3为抗菌肽XH-12C杀灭金黄色葡萄球菌(A)、李斯特菌(B)、大肠杆菌(C)的效果图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1、最小抑菌浓度(MIC)评估，采用微量肉汤稀释法。
A、抗菌药物和培养基制备
抗菌药物和培养基制备，同常量肉汤稀释法。
药敏试验用抗菌药物浓度范围：根据NCCLS抗菌药物敏感性试验操作标准，药物浓度范围应包含耐药、中介和敏感分界点值。
培养基：NCCLS推荐使用Mueller-Hinton(MH)肉汤，Ph7.2～7.4。需氧菌及兼性厌氧菌在此培养基中生长良好。接种物的制备,用接种环挑取形态相似待检菌落3～5个，接种于4～5mL的水解酪蛋白(MH)肉汤中，35℃孵育2～6h。增菌后的对数生长期菌液用生理盐水或MH肉汤校正浓度至0.5麦氏比浊标准，约含1～2×108CFU/mL。
B、MIC板制备
MIC板制备：无菌操作，将倍比稀释后不同浓度的抗菌药物溶液分别加到灭菌的96孔聚苯乙烯板中，第1至第11孔加药液，每孔10μL，第12孔不加药作为生长对照，冰冻干燥后密封，-20℃以下保存备用。
C、接种物制备
将用生长法或直接菌悬液法制备的浓度相当于0.5麦氏比浊标准的菌悬液，经MH肉汤1：1000稀释后，向每孔中加100μL，密封后置35℃普通空气孵箱中，孵育16～20h判断结果。此时，第1孔至第11孔药物浓度分别为128、64、32、16、8、4、2、1、0.5、0.25、0.125μg/mL。
D、结果判断
结果判断：以在小孔内完全抑制细菌生长的最低药物浓度为MIC。当阳性对照孔(即不含抗生素)内细菌明显生长试验才有意义。当在微量肉汤稀释法出现单一的跳孔时，应记录抑制细菌生长的最高药物浓度。
结果见下表1。
表1
多肽编号 金黄色葡萄球菌 单核细胞增生李斯特菌 大肠杆菌
XH-12A 4μg/ml 4μg/ml 32μg/ml
XH-12B 16μg/ml 16μg/ml 64μg/ml
XH-12C 16μg/ml 8μg/ml 32μg/ml
本实验中使用：大肠杆菌(Escherich)ATCC25922、李斯特菌(Monocute proliferation)ATCC19115、金黄色葡萄球菌(Staphylococcus aureus)ATCC29213。
实施例2、最低杀菌浓度测试(MBC)
最小杀菌浓度(minimum bactericidal concentration，MBC)：杀死99.9％(降低3个数量级)的供试微生物所需的最低药物浓度。
二倍稀释测最小杀菌浓度：就是将药物浓度依次减半(倍比稀释)，比如在96孔板的某行/列做MBC，每个孔均加入10μL不同浓度的药液，使第一个孔药液终浓度为128μg/mL，第二个孔是64μg/mL，第三个32μg/mL，……，依次类推。最低至哪个浓度，已经没有活细菌了，该浓度即为MBC。结果见图1～3。
其中，HR---小时，μg/ml---浓度，CFU reduction％---菌落减少百分比。
本实验中使用：大肠杆菌(Escherich)ATCC25922、李斯特菌(Monocute proliferation)ATCC19115、金黄色葡萄球菌(Staphylococcus aureus)ATCC29213。
实施例3、对多重耐药菌的抑制活性及最小抑菌浓度(MIC)的测定
采用二倍稀释法进行最小抑菌浓度(Minimal inhibitoral concentration,MIC)的检测，受试菌株为人多重耐药鲍曼不动杆菌、肺炎克雷伯菌、金黄色葡萄球菌和动物多重耐药阪崎氏肠杆菌、鸡伤寒沙门氏菌、无乳链球菌、粪肠球菌、鸭疫里默氏杆菌。各菌株接种于MH固体培养基上，37℃培养箱中倒置培养。待菌落长出后用接种环挑取单菌落转接到MH液体培养基中，37℃培养箱震荡培养至对数生长期，用紫外分光光度计检测菌液OD600，根据1OD600≈1×109cfu/ml，计算出菌液浓度，使用MH培养基将其稀释为2×105cfu/ml。向每孔中加90μL菌液和10μL多肽溶液，混均匀后置于37℃培养18h，使用酶标仪测OD 600。能抑制细菌生长的最小样品浓度就是该样品的最小抑菌浓度(MIC)。实验重复3次，取平均值。所得结果如下表2和表3所述。
表2、抗菌肽对人多重耐药菌的最小抑菌浓度
  多重耐药金黄色葡萄球菌 多重耐药鲍曼不动杆菌 多重耐药肺炎克雷伯菌
XH-12C 4μg/ml 4μg/ml 4μg/ml
XH-12B 4μg/ml 8μg/ml 4μg/ml
XH-12A 4μg/ml 8μg/ml 8μg/ml
表3、抗菌肽对动物多重耐药菌的最小抑菌浓度
  阪崎氏肠杆菌 鸡伤寒沙门氏菌 无乳链球菌 粪肠球菌 鸭疫里默氏杆菌
XH-12C 300μg/ml 300μg/ml 50μg/ml 200μg/ml 300μg/ml
XH-12B 300μg/ml 300μg/ml 50μg/ml 50μg/ml 300μg/ml
XH-12A 250μg/ml 300μg/ml 50μg/ml 50μg/ml 300μg/ml
实验结果表明：以上抗菌肽对人多重耐药菌和动物多重耐药菌都有抑制活性。
实施例4、对真菌的抑制活性及最小抑菌浓度(MIC)的测定
依据NCCLS的M27-A方案，先按二倍稀释将抗菌肽100μL加到96孔板中，再加入0.5～2.5×103个/mL白色念珠菌的混悬液100μL，37℃培养48h，以无菌生长的最小药物稀释度作为抗菌肽对白色念珠菌的MIC。实验重复3次，取平均值。所得结果如表4所述。
表4、抗菌肽对真菌的最小抑菌浓度
  白色念珠菌
XH-12C 4μM
XH-12B 146μM
XH-12A 20μM
实验结果表明：以上抗菌肽对白色念珠菌都有抑制活性，特别是抗菌肽XH-12C和XH-12A。
实施例5、对耐药癌细胞的抑制活性及半抑制浓度(IC50)的测定
用MTT法分析抗菌肽对癌细胞的毒性。细胞先用胰蛋白酶消化湿气悬浮在培养皿中，取180μL细胞液接种在96孔板，使细胞终浓度为5×103个细胞/孔，孵化24小时。然后加入20μL不同浓度的多肽，用拓扑替康、紫杉醇、长春新碱和阿霉素作为阳性对照，继续孵化68小时后，加入MTT试剂(4mg/mL)，再孵育4小时，然后弃去上清液，加入100μL DMSO溶解结晶物。在570nm波长处测量细胞存活率。根据生存曲线计算IC50值。实验重复3次， 取平均值。所得结果如表5所述。
表5、抗菌肽对癌细胞和耐药癌细胞的最小抑菌浓度
  H460 MX20 KB-3-1 KB-C2 KB-CV60
XH-12C 6μM 6μM 6μM 3μM 6μM
XH-12B 8μM 7μM 5μM 4μM 7μM
XH-12A 75μM 49μM 20μM 22μM 25μM
其中：MX20是用二羟葱二酮(Mitoxantrone)诱导的BCRP蛋白过表达的H460细胞耐药株；
KB-C2是用秋水仙碱(Colchicine)诱导的P-gp蛋白过表达KB-3-1细胞的耐药株；
KB-CV60是用千金藤素(Cepharanthine)和长春新碱(Vincritine)共同诱导的MRP1蛋白过表达的KB-3-1细胞耐药株。
实验结果表明：以上抗菌肽对癌细胞和耐药癌细胞均具有抑制活性。
实施例6、溶血活性的测定
1)、新鲜人血红细胞置于含肝素抗凝剂的离心管中，离心(1200rpm/15min)。弃去上清液，细胞用生理盐水冲洗几次。将所得红细胞用PBS缓冲液配制成2％(v/v)的混悬液。取100μL细胞混悬液和抗菌肽溶液100μL(浓度为16～3200μg/ml)置于96孔板，在37℃孵育2h后，离心(1200rpm/10min)，用酶标仪检测620nm处的吸光度D。并计算溶血率，实验重复3次，取平均值。溶血率(％)＝(D试-D阴性)/(D阳性-D阴性)×100％。参考医用输液、输血、注射器具检验方法中溶血实验判断标准溶血率小于5％，为阴性反应，无溶血现象；溶血率大于5％为阳性反应，有溶血现象。所得结果如表6所述。
表6、抗菌肽对人血红细胞的溶血活性
  溶血率小于5％的浓度
XH-12C 64μg/mL
XH-12B 80μg/mL
XH-12A 320μg/mL
实验结果表明：上述抗菌肽对人血红细胞无伤害。
2)、健康的家兔心脏采血6ml，按照1:1的比例立即将家兔与阿氏液混合置于离心管中，离心(1200rpm/15min)。弃去上清液，细胞用生理盐水冲洗几次。将所得红细胞用PBS缓冲液配制成2％(v/v)的混悬液。取100μL细胞混悬液和抗菌肽溶液100μL(浓度为16～3200μg/ml)置于96孔板，在37℃孵育2h后，离心(1200rpm/10min)，用酶标仪检测540nm处的吸光度D。并计算溶血率，实验重复3次，取平均值。溶血率(％)＝(D试-D阴性)/(D 阳性-D阴性)×100％。参考医用输液、输血、注射器具检验方法中溶血实验判断标准溶血率小于5％，为阴性反应，无溶血现象；溶血率大于5％为阳性反应，有溶血现象。所得结果如表7所述。
表7、抗菌肽对兔血红细胞的溶血活性
  溶血率小于5％的浓度
XH-12C 80μg/mL
XH-12B 160μg/mL
XH-12A 320μg/mL
实验结果表明：上述抗菌肽对哺乳动物血红细胞无伤害。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
Figure PCTCN2017109799-appb-000001
Figure PCTCN2017109799-appb-000002

Claims (8)

  1. 能抑制和杀灭多种耐药性细菌的抗菌肽，其特征是为以下任一：
    XH-12C：Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile-Trp-Arg；
    XH-12B：Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile-Phe；
    XH-12A：Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile。
  2. 如权利要求1所述的抗菌肽在制备抑制和/或杀灭真菌、革兰氏阳性菌、革兰氏阴性菌、耐药性细菌药物中的应用。
  3. 根据权利要求2所述的应用，其特征是：
    所述真菌为白色念珠菌；
    所述革兰氏阳性菌为金黄色葡萄球菌、李斯特菌，
    所述革兰氏阴性菌为大肠杆菌；
    所述耐药性细菌为耐药金黄色葡萄球、耐药鲍曼不动杆菌、耐药肺炎克雷伯菌、耐药阪崎氏肠杆菌、耐药鸡伤寒沙门氏菌、耐药无乳链球菌、耐药粪肠球菌、耐药鸭疫里默氏杆菌。
  4. 如权利要求1所述的抗菌肽在制备抗癌药物中的应用。
  5. 根据权利要求4所述的应用，其特征是：所述癌为H460、KB-3-1或耐药癌MX20、KB-C2、KB-CV60。
  6. 如权利要求1所述的抗菌肽在医用载体中的应用。
  7. 根据权利要求2～6任一所述的抗菌肽的应用，其特征是：所述药物包括XH-12C、XH-12B、XH-12A中的至少一个。
  8. 根据权利要求2～6任一所述的抗菌肽的应用，其特征是应用领域为以下任一：人用药、兽用药、食品添加剂、饲料添加剂、日化用品。
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