WO2017061732A1 - Nouvelle protéine antibactérienne bps13 dotée d'une fonction lytique spécifique à des souches de bacillus - Google Patents

Nouvelle protéine antibactérienne bps13 dotée d'une fonction lytique spécifique à des souches de bacillus Download PDF

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WO2017061732A1
WO2017061732A1 PCT/KR2016/010953 KR2016010953W WO2017061732A1 WO 2017061732 A1 WO2017061732 A1 WO 2017061732A1 KR 2016010953 W KR2016010953 W KR 2016010953W WO 2017061732 A1 WO2017061732 A1 WO 2017061732A1
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bacillus
bps13
protein
specific
bacteria
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PCT/KR2016/010953
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Korean (ko)
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윤성준
전수연
정기모
강상현
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주식회사 인트론바이오테크놀로지
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    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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 an antimicrobial protein BPS13 having a specific lytic activity against Bacillus species, more specifically, the ability to specifically lysate Bacillus bacteria that can cause disease by infecting animals including humans.
  • Pharmaceutical treatment for infection caused by Bacillus bacteria comprising a lytic protein BPS13 specific for Bacillus strains, and the Bacillus specific lytic protein BPS13 as an active ingredient, characterized by having an amino acid sequence represented by SEQ ID NO: 1 It relates to a composition.
  • Bacillus (Bacillus) bacteria is the most motile Gram-positive spore forming bacilli, and includes more than 260 kinds of different types. Bacillus subtilis or Bacillus laeblacticus Some beneficial Bacillus bacteria have been used as probiotics in real life, such as laevolacticus , while others are harmful Bacillus bacteria that clinically cause various infectious diseases such as bacteremia, sepsis and meningitis. Representative harmful bacillus species is Bacillus cereus (Bacillus cereus ), Bacillus licheniformis , Bacillus Circulans circulans ), Bacillus pumilus , and the like. In the case of Bacillus cereus, as well as E. coli, Salmonella, Staphylococcus aureus is known as a representative food poisoning cause, Bacillus cereus and Bacillus licheniformis is also well known as a causative agent of milk mastitis.
  • Antibiotics have been widely used to treat these harmful Bacillus infections. Recently, the resistance of antibiotics to Bacillus bacteria continues to be severe, resulting in seriously low effectiveness of treatment with antibiotics. The development of new antibiotics / antibacterial materials is needed to effectively cope with Bacillus infections that have acquired resistance to these existing antibiotics.
  • the present inventors provide an antimicrobial protein capable of selectively lysing Bacillus bacteria and a method for producing the same, and furthermore, to provide a pharmaceutical composition for treating Bacillus bacteria infection using lytic proteins.
  • an object of the present invention is to provide an antibacterial protein BPS13 that can specifically lyse Bacillus bacteria, which are the causative agents of infectious diseases in animals including humans.
  • Another object of the present invention is to provide a method for efficiently preparing the antibacterial protein BPS13 capable of specifically lysing the Bacillus bacteria.
  • Another object of the present invention to provide a pharmaceutical composition for treating Bacillus bacteria infection comprising an antibacterial protein BPS13 that can specifically lyse the Bacillus bacteria as an active ingredient.
  • the inventors of the present invention utilize the various genes and protein information known to achieve the above objects of the present invention to prepare various protein candidates in the form of recombinant proteins, and then investigate their lytic activity against Bacillus bacteria to achieve the expected level of lysis.
  • the present invention was completed by selecting a protein having an activity, developing a method for efficiently preparing the same, and finally, developing a pharmaceutical composition that can be used for the treatment of Bacillus infections using the same as an active ingredient.
  • the present invention provides an amino acid sequence of the antibacterial protein BPS13 capable of specifically lysing Bacillus bacteria.
  • the amino acid sequence of SEQ ID NO: 1 corresponds.
  • the antimicrobial protein BPS13 which can specifically lyse Bacillus bacteria, consists of 277 amino acids and has a molecular weight of about 31 kDa.
  • amino acid sequence may be partially modified by those skilled in the art using known techniques. Such modifications include some substitutions of amino acid sequences, some additions of amino acid sequences, and some deletions of amino acid sequences. However, it is most preferable to apply the amino acid sequence of SEQ ID NO: 1 disclosed in the present invention mutatis mutandis.
  • the present invention is a strain Escherichia that can be used for the production of the antibacterial protein BPS13 having the amino acid sequence of SEQ ID NO: 1 coli BL21-pBAD-BPS13 is provided.
  • This Escherichia coli BL21-pBAD-BPS13 was developed and produced by the present inventors and deposited in the Korea Institute of Biotechnology and Biotechnology Center on September 3, 2015 (Accession No. KCTC 12892BP).
  • the present invention can be effectively used for the treatment of Bacillus bacteria infection characterized by the antimicrobial protein BPS13 characterized by the amino acid sequence of SEQ ID NO: 1 having a specific lytic ability against Bacillus bacteria as an active ingredient It provides a pharmaceutical composition that can be.
  • the antimicrobial protein BPS13 having a specific lytic ability against the Bacillus bacteria characterized by the amino acid sequence of SEQ ID NO: 1 of the present invention, which is included in the pharmaceutical composition of the present invention, specifically lyses Bacillus bacteria, It is effective in the treatment of various diseases caused by Bacillus bacteria. Therefore, the pharmaceutical composition of the present invention can be used to treat diseases of animals and human diseases caused by Bacillus bacteria.
  • treatment refers to (1) inhibition of infection caused by Bacillus bacteria; And (2) alleviation of infection caused by Bacillus bacteria.
  • Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen.
  • the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
  • the pharmaceutical composition of the present invention may be administered through oral or parenteral administration, and may be administered using parenteral administration by intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration. It can also be used as a method of applying or spraying to the diseased area.
  • Suitable applications, sprays, and dosages of the pharmaceutical compositions of the present invention may be formulated by the method of formulation, mode of administration, age, weight, sex, degree of disease symptom, food, time of administration, route of administration, rate of excretion and Depending on factors such as response responsiveness, usually an experienced physician or veterinarian can readily determine and prescribe a dosage effective for the desired treatment.
  • compositions of the present invention are prepared in unit dosage form by being formulated with pharmaceutically acceptable carriers and / or excipients according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container.
  • the formulations here may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
  • the pharmaceutical composition of the present invention may be embodied as an antibiotic, a disinfectant, a bactericide, a therapeutic agent and the like.
  • the pharmaceutical composition of the present invention may be implemented in the form of food additives.
  • the present invention provides a Bacillus specific lysate protein BPS13 that can effectively treat Bacillus infection and a pharmaceutical composition comprising the same as an active ingredient, and further provides a food additive for the purpose of preventing and treating food poisoning.
  • the pharmaceutical composition or food additive of the present invention may effectively act on Bacillus bacteria that have obtained resistance to existing antibiotics or antibacterial substances.
  • the Bacillus specific lysate protein BPS13 of the present invention does not affect normal flora in the body other than Bacillus bacteria can minimize side effects due to the use of pharmaceutical compositions or food additives containing it as an active ingredient . Existing antibiotics used to adversely affect beneficial bacteria in the body, causing various side effects.
  • Figure 1 is an electrophoresis picture showing the results of the production of Bacillus bacteria-specific lytic protein BPS13 in recombinant protein form, characterized in that having the amino acid sequence represented by SEQ ID NO: 1, lane M is a protein size marker.
  • FIG. 2 shows the results of an experiment of a turbidity reduction assay in Bacillus cereus.
  • (-) Is the negative control without BPS13, and (+) is the case with BPS13 added.
  • the horizontal axis is time (minutes) and the vertical axis is absorbance at 600 nm.
  • Figure 3 is a result showing the results of the turbidity reduction assay (turbidity reduction assay) for Bacillus licheniformis bacteria.
  • (-) Is the negative control without BPS13, and (+) is the case with BPS13 added.
  • the horizontal axis is time (minutes) and the vertical axis is absorbance at 600 nm.
  • Figure 4 is a result showing the experimental results of the turbidity reduction assay (Turbidity reduction assay) for Bacillus sircurans bacteria.
  • (-) Is the negative control without BPS13, and (+) is the case with BPS13 added.
  • the horizontal axis is time (minutes) and the vertical axis is absorbance at 600 nm.
  • FIG. 5 shows the results of an experiment of a turbidity reduction assay of Bacillus megaterium bacteria.
  • (-) Is the negative control without BPS13, and (+) is the case with BPS13 added.
  • the horizontal axis is time (minutes) and the vertical axis is absorbance at 600 nm.
  • FIG. 6 shows the results of an experiment of a turbidity reduction assay in Bacillus fumirus.
  • FIG. (-) Is the negative control without BPS13, and (+) is the case with BPS13 added.
  • the horizontal axis is time (minutes) and the vertical axis is absorbance at 600 nm.
  • Bacillus specific lytic protein BPS13 having the amino acid sequence represented by SEQ ID NO: 1 selected as Bacillus specific lytic protein will be described below.
  • Escherichia a strain deposited by the present inventors at the Korea Research Institute of Bioscience and Biotechnology, as of September 3, 2015 coli BL21-pBAD-BPS13 (Accession No. KCTC 12892BP) was used as a production strain.
  • Escherichia in 20 ml of LB medium tryptone, 10 g / L; yeast extract, 5 g / L; sodium chloride, 10 g / L
  • Inoculate coli BL21-pBAD-BPS13 add 20 ⁇ l and incubate for 6-7 hours at 37 ° C.
  • 20 ⁇ l of the culture solution was inoculated into 200 ml of LB medium containing kanamycin to 50 ⁇ g / ml, followed by shaking culture at 37 ° C. for one night.
  • Expression of the bacterium specific lytic protein BPS13 was induced. After expression induction, an additional 4 hours of incubation was performed at 30 ° C. After the completion of the culture, the cell culture was taken and centrifuged at 6,000 rpm for 10 minutes at 4 ° C to recover cell precipitates. The recovered cell precipitate was suspended in 200 ml of 20 mM Tris-HCl (Tris-HCl, pH 7.0) buffer. The cell suspension prepared in this way was crushed by the ultrasonic grinding method.
  • Example 2 Bacillus Bacillus specific lytic protein BPS13 has Preparation of Included Pharmaceutical Compositions
  • composition comprising Bacillus specific lysate protein BPS13 prepared in Example 1 as an active ingredient was prepared.
  • the composition shown in the present embodiment is merely an applicable composition example, and is not limited thereto.
  • compositions can be prepared to provide a level of industrial stability when Bacillus-specific lysate protein BPS13 is added to each composition.
  • the composition was explored.
  • the pharmacologically acceptable components ie, drug limits
  • the isoelectric point of BPS13 were considered.
  • Stability investigations were conducted by comparing physical stress, such as agitation (2,500 rpm; 2 hours) and heating (40 ° C; 16 hours), with short-term storage stability of 4 weeks. Absorbance measurements and HPLC analysis were used for stability evaluation. As a result, the following four compositions could be selected as a suitable formulation for Bacillus specific lysate protein BPS13 (Table 1).
  • composition 1 10 mM L-Histidine, 0.1% Polysobate 20, 0.1% L-Cysteine hydrate, pH 6.0
  • Composition 2 10 mM L-Histidine, 0.1% Polysobate 20, 1% Sucrose, pH 6.0
  • Composition 3 10 mM L-Histidine, 0.1% Polysobate 80, 0.1% L-Cysteine hydrate, pH 6.0
  • Composition 4 10 mM L-Histidine, 0.1% Polysobate 80, 5% Mannitol, pH 6.0
  • the final pharmaceutical composition was first prepared by buffer exchange the protein BPS13 sample obtained in Example 1 with the composition shown in Table 1 above, and then adjusted to a final concentration of 1 mg / ml or 10 mg / ml. It was used in the field.
  • Example 3 Bacillus Bacillus specific lytic protein Of BPS13 Investigation of lytic activity
  • Bacillus bacteria used to investigate the lytic activity were pathogenic Bacillus cereus, Bacillus richeniformis, Bacillus sircurans, Bacillus megaterium, and Bacillus pumirus, the details of which are shown in Table 2.
  • Bacillus bacteria used to investigate the lytic activity were pathogenic Bacillus cereus, Bacillus richeniformis, Bacillus sircurans, Bacillus megaterium, and Bacillus pumirus, the details of which are shown in Table 2.
  • three strains of Salmonella, two strains of E. coli, two strains of Streptococcus mutans , and Enterococcus faecalis were also included as the test bacteria.
  • the lysis activity was investigated using a turbidity reduction assay.
  • the experimental method of the turbidity reduction investigation method is as follows. The test bacteria were suspended in physiological saline at 600 nm to have an absorbance of about 1.0, and then diluted with 0.9 ml of the suspension to which the composition 2 prepared in Example 2 was applied (BPS13 concentration: 5 ⁇ g / ml). After the addition of ml, the absorbance was performed at 600 nm in a manner of measuring 20 minutes.
  • Bacillus specific lysate protein BPS13 showed lytic activity only against Bacillus bacteria as expected and did not have lytic activity against other test bacteria. From this, it was confirmed that the lytic activity of the protein of the present invention is very specific for Bacillus bacteria. Experimental results for Bacillus bacteria are shown in FIGS. In the investigation of lysis activity through the turbidity reduction investigation, it was confirmed that lysis activity was exerted very quickly. This rapid lytic effect is a property that no existing antibiotics can provide. For reference, similar results were confirmed when the remaining compositions selected in Example 2 were applied.
  • Bacillus specific bacterium protein BPS13 of the present invention can be killed by lysing Bacillus bacteria.
  • the pharmaceutical composition containing Bacillus specific lysate protein BPS13 can be used for Bacillus killing for Bacillus infection, and can be used in the same way as conventional antibiotics for the treatment of Bacillus infection. Show that there is.
  • Example 4 Bacillus Bacillus specific lytic protein Of BPS13 Bacillus Investigation of therapeutic effects on bacterial infections
  • composition 2 prepared in Example 2 Using the pharmaceutical composition (10 mg / ml) to which composition 2 prepared in Example 2 was applied, the therapeutic effect of Bacillus specific lysate protein BPS13 on Bacillus infection was investigated using an infected animal model.
  • Bacillus cereus and Bacillus sircurans were used as model Bacillus bacterial infections.
  • Five-week-old ICR mice (specific pathogen-free (SPF) grade) of about 20 g body weight were used as experimental animals.
  • a total of 20 animals were divided into two groups (10 animals per group), followed by intravenous administration of 1 ⁇ 10 8 cfu (ie 1 ⁇ 10 8 cfu / mouse) each of Bacillus cereus and Bacillus sircurans per mouse. Dosing led to infection.
  • One group (treatment group) was administered the pharmaceutical composition (10 mg / ml) to which composition 2 prepared in Example 2 was applied at 30 minutes, 12 hours, and 24 hours after bacterial infection. It was. Dosage was set at 25 mg / kg.
  • excipients 10 mM L-Histidine, 0.1% Polysobate 20, 1% Sucrose, pH 6.0
  • Excipient administration was performed at 30 minutes, 12 hours, and 24 hours after the forced bacterial infection. The number of deaths was examined every day for five days after the forced infection of bacteria, and the occurrence of abnormalities was also examined twice a day, morning and afternoon.
  • Bacillus specific lysate protein BPS13 of the present invention is effective in treating Bacillus bacteria infection.
  • the pharmaceutical composition containing Bacillus specific lysate protein BPS13 can be used for the treatment of Bacillus infection, and can be used in the same way as conventional antibiotics for the treatment of Bacillus infection. .

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Abstract

La présente invention concerne une protéine antibactérienne BPS13 dotée d'une fonction lytique spécifique à des souches de Bacillus, et plus précisément : une protéine lytique BPS13 spécifique à des souches de Bacillus, qui est caractérisée en ce qu'elle possède une séquence d'acides aminés représentée par SEQ ID NO : 1 apte à tuer spécifiquement des souches de Bacillus pouvant être source d'infections et entraîner des maladies chez les animaux y compris les humains ; et une composition pharmaceutique contenant, en tant qu'ingrédient actif, la protéine lytique BPS13 spécifique à des souches de Bacillus, destinée au traitement d'infections provoquées par des souches de Bacillus.
PCT/KR2016/010953 2015-10-06 2016-09-30 Nouvelle protéine antibactérienne bps13 dotée d'une fonction lytique spécifique à des souches de bacillus WO2017061732A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019222502A1 (fr) * 2018-05-17 2019-11-21 University Of Maryland, College Park Endolysines actives contre les bactéries bacellus, produit pharmaceutique et méthodes associées
WO2021181235A1 (fr) * 2020-03-09 2021-09-16 Intron Biotechnology, Inc. Protéine antibactérienne ayant une activité lytique vis-à-vis du genre bacillus et son procédé de préparation

Citations (1)

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KR20140140477A (ko) * 2013-05-28 2014-12-09 한국생명공학연구원 용균 또는 항균 능력이 개선된 리신 효소 변이체

Patent Citations (1)

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KR20140140477A (ko) * 2013-05-28 2014-12-09 한국생명공학연구원 용균 또는 항균 능력이 개선된 리신 효소 변이체

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KONG ET AL.: "Bacteriophage PBC1 and Its Endolysin as an Antimicrobial Agent Against Bacillus Cereus", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 81, no. 7, 1 April 2015 (2015-04-01), pages 2274 - 2283, XP055374041 *
PARK ET AL.: "Characterization of an Endolysin, LysBPS 13, from a Bacillus Cereus Bacteriophage", FEMS MICROBIOLOGY LETTERS, vol. 332, no. 1, July 2012 (2012-07-01), pages 76 - 83, XP055374023 *
SHIN ET AL.: "Characterization and Genome Analysis of the Bacillus Cereus-infecting Bacteriophages BPS10C and BPS13", ARCHIVES OF VIROLOGY, vol. 159, 2014, pages 2171 - 2175, XP055374038 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019222502A1 (fr) * 2018-05-17 2019-11-21 University Of Maryland, College Park Endolysines actives contre les bactéries bacellus, produit pharmaceutique et méthodes associées
US11890330B2 (en) 2018-05-17 2024-02-06 University Of Maryland, College Park Endolysins active against Bacillus bacteria, pharmaceutical compositions, and methods relating thereto
WO2021181235A1 (fr) * 2020-03-09 2021-09-16 Intron Biotechnology, Inc. Protéine antibactérienne ayant une activité lytique vis-à-vis du genre bacillus et son procédé de préparation
CN115427429A (zh) * 2020-03-09 2022-12-02 尹特荣生物科技株式会社 对芽孢杆菌属具有裂解活性的抗菌蛋白及其制备方法
CN115427429B (zh) * 2020-03-09 2023-09-12 尹特荣生物科技株式会社 对芽孢杆菌属具有裂解活性的抗菌蛋白及其制备方法

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