WO2016110177A1 - Alkaline antibacterial peptide and targeting design and use thereof - Google Patents

Alkaline antibacterial peptide and targeting design and use thereof Download PDF

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WO2016110177A1
WO2016110177A1 PCT/CN2015/097734 CN2015097734W WO2016110177A1 WO 2016110177 A1 WO2016110177 A1 WO 2016110177A1 CN 2015097734 W CN2015097734 W CN 2015097734W WO 2016110177 A1 WO2016110177 A1 WO 2016110177A1
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antimicrobial peptide
peptide
targeted
alkaline
antimicrobial
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French (fr)
Chinese (zh)
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刘秋云
何建国
周文良
翁少萍
邢梦
付敏
彭静立
兰崇峰
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中山大学
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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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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 the field of biotechnology, and more particularly to alkaline antimicrobial peptides and their targeted design and applications.
  • Antibiotics are a class of natural or synthetic compounds that kill bacteria or inhibit the growth of bacteria. With the continuous development of science and technology, the definition of antibiotics has been continuously expanded, and anti-microbial compounds, including antifungal compounds, have been included in the scope of antibiotics.
  • antibiotic resistance refers to a decrease in sensitivity to chemotherapeutic drugs such as pathogens and cancer cells.
  • the antibiotic resistance mainly refers to the phenomenon that when the microorganism is exposed to the antibiotic environment, it can still survive and reproduce. The reason for the emergence of drug resistance is that under the pressure of natural selection, the strain with the resistance gene will survive as the dominant strain.
  • These resistance genes are usually present in the plasmid, and for microorganisms (especially bacteria), the resistance gene can be transferred and rapidly replicated by transformation, transduction, etc., so that one colony can quickly acquire resistance.
  • Antibacterial peptides generally carry a positive charge, which has a The bacteria have strong activity and are not easy to produce drug resistance.
  • Antibacterial peptides are generally 10 to 40 amino acids in length, often form membrane channels and often have hemolytic and toxic properties, and lack of targeted antibacterial and anticancer properties. If artificially designed antibacterial peptides, new antibiotic resources can be developed to effectively solve the current medical antibiotic resistance problem.
  • the technical problem to be solved by the present invention is to overcome the defects of hemolytic, membrane permeability and toxicity which the conventional antimicrobial peptide often has, and to provide an alkaline antimicrobial peptide.
  • a second object of the present invention is to provide a targeted design method for the above basic antimicrobial peptide.
  • a third object of the present invention is to provide a targeted antimicrobial peptide designed from the above alkaline antimicrobial peptide, which can be targeted against cancer.
  • a fourth object of the present invention is to provide an application of the above basic antimicrobial peptide or a targeted antimicrobial peptide.
  • alkaline antimicrobial peptide consisting of leucine and lysine, the basic antimicrobial peptide having 12 to 24 amino acids, wherein the proportion of lysine is greater than 33.3% .
  • the basic amino acid (lysine) has a positive charge. Generally, it can bind negative ions such as chloride ions, and some hydrophobic amino acids can bind to the cell membrane, destroy the integrity of the cell membrane, and produce antibacterial and bactericidal effects by changing the membrane permeability. .
  • antibacterial peptides obtained by studying basic amino acids (lysine) and hydrophobic amino acids (leucine) in the prior art, but the design of the antibacterial peptide usually requires consideration of the secondary structure of the peptide, and the secondary structure is also It will affect the activity of the finally synthesized alkaline antimicrobial peptide, which not only increases the synthesis cost of the antimicrobial peptide, but also affects the practical application of the antimicrobial peptide; in addition, the alkaline antimicrobial peptide synthesized by the prior art usually forms a membrane channel, and more It is hemolytic and is not safe to the human body.
  • the ratio of the basic amino acid of the antimicrobial peptide obtained by the invention is greater than 33.3%; at the same time, the alkaline antimicrobial peptide of the present invention is a non-high permeability type peptide (usually low permeability type) and does not cleave human red blood cells. , no hemolysis activity, generally does not form a high-efficiency membrane channel, so it is safe for the human body.
  • the hydrophobic amino acid when the hydrophobic amino acid is placed at one end of the antimicrobial peptide, the antibacterial effect of the alkaline antimicrobial peptide is very good; specifically, the hydrophobic amino acid is located at one end of the alkaline antimicrobial peptide, and the hydrophobic amino acid thereof The number is 3-8.
  • the basic antibacterial peptide of the invention has the composition of trillions, and the design of the antibacterial peptide does not need to consider the secondary structure and physicochemical properties of the amino acid, as long as the amino acid composition and the high basic amino acid ratio are maintained, the activity can be formed high.
  • Alkaline antimicrobial peptide Alkaline antimicrobial peptide.
  • amino acid sequence of the basic antimicrobial peptide is as shown in SEQ ID NOS: 1 to 23.
  • the sequence of the basic antimicrobial peptide is L m (L/K) n , (L/K) n L m , wherein 12 ⁇ n ⁇ 24, m ⁇ 8.
  • the amino acid in the antimicrobial peptide may be D-form or L-form, and may have both L-form and D-form.
  • a targeted antimicrobial peptide obtained by linking any one of the above basic antimicrobial peptides to an antibody complementarity determining region through a plurality of leucine; specifically, the targeted antimicrobial peptide is composed of an antimicrobial peptide -Lo-antibody complementarity determining region; wherein o is the number of leucine, o ⁇ 4.
  • the sequence of the targeted antimicrobial peptide is set forth in SEQ ID NOS: 25-31.
  • antibacterial peptides attached to certain antibody complementarity determining regions become targeted antibacterial peptides, and the antibacterial peptides that do not cleave human red blood cells become targeted antibacterial peptides for lysing human red blood cells. Therefore, appropriate screening should be carried out when developing antibacterial peptide drugs.
  • alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention have anti-Escherichia coli (MG1655), Pseudomonas aeruginosa (1.2464, Beijing China General Microbial Culture Collection Management Center), Staphylococcus aureus (ATCC6538), multiple resistance Staphylococcus aureus Y5 (Zhang Ying et al., Molecular typing of Staphylococcus aureus in a food poisoning event, Chinese Journal of Preventive Medicine, 2008, 42(9): 672-676; Ran He et al.
  • MG1655 anti-Escherichia coli
  • Pseudomonas aeruginosa (1.2464, Beijing China General Microbial Culture Collection Management Center
  • Staphylococcus aureus ATCC6538
  • multiple resistance Staphylococcus aureus Y5 Zhang Ying et al., Molecular typing of Staphylococcus aureus
  • the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention can be used for bacteriostatic, and can also be used for preparing bacteriostatic drugs.
  • the above antimicrobial peptides when the number of lysines is equal to or exceeds 50% of the peptide, it is effective against Escherichia coli. Above or below 50%, it is effective against both Staphylococcus aureus and Pseudomonas aeruginosa; antimicrobial peptides between 16 and 24 amino acids in length are effective against the above three bacteria.
  • the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention also have an anticancer effect, and the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention have activity against lung cancer cell A549 and immortalization of SV40 virus.
  • the targeted antibacterial peptide has stronger anticancer activity than the control antibacterial peptide at a lower concentration, especially the antibacterial peptide numbered 59, which kills lung cancer cells in 0.1 mM
  • the efficiency is as high as 100.3%; therefore, the targeted antimicrobial peptide of the present invention can be used for targeting bacteriostatic or directed anticancer, and can also be used for preparing bacteriostatic or anticancer drugs.
  • the number of intermediate leucines was 4. These peptides have stronger carcinostatic activity than the control antimicrobial peptide 2 or the control antimicrobial peptide 11 at a low concentration, and the antibody complementarity determining region is the complementarity determining region 3 and 2 of the antibody of the CD47 protein (Yasufumi Kikuchi, Shinsuke) Uno, Yasuko Kinoshita, et al. HUMANIZED ANTI-C D47ANTIBODY. European Patent Application EP1693385), the sequences are: ARGGYYTYDDWG and YIYPYNDGTKYNEKFKD, respectively.
  • CD47 Human cancer cells tested by scientists all express CD47, which is usually expressed at higher levels (on average about three times higher) than normal cells.
  • the antimicrobial peptide of the present invention is linked to the antibody complementarity determining region of the CD47 protein, and the antimicrobial peptide can be brought to the cancer cell site.
  • the antibody complementarity determining region of the antibacterial peptide of the present invention and the CD47 protein must be linked by a leucine phase, and the inventors have found through experiments that the target formed by the complementarity determining region of the antibody directly linked to the CD47 protein by the basic antimicrobial peptide numbered 2 The antimicrobial peptide (No. 63) completely lost its antibacterial activity.
  • the present invention also provides the use of any of the above alkaline antimicrobial peptides for the preparation of a bacteriostatic or/and anticancer drug; the bacteriostatic effect is inhibition of fungi or/and bacteria; the anticancer is to kill lung cancer cells or Kill immortalized cells.
  • the present invention also provides the use of the targeted antimicrobial peptide for the preparation of a bacteriostatic or/and anticancer drug; the bacteriostatic effect is inhibition of fungi or/and bacteria; the anticancer is to kill lung cancer cells or kill immortalization cell.
  • the present invention has the following beneficial effects:
  • the present invention provides an alkaline antimicrobial peptide composed of leucine and lysine, and the number of amino acids of the basic antimicrobial peptide is 12 to 24, wherein lysine The ratio is greater than 33.3%; the alkaline antimicrobial peptide of the present invention does not consider the secondary structure and physicochemical properties of the amino acid, and the antimicrobial peptide can be arbitrarily combined; it is a non-high permeability type peptide (usually low permeability type) and does not cleave the human body.
  • the bacteriostasis effect the above-mentioned alkaline antimicrobial peptide is made into a targeted antimicrobial peptide, and the efficiency of the targeted antimicrobial peptide in the 0.1 mM lung cancer-killing cell is as high as 100.3%; the antimicrobial peptide or the targeted antimicrobial peptide of the present invention can be used for inhibiting Bacteria and anti-cancer, can be widely used in medical, agricultural, food preservatives and other fields.
  • Figure 1 shows the hemolytic activity of antimicrobial peptides and targeted antimicrobial peptides.
  • Figure 2 is the rate at which 80 ⁇ M different antimicrobial peptides hydrolyze ONPG.
  • Figure 3 is a graph showing the rate at which 80 ⁇ M of different antimicrobial peptides hydrolyze ONPG.
  • Figure 4 shows the results of antibacterial and antimicrobial peptides against multi-drug resistant S. aureus Y5.
  • Figure 5 shows the results of antibacterial activity of antibacterial peptides and targeted antibacterial peptides against Pseudomonas aeruginosa.
  • Figure 6 shows the results of an antibacterial peptide and a targeted antimicrobial peptide against S. cerevisiae INVSc1 inhibition zone.
  • Figure 7 shows the killing effect of antimicrobial peptides and targeted antimicrobial peptides on lung cancer cell line A549.
  • Figure 8 shows the killing effect of antimicrobial peptides and targeted antimicrobial peptides on the human bronchial epithelial cell line 16HBE14o– immortalized with SV40 virus.
  • Figure 9 is a graph showing the killing effect of a targeted antimicrobial peptide on the human bronchial epithelial cell line 16HBE14o- immortalized with the SV40 virus.
  • polypeptide sequence of Table 1 various peptides were synthesized by Shanghai Botai Biotechnology Co., Ltd. by fmoc solid phase synthesis, and purified by RP-HPLC; the polypeptide includes an antimicrobial peptide and a targeted antimicrobial peptide, wherein the targeted antimicrobial peptide is The anti-peptide and antibody complementarity determining regions are obtained by several leucine linkages.
  • the bacteria used were Escherichia coli (MG1655), Pseudomonas aeruginosa (1.2464, Beijing China General Microbial Culture Collection Management Center), Staphylococcus aureus (ATCC6538), multi-drug resistant Staphylococcus aureus Y5 (Zhang Ying et al, together with food Molecular typing of Staphylococcus aureus in poisoning events, Chinese Journal of Preventive Medicine, 2008, 42(9): 672-676; Ran He et al. A combinatorial yeast overlay method for the isolation of antibacterial oligopeptides, Proceedings of the National Academy Of Sciences, India Section B: Biological Sciences, 2014, 84(4): 1069–1075),
  • the minimum inhibitory concentration (MIC) determination process is as follows:
  • Bacterial single colonies were inoculated in a conventional LB liquid medium, cultured at 37 ° C, shaking at 200 rpm for 11 hours overnight, and colony forming units were determined.
  • Each peptide (antibacterial peptide and targeted antimicrobial peptide) was set to four final concentrations: 40 ⁇ M, 80 ⁇ . M, 160 ⁇ M and 320 ⁇ M; each peptide was set to 3 parallels per concentration.
  • control group 2 50 ⁇ l of sterile medium and bacterial solution
  • control group 3 50 ⁇ l of bacterial solution + corresponding amount of DMSO and medium corresponding to the sample
  • control group 4 50 ⁇ l Bacterial solution + corresponding amount of water and medium corresponding to the sample.
  • the OD value was measured at 492 nm and 620 nm with a microplate reader.
  • Inhibition rate% 100 ⁇ [1-(peptide and bacterial liquid experimental group - no bacterial culture control group) / (water or DMSO and bacterial liquid control group - no bacterial culture control group)]
  • Antibacterial peptide treatment The antimicrobial peptide was dissolved in PBS, and finally an antibacterial peptide solution having a final concentration of 40 ⁇ M, 80 ⁇ M, 160 ⁇ M, and 320 ⁇ M was obtained.
  • cecropin 20 ⁇ M, 40 ⁇ M, 80 ⁇ M and 160 ⁇ M cecropin (CecA for short) were mixed with the red blood cell suspension according to the above method, the positive control was 1% Triton X-100; the negative control was PBS solution.
  • the 96-well plate after shaking culture was centrifuged at 2000 rpm for 8 minutes, and 60 ⁇ l of the supernatant was aspirated and transferred to a new 96-well plate, and the OD value at a wavelength of 540 nm was detected by a microplate reader.
  • Permeation percentage (A 540 sample - A 540 negative control) / (A 540 positive control - A 540 negative control) x 100%.
  • --galactosidase is a hydrolase located in the cytoplasm of bacteria that hydrolyzes o-nitrophenyl ⁇ -D-galactopyranoside (ONPG) to galactose and o-nitrophenol (yellow).
  • ONPG o-nitrophenyl ⁇ -D-galactopyranoside
  • a certain amount of ONPG was added to the system, and the degree of hydrolysis of ONPG was determined by measuring the change in the absorbance of the culture solution at 420 nm to determine whether ⁇ -galactosidase hydrolyzes ONPG.
  • the ONPG cannot enter the cell.
  • the culture solution rapidly turns yellow, and the A 420 value rises rapidly in a short time. Therefore, this method can be used to detect the effect of antimicrobial peptides on cell membrane permeability. High membrane permeability indicates that the antimicrobial peptide can form a membrane channel.
  • the permeability experiment process of the present invention is as follows:
  • the final experimental group has a volume of 100 ⁇ l per well, a final peptide concentration of 80 ⁇ M, a final concentration of ONPG of 1.5 mM, and a bacterial concentration of 1 ⁇ 10 7 colony forming units; and the final concentration of the peptide in the experimental group is 40 ⁇ M per well.
  • Peptide-to-multi-drug resistant S. aureus Y5 and Pseudomonas aeruginosa inhibition zone experiments Inoculation of bacterial single colonies in conventional LB liquid medium, shaking at 37 ° C, 200 rpm overnight for 11 hours, and determination of colony forming units .
  • the counted S. aureus Y5 and Pseudomonas aeruginosa (1.2464) bacteria are respectively added, so that the concentration of the bacterial liquid is about 1 ⁇ 10 8 colony forming units/ml. Pour into the plate and cool until completely solidified.
  • S. aureus plates were filled with 40 microliters of 5 mM peptide per well, the negative control was 40 microliters of DMSO/water, and the positive control was 10 microliters of 5 mg/ml Amp + , 30 microliters of water.
  • P. aeruginosa plates were supplemented with 25 microliters of 5 mM peptide per well, the negative control was 25 microliters of water per DMSO, and the positive control was 10 microliters, 5 milligrams per milliliter of Amp + , 15 microliters of water.
  • Table 2 The inhibition zone size of the polypeptide against Staphylococcus aureus and Pseudomonas aeruginosa
  • the diameter of the zone of inhibition does not include the diameter of the puncher; a: ARGGYYTYDDWG is the complementarity determining region of the antibody of CD47 protein; b: YIYPYNDGTKYNEKFKD is the complementarity determining region of the antibody of CD47 protein 2
  • YPD medium glucose 2%, peptone 2%, yeast extract 1%, solid medium plus agar 2%; streak culture S. cerevisiae INVSc1 single colony; inoculate single colonies at 30 ° C shaker for 24 hours and remove Store at 4 ° C; measure colony forming units.
  • YPD medium 1:20
  • the yeast liquid concentration is 1 ⁇ 10 6 /ml.
  • the positive control was 10 microliters, 10 mg/l benomyl + 30 microliters of water.
  • the plate was sealed with a parafilm and placed in a 30 ° C incubator for about 18 hours, photographed and the size of the zone of inhibition was recorded. The results are shown in Figure 6 and Table 3.
  • the diameter of the zone of inhibition does not include the diameter of the puncher; a: ARGGYYTYDDWG is the complementarity determining region of the antibody of CD47 protein; b: YIYPYNDGTKYNEKFKD is the complementarity determining region of the antibody of CD47 protein 2
  • the lung cancer cell line A549 is described in the literature (Lieber M, Smith B, Szakal A et al. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer, 2006, 17(1): 62-70). Cell culture methods are as described by the American Type Culture Collection. Immortalized human bronchial epithelial cell line 16HBE14o– cells are found in references (Cozens A L, Yezzi M J, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner W E, Widdicombe J H, and Gruenert D C.” CFTR Expression and chloride secretion in polarized immortal human bronchial epithelial cells.
  • cell viability inhibition rate (%) (control group 492 nm absorbance - experimental group 492 nm absorbance value / control group 492 nm absorbance ⁇ 00%) to calculate the inhibition rate of various peptides on cell viability The results are shown in Figures 7-9.

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Abstract

Disclosed is an alkaline antibacterial peptide consisting of leucine and lysine. The alkaline antibacterial peptide comprises 12-24 amino acids, wherein the proportion of lysine is more than 33.3%. Also disclosed is a targeted antibacterial peptide formed by the connection of the alkaline antibacterial peptide and the complementarity determining region of an antibody through leucine. The alkaline antibacterial peptide and the targeted antibacterial peptide can be used as an antibacterial and for cancer resistance.

Description

碱性抗菌肽及其靶向设计和应用Alkaline antimicrobial peptide and its targeted design and application 技术领域Technical field
本发明涉及生物技术领域，更具体地，涉及碱性抗菌肽及其靶向设计和应用。The present invention relates to the field of biotechnology, and more particularly to alkaline antimicrobial peptides and their targeted design and applications.
背景技术Background technique
抗生素(antibiotics)是一类天然或人工合成的化合物，能够杀死细菌或者可以抑制细菌的生长。随着科学技术的不断发展，抗生素的定义也不断被扩充，其中抗微生物，包括抗真菌等化合物都被纳入到抗生素的范围。Antibiotics are a class of natural or synthetic compounds that kill bacteria or inhibit the growth of bacteria. With the continuous development of science and technology, the definition of antibiotics has been continuously expanded, and anti-microbial compounds, including antifungal compounds, have been included in the scope of antibiotics.
人类自1940年发现第一种抗生素青霉素(penicillin)并将其应用于临床之后，就开始了抗生素治疗的新时代。许多曾经严重危害人类生命健康的感染性疾病因抗生素的使用而得到了有效的控制，并大幅降低了婴儿出生的死亡率和手术后的感染率，人类的平均寿命也得以延长15～20年。因此，各种各样的抗生素已经成为多数疾病的治疗中必不可少的药物。Since the discovery of the first antibiotic penicillin in 1940 and its application in clinical practice, humans have embarked on a new era of antibiotic treatment. Many infectious diseases that have been seriously endangering human life and health have been effectively controlled by the use of antibiotics, and have greatly reduced the birth mortality rate and post-operative infection rate, and the average life expectancy of humans has been extended by 15 to 20 years. Therefore, a variety of antibiotics have become essential drugs in the treatment of most diseases.
然而，随之而来的是抗生素的滥用导致的严重的问题：抗生素的耐药性(Drug resistance)。在临床上，耐药性是指病原体及癌细胞等对化学治疗药物敏感性降低。而抗生素的耐药性主要指当微生物暴露在抗生素环境中时，仍然能够生存并进行繁殖的现象。出现耐药性的原因是，在自然选择的压力下，拥有抗性基因的菌株会成为优势菌株存活下来。这些抗性基因通常存在于质粒中，而对于微生物(尤其是细菌)，抗性基因可以通过转化、转导等现象进行转移并迅速复制，使一个菌落迅速获得抗性。However, it is followed by a serious problem caused by the abuse of antibiotics: antibiotic resistance (Drug resistance). Clinically, drug resistance refers to a decrease in sensitivity to chemotherapeutic drugs such as pathogens and cancer cells. The antibiotic resistance mainly refers to the phenomenon that when the microorganism is exposed to the antibiotic environment, it can still survive and reproduce. The reason for the emergence of drug resistance is that under the pressure of natural selection, the strain with the resistance gene will survive as the dominant strain. These resistance genes are usually present in the plasmid, and for microorganisms (especially bacteria), the resistance gene can be transferred and rapidly replicated by transformation, transduction, etc., so that one colony can quickly acquire resistance.
研究表明，研制一种新型抗生素大约需要十年或更长的时间，而细菌产生耐药性的时间却不足两年，新药的研制速度远远跟不上细菌耐药性产生的速度。而一旦出现拥有多种抗性基因的“超级细菌”，人们将对其无药可用。早在1976年，肺炎链球菌就被发现对青霉素产生了耐药。而在2010年在南亚地区发现的新型超级病菌NDM-1，所有现存的抗生素都对其不起作用。目前为止，NDM-1引起的疾病还找不到有效的治疗方法，而且有不断蔓延的趋势，引起了广泛关注。Studies have shown that it takes about a decade or more to develop a new type of antibiotic, while bacteria produce resistance for less than two years. The speed of development of new drugs is far behind the rate of bacterial resistance. Once there is a "superbug" with multiple resistance genes, people will have no medicine available. As early as 1976, Streptococcus pneumoniae was found to be resistant to penicillin. In the new superbug NDM-1 discovered in South Asia in 2010, all existing antibiotics have no effect on it. So far, the disease caused by NDM-1 has not found an effective treatment, and it has a tendency to spread and has attracted widespread attention.
因此在医疗卫生上迫切需要高效、低毒、高选择性广谱抑菌药物的研究和开发以保护人类的健康。而研究表明许多生物的基因组中也有编码抗生素的基因。这些基因编码的多为一些短肽，称为抗菌肽。抗菌肽一般携带正电荷，其具有抑 菌活性强、不易产生耐药性等特点。抗菌肽一般长为10到40个氨基酸，经常形成过膜通道并经常具有溶血性、毒性，缺乏靶向抗菌抗癌特性。如果能够人工设计抗菌肽，则能够开发新的抗生素的资源，有效地解决当前医学上抗生素耐药性的问题。Therefore, there is an urgent need for research and development of high-efficiency, low-toxicity, high-selective broad-spectrum antibacterial drugs to protect human health. Studies have shown that many organisms also have genes encoding antibiotics in their genomes. These genes encode mostly short peptides called antimicrobial peptides. Antibacterial peptides generally carry a positive charge, which has a The bacteria have strong activity and are not easy to produce drug resistance. Antibacterial peptides are generally 10 to 40 amino acids in length, often form membrane channels and often have hemolytic and toxic properties, and lack of targeted antibacterial and anticancer properties. If artificially designed antibacterial peptides, new antibiotic resources can be developed to effectively solve the current medical antibiotic resistance problem.
发明内容Summary of the invention
本发明所要解决的技术问题是克服现有抗菌肽经常具有的溶血性、膜通透性和毒性的缺陷，提供一种碱性抗菌肽。The technical problem to be solved by the present invention is to overcome the defects of hemolytic, membrane permeability and toxicity which the conventional antimicrobial peptide often has, and to provide an alkaline antimicrobial peptide.
本发明的第二个目的是提供上述碱性抗菌肽的靶向设计方法。A second object of the present invention is to provide a targeted design method for the above basic antimicrobial peptide.
本发明的第三个目的是提供一种由上述碱性抗菌肽设计得到的靶向抗菌肽，所述靶向抗菌肽可靶向抗癌。A third object of the present invention is to provide a targeted antimicrobial peptide designed from the above alkaline antimicrobial peptide, which can be targeted against cancer.
本发明的第四个目的是提供上述碱性抗菌肽或靶向抗菌肽的应用。A fourth object of the present invention is to provide an application of the above basic antimicrobial peptide or a targeted antimicrobial peptide.
本发明的目的是通过以下技术方案予以实现的：The object of the present invention is achieved by the following technical solutions:
一种碱性抗菌肽，所述碱性抗菌肽由亮氨酸和赖氨酸组成，所述碱性抗菌肽的氨基酸的个数为12～24个，其中，赖氨酸的比例大于33.3％。An alkaline antimicrobial peptide consisting of leucine and lysine, the basic antimicrobial peptide having 12 to 24 amino acids, wherein the proportion of lysine is greater than 33.3% .
碱性氨基酸(赖氨酸)带有正电荷，一般可结合氯离子等负离子，加上一些疏水氨基酸，就可以结合到细胞膜上，破坏细胞膜的完整性，通过改变膜透性产生抑菌杀菌作用。现有技术中也有研究碱性氨基酸(赖氨酸)和疏水氨基酸(亮氨酸)所得到的抗菌肽，但所述抗菌肽的设计通常都需要考虑肽的二级结构，二级结构同时还会影响最终合成的碱性抗菌肽的活性，这不仅会增加抗菌肽的合成成本，也会影响抗菌肽的实际应用；另外，现有技术所合成的碱性抗菌肽通常形成过膜通道，多有溶血性，对人体是不安全的。The basic amino acid (lysine) has a positive charge. Generally, it can bind negative ions such as chloride ions, and some hydrophobic amino acids can bind to the cell membrane, destroy the integrity of the cell membrane, and produce antibacterial and bactericidal effects by changing the membrane permeability. . There are also antibacterial peptides obtained by studying basic amino acids (lysine) and hydrophobic amino acids (leucine) in the prior art, but the design of the antibacterial peptide usually requires consideration of the secondary structure of the peptide, and the secondary structure is also It will affect the activity of the finally synthesized alkaline antimicrobial peptide, which not only increases the synthesis cost of the antimicrobial peptide, but also affects the practical application of the antimicrobial peptide; in addition, the alkaline antimicrobial peptide synthesized by the prior art usually forms a membrane channel, and more It is hemolytic and is not safe to the human body.
本发明所得到的抗菌肽碱性氨基酸的比例大于33.3％时效果很好；同时，本发明所述碱性抗菌肽是非高通透型的肽(通常是低通透型)且不裂解人体红细胞，无溶血活性，一般不形成高效过膜通道，因此对人体是安全的。The ratio of the basic amino acid of the antimicrobial peptide obtained by the invention is greater than 33.3%; at the same time, the alkaline antimicrobial peptide of the present invention is a non-high permeability type peptide (usually low permeability type) and does not cleave human red blood cells. , no hemolysis activity, generally does not form a high-efficiency membrane channel, so it is safe for the human body.
申请人通过大量的筛选和研究发现，将疏水氨基酸置于抗菌肽的一端时，碱性抗菌肽的抗菌效果很好；具体地，所述疏水氨基酸位于碱性抗菌肽的一端，其疏水氨基酸的个数为3～8个。Applicants have found through extensive screening and research that when the hydrophobic amino acid is placed at one end of the antimicrobial peptide, the antibacterial effect of the alkaline antimicrobial peptide is very good; specifically, the hydrophobic amino acid is located at one end of the alkaline antimicrobial peptide, and the hydrophobic amino acid thereof The number is 3-8.
本发明所述碱性抗菌肽有上万亿种组成，且抗菌肽的设计不用考虑氨基酸的二级结构和物化性质，只要由上述氨基酸组成和保持高的碱性氨基酸比例，就可以形成活性高的碱性抗菌肽。 The basic antibacterial peptide of the invention has the composition of trillions, and the design of the antibacterial peptide does not need to consider the secondary structure and physicochemical properties of the amino acid, as long as the amino acid composition and the high basic amino acid ratio are maintained, the activity can be formed high. Alkaline antimicrobial peptide.
优选地，所述碱性抗菌肽的氨基酸序列如SEQ ID NO：1～23所示。Preferably, the amino acid sequence of the basic antimicrobial peptide is as shown in SEQ ID NOS: 1 to 23.
更优选地，所述碱性抗菌肽的序列为Lm(L/K)n、(L/K)n Lm，其中，12≤n≤24，m≤8。More preferably, the sequence of the basic antimicrobial peptide is L m (L/K) n , (L/K) n L m , wherein 12 ≤ n ≤ 24, m ≤ 8.
优选地，所述抗菌肽中氨基酸可以是D型，也可以是L型，还可以同时具有L型和D型。Preferably, the amino acid in the antimicrobial peptide may be D-form or L-form, and may have both L-form and D-form.
一种靶向抗菌肽，所述靶向抗菌肽由上述任意一种碱性抗菌肽与抗体互补决定区通过若干个亮氨酸连接获得；具体得，所述靶向抗菌肽结构组成为抗菌肽-Lo-抗体互补决定区；其中o为亮氨酸的个数，o≥4。A targeted antimicrobial peptide obtained by linking any one of the above basic antimicrobial peptides to an antibody complementarity determining region through a plurality of leucine; specifically, the targeted antimicrobial peptide is composed of an antimicrobial peptide -Lo-antibody complementarity determining region; wherein o is the number of leucine, o ≥ 4.
优选地，所述靶向抗菌肽的序列如SEQ ID NO：25～31所示。Preferably, the sequence of the targeted antimicrobial peptide is set forth in SEQ ID NOS: 25-31.
需要说明的是，仅仅有个别抗菌肽接上某些抗体互补决定区成为靶向抗菌肽后，会使不裂解人体红细胞的抗菌肽变为裂解人体红细胞的靶向抗菌肽。因此，开发靶向抗菌肽药物时要进行适当筛选。It should be noted that only a few antibacterial peptides attached to certain antibody complementarity determining regions become targeted antibacterial peptides, and the antibacterial peptides that do not cleave human red blood cells become targeted antibacterial peptides for lysing human red blood cells. Therefore, appropriate screening should be carried out when developing antibacterial peptide drugs.
另外，本发明的碱性抗菌肽和靶向抗菌肽均具有抗大肠杆菌(MG1655)，绿脓杆菌(1.2464，北京中国普通微生物菌种保藏管理中心)，金黄色葡萄球菌(ATCC6538)，多重耐药金黄色葡萄球菌Y5(张颖等，一起食物中毒事件的金黄色葡萄球菌分子分型研究，中华预防医学杂志，2008，42(9):672-676；Ran He et al.A combinatorial yeast overlay method for the isolation of antibacterial oligopeptides,Proceedings of the National Academy of Sciences,India Section B:Biological Sciences,2014,84(4):1069–1075)，酿酒酵母(INVSc1)等的效果。In addition, the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention have anti-Escherichia coli (MG1655), Pseudomonas aeruginosa (1.2464, Beijing China General Microbial Culture Collection Management Center), Staphylococcus aureus (ATCC6538), multiple resistance Staphylococcus aureus Y5 (Zhang Ying et al., Molecular typing of Staphylococcus aureus in a food poisoning event, Chinese Journal of Preventive Medicine, 2008, 42(9): 672-676; Ran He et al. A combinatorial yeast overlay Method for the isolation of antibacterial oligopeptides, Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 2014, 84(4): 1069-1075), the effect of Saccharomyces cerevisiae (INVSc1) and the like.
因此，本发明所述碱性抗菌肽和靶向抗菌肽可用于抑菌，也可用于制备抑菌药物。上述抗菌肽中，当赖氨酸个数等于或超过肽的50％时，对大肠杆菌较为有效。高于或低于50％，对金黄色葡萄球菌和绿脓杆菌都有效；长度为16至24个氨基酸之间的抗菌肽对上述3个菌都有效。Therefore, the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention can be used for bacteriostatic, and can also be used for preparing bacteriostatic drugs. Among the above antimicrobial peptides, when the number of lysines is equal to or exceeds 50% of the peptide, it is effective against Escherichia coli. Above or below 50%, it is effective against both Staphylococcus aureus and Pseudomonas aeruginosa; antimicrobial peptides between 16 and 24 amino acids in length are effective against the above three bacteria.
同时，本发明所述碱性抗菌肽和靶向抗菌肽同样具有抗癌的作用，本发明所述碱性抗菌肽和靶向抗菌肽具有抗肺癌细胞A549的活性和对SV40病毒永生化的人支气管上皮细胞株16HBE14o–的杀伤作用；所述靶向抗菌肽在较低的浓度下具有比对照抗菌肽更强的抗癌活性，特别是编号为59的抗菌肽，在0.1mM杀肺癌细胞的效率高达100.3％；因此本发明所述靶向抗菌肽可以用于导向抑菌或导向抗癌，也可用于制备抑菌或抗癌药物。 Meanwhile, the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention also have an anticancer effect, and the alkaline antimicrobial peptide and the targeted antimicrobial peptide of the present invention have activity against lung cancer cell A549 and immortalization of SV40 virus. The killing effect of bronchial epithelial cell line 16HBE14o-; the targeted antibacterial peptide has stronger anticancer activity than the control antibacterial peptide at a lower concentration, especially the antibacterial peptide numbered 59, which kills lung cancer cells in 0.1 mM The efficiency is as high as 100.3%; therefore, the targeted antimicrobial peptide of the present invention can be used for targeting bacteriostatic or directed anticancer, and can also be used for preparing bacteriostatic or anticancer drugs.
对于靶向抗菌肽(编号为49，50，59，60)而言，中间的亮氨酸个数为4。这几个肽在低浓度具有比对照抗菌肽2或对照抗菌肽11更强的杀癌活性，其用到的抗体互补决定区为CD47蛋白质的抗体的互补决定区3和2(Yasufumi Kikuchi,Shinsuke Uno,Yasuko Kinoshita,et al.HUMANIZED ANTI-C D47ANTIBODY.European Patent Application EP1693385)，其序列分别为：ARGGYYTYDDWG和YIYPYNDGTKYNEKFKD。科学家检测过的人类癌细胞都表达CD47，相比于正常细胞通常以较高水平表达(平均大约高3倍多)。将本发明所述抗菌肽与CD47蛋白的抗体互补决定区相连接，可以将抗菌肽带到癌细胞位置。For targeted antimicrobial peptides (numbered 49, 50, 59, 60), the number of intermediate leucines was 4. These peptides have stronger carcinostatic activity than the control antimicrobial peptide 2 or the control antimicrobial peptide 11 at a low concentration, and the antibody complementarity determining region is the complementarity determining region 3 and 2 of the antibody of the CD47 protein (Yasufumi Kikuchi, Shinsuke) Uno, Yasuko Kinoshita, et al. HUMANIZED ANTI-C D47ANTIBODY. European Patent Application EP1693385), the sequences are: ARGGYYTYDDWG and YIYPYNDGTKYNEKFKD, respectively. Human cancer cells tested by scientists all express CD47, which is usually expressed at higher levels (on average about three times higher) than normal cells. The antimicrobial peptide of the present invention is linked to the antibody complementarity determining region of the CD47 protein, and the antimicrobial peptide can be brought to the cancer cell site.
本发明所述抗菌肽与CD47蛋白的抗体互补决定区必须通过亮氨酸相连接，发明人通过实验发现，由编号为2的碱性抗菌肽直接连接CD47蛋白的抗体互补决定区形成的靶向抗菌肽(编号为63)完全丧失抑菌活性。The antibody complementarity determining region of the antibacterial peptide of the present invention and the CD47 protein must be linked by a leucine phase, and the inventors have found through experiments that the target formed by the complementarity determining region of the antibody directly linked to the CD47 protein by the basic antimicrobial peptide numbered 2 The antimicrobial peptide (No. 63) completely lost its antibacterial activity.
因此，本发明还提供上述任一种碱性抗菌肽在制备抑菌或/和抗癌药物方面的应用；所述抑菌为抑制真菌或/和细菌；所述抗癌为杀死肺癌细胞或杀死永生化细胞。Accordingly, the present invention also provides the use of any of the above alkaline antimicrobial peptides for the preparation of a bacteriostatic or/and anticancer drug; the bacteriostatic effect is inhibition of fungi or/and bacteria; the anticancer is to kill lung cancer cells or Kill immortalized cells.
本发明还提供所述靶向抗菌肽在制备抑菌或/和抗癌药物方面的应用；所述抑菌为抑制真菌或/和细菌；所述抗癌为杀死肺癌细胞或杀死永生化细胞。The present invention also provides the use of the targeted antimicrobial peptide for the preparation of a bacteriostatic or/and anticancer drug; the bacteriostatic effect is inhibition of fungi or/and bacteria; the anticancer is to kill lung cancer cells or kill immortalization cell.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明提供了一种碱性抗菌肽，所述碱性抗菌肽由亮氨酸和赖氨酸组成，所述碱性抗菌肽的氨基酸的个数为12～24个，其中，赖氨酸的比例大于33.3％；本发明所述碱性抗菌肽不用考虑氨基酸的二级结构和物化性质，所述抗菌肽可任意组合；是非高通透型的肽(通常低通透型)且不裂解人体红细胞，无溶血活性，一般不形成高效过膜通道，因此对人体是安全的；同时对大肠杆菌、绿脓杆菌、金黄色葡萄球菌、多重耐药性金黄色葡萄球菌、酿酒酵母等具有广谱的抑菌作用；将上述碱性抗菌肽制成靶向抗菌肽，所述靶向抗菌肽在0.1mM杀肺癌细胞的效率高达100.3％；本发明所述抗菌肽或靶向抗菌肽可用于抑菌和抗癌，可广泛用于医疗、农业、食品防腐剂等领域。The present invention provides an alkaline antimicrobial peptide composed of leucine and lysine, and the number of amino acids of the basic antimicrobial peptide is 12 to 24, wherein lysine The ratio is greater than 33.3%; the alkaline antimicrobial peptide of the present invention does not consider the secondary structure and physicochemical properties of the amino acid, and the antimicrobial peptide can be arbitrarily combined; it is a non-high permeability type peptide (usually low permeability type) and does not cleave the human body. Red blood cells, no hemolytic activity, generally do not form a high-efficiency membrane channel, so it is safe for the human body; at the same time, it has a broad spectrum for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, multi-drug resistant Staphylococcus aureus, Saccharomyces cerevisiae, etc. The bacteriostasis effect; the above-mentioned alkaline antimicrobial peptide is made into a targeted antimicrobial peptide, and the efficiency of the targeted antimicrobial peptide in the 0.1 mM lung cancer-killing cell is as high as 100.3%; the antimicrobial peptide or the targeted antimicrobial peptide of the present invention can be used for inhibiting Bacteria and anti-cancer, can be widely used in medical, agricultural, food preservatives and other fields.
附图说明DRAWINGS
图1为抗菌肽和靶向抗菌肽的溶血活性。 Figure 1 shows the hemolytic activity of antimicrobial peptides and targeted antimicrobial peptides.
图2为80μM不同的抗菌肽水解ONPG的速率。Figure 2 is the rate at which 80 μM different antimicrobial peptides hydrolyze ONPG.
图3为80μM不同的抗菌肽水解ONPG的速率。Figure 3 is a graph showing the rate at which 80 μM of different antimicrobial peptides hydrolyze ONPG.
图4为抗菌肽和靶向抗菌肽对多重耐药金黄色葡萄球菌Y5抑菌实验结果。Figure 4 shows the results of antibacterial and antimicrobial peptides against multi-drug resistant S. aureus Y5.
图5为抗菌肽和靶向抗菌肽对绿脓杆菌抑菌实验结果。Figure 5 shows the results of antibacterial activity of antibacterial peptides and targeted antibacterial peptides against Pseudomonas aeruginosa.
图6为抗菌肽和靶向抗菌肽对酿酒酵母INVSc1抑菌圈实验结果。Figure 6 shows the results of an antibacterial peptide and a targeted antimicrobial peptide against S. cerevisiae INVSc1 inhibition zone.
图7为抗菌肽和靶向抗菌肽对肺癌细胞系A549的杀伤作用。Figure 7 shows the killing effect of antimicrobial peptides and targeted antimicrobial peptides on lung cancer cell line A549.
图8为抗菌肽和靶向抗菌肽对SV40病毒永生化的人支气管上皮细胞株16HBE14o–的杀伤作用。Figure 8 shows the killing effect of antimicrobial peptides and targeted antimicrobial peptides on the human bronchial epithelial cell line 16HBE14o– immortalized with SV40 virus.
图9为靶向抗菌肽对SV40病毒永生化的人支气管上皮细胞株16HBE14o–的杀伤作用。Figure 9 is a graph showing the killing effect of a targeted antimicrobial peptide on the human bronchial epithelial cell line 16HBE14o- immortalized with the SV40 virus.
具体实施方式detailed description
下面结合说明书附图和具体实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的简单修改或替换，均属于本发明的范围；若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。The contents of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments, but are not to be construed as limiting. Modifications or substitutions of the methods, procedures, or conditions of the present invention are intended to be within the scope of the present invention without departing from the spirit and scope of the invention; Conventional means well known.
实施例1最小抑菌浓度(MIC)测定Example 1 Determination of minimum inhibitory concentration (MIC)
按表1的多肽序列，由上海波泰生物科技有限公司采用fmoc固相合成法合成各种多肽，使用RP-HPLC纯化；所述多肽包括抗菌肽和靶向抗菌肽，其中靶向抗菌肽是由抗菌肽与抗体互补决定区通过若干个亮氨酸连接获得。According to the polypeptide sequence of Table 1, various peptides were synthesized by Shanghai Botai Biotechnology Co., Ltd. by fmoc solid phase synthesis, and purified by RP-HPLC; the polypeptide includes an antimicrobial peptide and a targeted antimicrobial peptide, wherein the targeted antimicrobial peptide is The anti-peptide and antibody complementarity determining regions are obtained by several leucine linkages.
所用的细菌为大肠杆菌(MG1655)，绿脓杆菌(1.2464，北京中国普通微生物菌种保藏管理中心)，金黄色葡萄球菌(ATCC6538)，多重耐药金黄色葡萄球菌Y5(张颖等，一起食物中毒事件的金黄色葡萄球菌分子分型研究，中华预防医学杂志，2008，42(9):672-676；Ran He et al.A combinatorial yeast overlay method for the isolation of antibacterial oligopeptides,Proceedings of the National Academy of Sciences,India Section B:Biological Sciences,2014,84(4):1069–1075)，The bacteria used were Escherichia coli (MG1655), Pseudomonas aeruginosa (1.2464, Beijing China General Microbial Culture Collection Management Center), Staphylococcus aureus (ATCC6538), multi-drug resistant Staphylococcus aureus Y5 (Zhang Ying et al, together with food Molecular typing of Staphylococcus aureus in poisoning events, Chinese Journal of Preventive Medicine, 2008, 42(9): 672-676; Ran He et al. A combinatorial yeast overlay method for the isolation of antibacterial oligopeptides, Proceedings of the National Academy Of Sciences, India Section B: Biological Sciences, 2014, 84(4): 1069–1075),
最小抑菌浓度(MIC)测定过程如下：The minimum inhibitory concentration (MIC) determination process is as follows:
(1)接种细菌单菌落于常规LB液体培养基中，37℃，200转/分钟摇床过夜培养11小时，并测定菌落形成单位。(1) Bacterial single colonies were inoculated in a conventional LB liquid medium, cultured at 37 ° C, shaking at 200 rpm for 11 hours overnight, and colony forming units were determined.
(2)将各肽(抗菌肽和靶向抗菌肽)分别设置四个终浓度：40μM、80μ M、160μM和320μM；每个肽每个浓度设置3个平行。(2) Each peptide (antibacterial peptide and targeted antimicrobial peptide) was set to four final concentrations: 40 μM, 80 μ. M, 160 μM and 320 μM; each peptide was set to 3 parallels per concentration.
(3)用常规LB液体培养基稀释培养过夜的菌液得到106菌落形成单位的使用菌液；实验组：每孔多肽和菌液各50微升，对照组1：100微升无菌培养基；对照组2：无菌培养基和菌液各50微升；对照组3：50微升的菌液+与样品相对应的相应量的DMSO和培养基；对照组4：50微升的菌液+与样品相对应的相应量的水和培养基。(3) Dilute the culture solution overnight with a conventional LB liquid medium to obtain a bacterial solution using 10 6 colony forming units; experimental group: 50 μl of each peptide and bacterial solution, and 1:100 μl of sterile culture in the control group. Base 2; control group 2: 50 μl of sterile medium and bacterial solution; control group 3: 50 μl of bacterial solution + corresponding amount of DMSO and medium corresponding to the sample; control group 4: 50 μl Bacterial solution + corresponding amount of water and medium corresponding to the sample.
(4)将加好样品的96孔板在37℃恒温箱培养20小时后，用酶标仪在492纳米及620纳米处测OD值。(4) After culturing the 96-well plate with the sample in the incubator at 37 ° C for 20 hours, the OD value was measured at 492 nm and 620 nm with a microplate reader.
(5)数据分析(5) Data analysis
计算公式：用492纳米处的OD值计算。Calculation formula: Calculated using the OD value at 492 nm.
抑制率％＝100×[1-(肽与菌液实验组-不加菌液对照组)/(水或者DMSO与菌液对照组-不加菌液对照组)]Inhibition rate%=100×[1-(peptide and bacterial liquid experimental group - no bacterial culture control group) / (water or DMSO and bacterial liquid control group - no bacterial culture control group)]
当抑菌率达到79.5％时，记录为MIC(最小抑菌浓度)，结果见表1。When the inhibition rate reached 79.5%, it was recorded as MIC (minimum inhibitory concentration), and the results are shown in Table 1.
表1抗菌肽对多种细菌的最小抑菌浓度Table 1 Minimum inhibitory concentration of antimicrobial peptides against various bacteria
Figure PCTCN2015097734-appb-000001
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Figure PCTCN2015097734-appb-000009
Figure PCTCN2015097734-appb-000009
*-表示未测定；编号的括号里标明DMSO，表示为DMSO溶解的肽。未标注为水溶肽；无活性：所有4个浓度抑菌率低于15％；促进生长：所有4个浓度抑菌率低于-20％；括号内表示为最大抑菌率。氨基酸小写为D型。a：ARGGYYTYDDWG(CD47蛋白质的抗体的互补决定区3)。b：YIYPYNDGTKYNEKFKD(CD47蛋白质的抗体的互补决定区2)。*- indicates no determination; the numbered parentheses indicate DMSO, indicating the peptide dissolved in DMSO. Not labeled as water-soluble peptide; no activity: all four concentrations of inhibition rate less than 15%; promote growth: all four concentrations of inhibition rate is less than -20%; brackets expressed as the maximum inhibition rate. The amino acid is lowercased to D type. a: ARGGYYTYDDWG (complementarity determining region 3 of the antibody of CD47 protein). b: YIYPYNDGTKYNEKFKD (complementarity determining region 2 of the antibody of CD47 protein).
实施例2溶血性实验Example 2 Hemolysis Experiment
1.血液处理Blood treatment
(1)采集新鲜人血5毫升放入含有0.5毫升3.8％柠檬酸钠管中，吹打抗凝管中的血液，使其与抗凝剂充分混匀。(1) Collect 5 ml of fresh human blood into a tube containing 0.5 ml of 3.8% sodium citrate, and blow the blood in the anticoagulant tube to mix well with the anticoagulant.
(2)将上述血液在2000转/分钟条件下离心8分钟，去上清。用10mM，pH＝7.4的PBS漂洗血液，2000转/分钟离心5分钟，弃上清，重复操作，直到红细胞悬液清亮透彻无血清等杂质为止，弃去上清。然后按照5％(v/v)溶于PBS中，得到红细胞悬浮液。(2) The above blood was centrifuged at 2000 rpm for 8 minutes, and the supernatant was removed. The blood was rinsed with 10 mM, pH=7.4 PBS, centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and the operation was repeated until the red blood cell suspension was clear and free of impurities such as serum, and the supernatant was discarded. It was then dissolved in PBS at 5% (v/v) to give a red blood cell suspension.
2.抗菌肽处理：将抗菌肽溶于PBS，最后得到终浓度为40μM、80μM、160μM、320μM的抗菌肽溶液。2. Antibacterial peptide treatment: The antimicrobial peptide was dissolved in PBS, and finally an antibacterial peptide solution having a final concentration of 40 μM, 80 μM, 160 μM, and 320 μM was obtained.
3.体系混合3. System mixing
吸取50微升处理过的抗菌肽溶液加入到96孔板中，每个肽每个浓度3个平行，之后用排枪吸取倒在培养皿上的红细胞悬液，每孔50微升加入到96孔板中，全部加完后密封96孔板，放入摇床，37℃，200转/分钟震荡1小时。 Pipette 50 μl of the treated antimicrobial peptide solution into a 96-well plate at a concentration of 3 parallels per peptide. Then, use a lance to draw the red blood cell suspension poured onto the culture dish, and add 50 μl per well to the 96-well. In the plate, after the addition was completed, the 96-well plate was sealed and placed in a shaker at 37 ° C for 2 hours at 200 rpm.
将20μM、40μM、80μM和160μM的天蚕素(简称CecA)按照上述方法同红细胞悬浮液混合，阳性对照为1％Triton X-100；阴性对照为PBS溶液。20 μM, 40 μM, 80 μM and 160 μM cecropin (CecA for short) were mixed with the red blood cell suspension according to the above method, the positive control was 1% Triton X-100; the negative control was PBS solution.
4.数据分析4. Data analysis
将震荡培养后的96孔板在2000转/分钟条件下离心8分钟，吸取60微升上清液平行转移到新的96孔板中，酶标仪检测540纳米波长处的OD值。The 96-well plate after shaking culture was centrifuged at 2000 rpm for 8 minutes, and 60 μl of the supernatant was aspirated and transferred to a new 96-well plate, and the OD value at a wavelength of 540 nm was detected by a microplate reader.
溶血百分值＝(A540样品-A540阴性对照)/(A540阳性对照-A540阴性对照)×100％。 Permeation percentage = (A 540 sample - A 540 negative control) / (A 540 positive control - A 540 negative control) x 100%.
实验结果如图1。The experimental results are shown in Figure 1.
实施例3大肠杆菌膜通透性实验Example 3 Escherichia coli membrane permeability test
β-半乳糖苷酶是一种水解酶，位于细菌的细胞质中，可以将邻硝基苯β-D-半乳吡喃糖苷(ONPG)水解成半乳糖和邻硝基苯酚(呈黄色)。在体系中加入一定量的ONPG，通过测量培养液在420纳米的吸光值的变化，可以判断ONPG的水解度，从而测知β-半乳糖苷酶是否水解ONPG。一般情况下，由于酶位于细胞内部，而ONPG不能进入细胞内。但一旦细胞膜通透性改变时，ONPG进入细胞内并水解，培养液迅速变成黄色，A420值在短时间内快速升高。因此，可以利用此法来检测抗菌肽对细胞膜通透性的影响。膜通透性高说明抗菌肽能形成过膜通道。--galactosidase is a hydrolase located in the cytoplasm of bacteria that hydrolyzes o-nitrophenyl β-D-galactopyranoside (ONPG) to galactose and o-nitrophenol (yellow). A certain amount of ONPG was added to the system, and the degree of hydrolysis of ONPG was determined by measuring the change in the absorbance of the culture solution at 420 nm to determine whether β-galactosidase hydrolyzes ONPG. In general, since the enzyme is located inside the cell, the ONPG cannot enter the cell. However, once the cell membrane permeability changes, ONPG enters the cell and hydrolyzes, the culture solution rapidly turns yellow, and the A 420 value rises rapidly in a short time. Therefore, this method can be used to detect the effect of antimicrobial peptides on cell membrane permeability. High membrane permeability indicates that the antimicrobial peptide can form a membrane channel.
本发明所述通透性实验过程如下：The permeability experiment process of the present invention is as follows:
(1)挑取大肠杆菌ML-35单菌落过夜培养，并测量其菌落形成单位；根据测量的菌落形成单位数值，取适量体积的过夜培养物于EP管中，10000转离心1分钟，并用10mM磷酸钠(含0.1M NaCl)缓冲液重悬。重复3次，用足量磷酸钠缓冲液将菌体重悬至菌落形成单位＝2×107。(1) Pick up E. coli ML-35 single colony overnight culture, and measure its colony forming unit; according to the measured colony forming unit value, take an appropriate volume of the overnight culture in EP tube, centrifuge at 10,000 rpm for 1 minute, and use 10 mM Resuspend in sodium phosphate (containing 0.1 M NaCl) buffer. The reaction was repeated 3 times, and the bacterial body weight was suspended to a colony forming unit = 2 × 10 7 with a sufficient amount of sodium phosphate buffer.
(2)取10微升10mM肽(包括抗菌肽和靶向抗菌肽)，加入490微升磷酸钠缓冲液，得到200μM、500微升的肽稀释液；取10微升水或DMSO代替肽，加入90微升磷酸钠缓冲液作为对照组。(2) Take 10 μl of 10 mM peptide (including antimicrobial peptide and targeted antimicrobial peptide), add 490 μl of sodium phosphate buffer to obtain 200 μM, 500 μl of peptide dilution; take 10 μl of water or DMSO instead of peptide, add 90 μl of sodium phosphate buffer was used as a control group.
(3)取200微升肽稀释液，加入50微升ONPG溶液，得到肽-ONPG混合液；在对照组中也加入50微升ONPG；用肽-ONPG混合液加96孔板，每孔50微升，每个样三个重复；取4℃保存的菌液，用排枪吸取50微升加入每孔。按此方法，最终实验组每孔溶液体积100微升，肽终浓度80μM，ONPG终浓度1.5mM，菌浓度1x107菌落形成单位；形成实验组每孔肽终浓度为40μM时方法同上。(3) Take 200 μl of peptide dilution, add 50 μl of ONPG solution to obtain peptide-ONPG mixture; add 50 μl of ONPG to the control group; add 96-well plate with peptide-ONPG mixture, 50 per well. Microliters, three replicates per sample; take the bacterial solution stored at 4 °C, and add 50 μl to each well with a lance. According to this method, the final experimental group has a volume of 100 μl per well, a final peptide concentration of 80 μM, a final concentration of ONPG of 1.5 mM, and a bacterial concentration of 1×10 7 colony forming units; and the final concentration of the peptide in the experimental group is 40 μM per well.
室温加完菌液后迅速转移至多功能酶标仪，测量其在37℃下420纳米的吸光值，从第一次测量起，每隔10分钟测量一次。测量13次。记录数据并分析，结果如图2、图3。After adding the bacterial solution at room temperature, it was quickly transferred to a multi-plate reader, and its absorbance at 420 nm at 37 ° C was measured, and it was measured every 10 minutes from the first measurement. Measured 13 times. The data was recorded and analyzed, and the results are shown in Figures 2 and 3.
实施例4抑菌圈实验Example 4 inhibition zone experiment
肽对多重耐药金黄色葡萄球菌Y5和绿脓杆菌抑菌圈实验：接种细菌单菌落 于常规LB液体培养基中，37℃，200转/分钟摇床过夜培养11小时，并测定菌落形成单位。待LB固体培养基冷却至不烫手但未凝固时分别接入计数好的金黄色葡萄球菌Y5和绿脓杆菌(1.2464)菌液，使菌液浓度约为1×108菌落形成单位/毫升，倒入平板，冷却至完全凝固。Peptide-to-multi-drug resistant S. aureus Y5 and Pseudomonas aeruginosa inhibition zone experiments: Inoculation of bacterial single colonies in conventional LB liquid medium, shaking at 37 ° C, 200 rpm overnight for 11 hours, and determination of colony forming units . When the LB solid medium is cooled to a non-hot but not solidified state, the counted S. aureus Y5 and Pseudomonas aeruginosa (1.2464) bacteria are respectively added, so that the concentration of the bacterial liquid is about 1×10 8 colony forming units/ml. Pour into the plate and cool until completely solidified.
1.样品处理：1. Sample processing:
金黄色葡萄球菌平板每个孔加入40微升、5mM肽，阴性对照为40微升DMSO/水，阳性对照为10微升5毫克/毫升Amp+，30微升水。S. aureus plates were filled with 40 microliters of 5 mM peptide per well, the negative control was 40 microliters of DMSO/water, and the positive control was 10 microliters of 5 mg/ml Amp + , 30 microliters of water.
绿脓杆菌平板每个孔加入25微升、5mM肽，阴性对照为25微升水/DMSO，阳性对照为10微升、5毫克/毫升Amp+，15微升水。P. aeruginosa plates were supplemented with 25 microliters of 5 mM peptide per well, the negative control was 25 microliters of water per DMSO, and the positive control was 10 microliters, 5 milligrams per milliliter of Amp + , 15 microliters of water.
将处理好的样品在37℃恒温培养16小时后记录是否有抑菌圈及其大小结果(如图4、5和表2)。The treated samples were incubated at 37 ° C for 16 hours at constant temperature to record whether there were inhibition zones and their size results (Figures 4, 5 and Table 2).
表2多肽对金黄色葡萄球菌和绿脓杆菌的抑菌圈大小Table 2: The inhibition zone size of the polypeptide against Staphylococcus aureus and Pseudomonas aeruginosa
Figure PCTCN2015097734-appb-000010
Figure PCTCN2015097734-appb-000010
Figure PCTCN2015097734-appb-000011
Figure PCTCN2015097734-appb-000011
注：表中抑菌圈直径都不包括打孔器直径；a：ARGGYYTYDDWG是CD47蛋白质的抗体的互补决定区3；b：YIYPYNDGTKYNEKFKD是CD47蛋白质的抗体的互补决定区2Note: The diameter of the zone of inhibition does not include the diameter of the puncher; a: ARGGYYTYDDWG is the complementarity determining region of the antibody of CD47 protein; b: YIYPYNDGTKYNEKFKD is the complementarity determining region of the antibody of CD47 protein 2
实施例5酿酒酵母INVSc1抑菌圈实验Example 5 Saccharomyces Cerevisiae INVSc1 inhibition zone experiment
配置YPD培养基:葡萄糖2％，蛋白胨2％，酵母膏1％，固体培养基还要加琼脂2％；划线培养酿酒酵母INVSc1单菌落；接种单菌落30℃摇床培养24小时后取出放4℃保存；测菌落形成单位。Configure YPD medium: glucose 2%, peptone 2%, yeast extract 1%, solid medium plus agar 2%; streak culture S. cerevisiae INVSc1 single colony; inoculate single colonies at 30 ° C shaker for 24 hours and remove Store at 4 ° C; measure colony forming units.
融化YPD固体培养基，等培养基不太热的时候加入上述测过菌落形成单位的菌液，按照菌液：YPD培养基＝1：20，使酵母菌液浓度为1×106个/毫升；倒入直径为15厘米的平板约50毫升/板；冷却后用直径为5毫米打孔器打孔，每孔加入40微升、10mM肽，阴性对照为40微升的水/DMSO阴性对照，阳性对照为10微升、10毫克/升的苯菌灵+30微升水。用封口膜封好平板之后放入30℃恒温箱培养约18小时后拍照并记录抑菌圈大小。结果如图6和表3。Melt the YPD solid medium, and add the above-mentioned bacterial colony forming unit when the medium is not too hot. According to the bacterial solution: YPD medium = 1:20, the yeast liquid concentration is 1×10 6 /ml. Pour into a plate of 15 cm in diameter and about 50 ml/plate; after cooling, punch with a 5 mm diameter puncher, add 40 μl of peptide per well, 10 mM peptide, and a negative control of 40 μl of water/DMSO negative control The positive control was 10 microliters, 10 mg/l benomyl + 30 microliters of water. The plate was sealed with a parafilm and placed in a 30 ° C incubator for about 18 hours, photographed and the size of the zone of inhibition was recorded. The results are shown in Figure 6 and Table 3.
表3酿酒酵母抑菌圈大小Table 3 Saccharomyces cerevisiae inhibition zone size
Figure PCTCN2015097734-appb-000012
Figure PCTCN2015097734-appb-000012
Figure PCTCN2015097734-appb-000013
Figure PCTCN2015097734-appb-000013
注：表中抑菌圈直径都不包括打孔器直径；a：ARGGYYTYDDWG是CD47蛋白质的抗体的互补决定区3；b：YIYPYNDGTKYNEKFKD是CD47蛋白质的抗体的互补决定区2Note: The diameter of the zone of inhibition does not include the diameter of the puncher; a: ARGGYYTYDDWG is the complementarity determining region of the antibody of CD47 protein; b: YIYPYNDGTKYNEKFKD is the complementarity determining region of the antibody of CD47 protein 2
实施例6肽对细胞活力的影响Effect of the peptide of Example 6 on cell viability
肺癌细胞系A549见参考文献(Lieber M,Smith B,Szakal A et al.A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells.Int J Cancer,2006,17(1):62-70)。细胞培养方法如美国菌种保藏中心所述。永生化的人支气管上皮细胞株16HBE14o–细胞见参考文献(Cozens A L,Yezzi M J,Kunzelmann K,Ohrui T,Chin L,Eng K,Finkbeiner W E,Widdicombe J H,and Gruenert D C."CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells."American Journal of Respiratory Cell and Molecular Biology,1994,10(1):38-47)培养于DMEM/F12培养基中，A549细胞培养于DMEM(高糖)培养基中，并且两种培养基中都加入10％的胎牛血清。The lung cancer cell line A549 is described in the literature (Lieber M, Smith B, Szakal A et al. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer, 2006, 17(1): 62-70). Cell culture methods are as described by the American Type Culture Collection. Immortalized human bronchial epithelial cell line 16HBE14o– cells are found in references (Cozens A L, Yezzi M J, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner W E, Widdicombe J H, and Gruenert D C.” CFTR Expression and chloride secretion in polarized immortal human bronchial epithelial cells. "American Journal of Respiratory Cell and Molecular Biology, 1994, 10 (1): 38-47) cultured in DMEM/F12 medium, A549 cells cultured in DMEM (high sugar) In the medium, 10% fetal calf serum was added to both mediums.
细胞活力采用3–(4,5dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide(MTT；MBCHAM)比色法测定，具体步骤参见参考文献(Warshamana-Greene GS,Litz J,Buchdunger E et al.The insulin-like growth factor-I receptor kinase inhibitor,NVP-ADW742,sensitizes small cell lung cancer cell lines to the effects of  chemotherapy.Clin Cancer Res,2005,11(4):1563-1571)。主要步骤：16HBE14o–和A549细胞培养于96孔板中至指数生长期。然后对各孔细胞施以指定的不同浓度的肽。处理24小时后，用MTT法测定在492纳米的吸光度值。最后根据公式：细胞活力抑制率(％)＝(对照组492纳米吸光值-实验组492纳米吸光值)/对照组492纳米吸光值×00％)来计算出各种肽对细胞活力的抑制率，结果如图7～9。 Cell viability was determined by 3–(4,5dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT; MBCHAM) colorimetric assay. For specific procedures, see references (Warshamana-Greene GS, Litz J, Buchdunger E et al. The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of Chemotherapy. Clin Cancer Res, 2005, 11(4): 1563-1571). Main steps: 16HBE14o– and A549 cells were cultured in 96-well plates to exponential growth phase. Each well cell is then subjected to the indicated different concentrations of peptide. After 24 hours of treatment, the absorbance at 492 nm was measured by the MTT method. Finally, according to the formula: cell viability inhibition rate (%) = (control group 492 nm absorbance - experimental group 492 nm absorbance value / control group 492 nm absorbance × 00%) to calculate the inhibition rate of various peptides on cell viability The results are shown in Figures 7-9.

Claims (9)

  1. 一种碱性抗菌肽，其特征在于，所述碱性抗菌肽由亮氨酸和赖氨酸组成，所述碱性抗菌肽的氨基酸的个数为12～24个，其中，赖氨酸的比例大于33.3％。An alkaline antimicrobial peptide characterized in that the basic antimicrobial peptide is composed of leucine and lysine, and the number of amino acids of the basic antimicrobial peptide is 12 to 24, wherein lysine The ratio is greater than 33.3%.
  2. 根据权利要求1所述的碱性抗菌肽，其特征在于，所述碱性抗菌肽的氨基酸序列如SEQ ID NO：1～23所示。The alkaline antimicrobial peptide according to claim 1, wherein the amino acid sequence of the basic antimicrobial peptide is as shown in SEQ ID NOS: 1 to 23.
  3. 根据权利要求1所述的碱性抗菌肽，其特征在于，所述氨基酸是D型或/和L型。The alkaline antimicrobial peptide according to claim 1, wherein the amino acid is D-form or/and L-form.
  4. 一种靶向抗菌肽，其特征在于，所述靶向抗菌肽结构组成为抗菌肽-Lo-抗体互补决定区；其中o为亮氨酸的个数，o≥4；所述抗菌肽为权利要求1至3任一项所述的抗菌肽。A targeted antimicrobial peptide, characterized in that the structure of the targeted antimicrobial peptide is an antimicrobial peptide-Lo-antibody complementarity determining region; wherein o is the number of leucine, o≥4; the antimicrobial peptide is a right The antimicrobial peptide of any one of 1 to 3 is claimed.
  5. 根据权利要求4所述的靶向抗菌肽，其特征在于，所述靶向抗菌肽的序列如SEQ ID NO：25～31所示。The targeted antimicrobial peptide according to claim 4, wherein the sequence of the targeted antimicrobial peptide is as shown in SEQ ID NOS: 25 to 31.
  6. 权利要求1至3任一项所述碱性抗菌肽在制备抑菌或/和抗癌药物方面的应用。Use of the alkaline antimicrobial peptide according to any one of claims 1 to 3 for the preparation of a bacteriostatic or/and anticancer drug.
  7. 权利要求4或5所述靶向抗菌肽在制备抑菌或/和抗癌药物方面的应用。Use of the targeted antimicrobial peptide according to claim 4 or 5 for the preparation of a bacteriostatic or/and anticancer drug.
  8. 根据权利要求6所述的应用，其特征在于，所述抑菌为抑制真菌或/和细菌；所述抗癌为杀死肺癌细胞或杀死永生化细胞。The use according to claim 6, wherein the bacteriostatic action is inhibition of fungi or/and bacteria; the anti-cancer is killing lung cancer cells or killing immortalized cells.
  9. 根据权利要求7所述的应用，其特征在于，所述抑菌为抑制真菌或/和细菌；所述抗癌为杀死肺癌细胞或杀死永生化细胞。 The use according to claim 7, wherein the bacteriostatic action is inhibition of fungi or/and bacteria; the anti-cancer is killing lung cancer cells or killing immortalized cells.
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