WO2017061732A1 - Novel antibacterial protein bps13 having lytic capability specific to bacillus strains - Google Patents

Abstract

The present invention relates to an antibacterial protein BPS13 having a lytic capability specific to Bacillus strains, and more specifically, to: a lytic protein BPS13 specific to Bacillus strains, which is characterized by having an amino acid sequence represented by SEQ ID NO:1 capable of specifically killing Bacillus strains that can infect and cause diseases in animals including humans; and a pharmaceutical composition containing, as an active ingredient, the lytic protein BPS13 specific to Bacillus strains, for treating infections caused the Bacillus strains.

Description

Novel Antibacterial Protein JPPS13 with Bacillus Species Specific Lysis Activity

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.

Accordingly, 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.

Accordingly, 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.

In addition, 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.

In addition, 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.

Therefore, according to one aspect of the present invention, the present invention provides an amino acid sequence of the antibacterial protein BPS13 capable of specifically lysing Bacillus bacteria. Specifically, 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.

It is apparent that this 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.

In addition, 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).

In addition, according to another aspect of the present invention, 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.

Since 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.

As used herein, the term '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. In addition to the above components, 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.

The pharmaceutical 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. In addition, 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. On the other hand, 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.

Hereinafter, although an Example demonstrates this invention more concretely, these Examples are only illustrations of this invention, The scope of the present invention is not limited to these Examples.

Example  One: Bacillus  Bacillus specific lytic protein Of BPS13  Produce

The production of 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. In this example, 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) containing kanamycin to 50 μg / ml Inoculate coli BL21-pBAD-BPS13 (add 20 μl) and incubate for 6-7 hours at 37 ° C. 20 μl of the culture solution was inoculated into 200 ml of LB medium containing kanamycin to 50 μg / ml, followed by shaking culture at 37 ° C. for one night. The next day, overnight culture was added to an incubator containing 5 L of LB medium containing kanamycin to 50 μg / ml so that the OD ₆₀₀ (absorbance at 600 nm) was 0.1. Incubate at 200 rpm, 5 L / min aeration, 37 ° C. When the cell concentration was 0.3-0.35 based on the absorbance at 600 nm, the culture temperature was lowered to 30 ° C. When the cell concentration reached 0.5 based on the absorbance at 600 nm (about 30 minutes after the change of the culture temperature), the Bacillus of the amino acid sequence represented by SEQ ID NO: 1 by adding L-arabinose to give a final concentration of 0.2% 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. Application conditions of the ultrasonic grinding method was repeated for 15 minutes to break the cells and stop for 3 seconds by applying ultrasonic waves for 3 seconds. At this time, it was carried out in an ice bath. After cell lysing, the cell lysate was centrifuged at 13,000 rpm for 20 minutes at 4 ° C to recover the supernatant. The obtained supernatant was purified through a conventional cation-exchange chromatography purification process. Brief description of the purification process is as follows. 5 ml of HiTrap ^™ SP HP (GE Healthcare, Inc.) was used as the cation-exchange resin. Chromatography was performed after the column was equilibrated with Buffer A (20 mM Tris-HCl, pH 7.0) in advance, and after dropping the sample onto the column, 5 CV (column volume) of Buffer A was added at a flow rate of 5 ml / min. Dripping was performed to wash. After washing, chromatography was performed under conditions such that the gradient from buffer A to buffer B (20 mM Tris-HCl, 500 mM NaCl, pH 7.0) was 0% to 100% at a flow rate of 5 ml / min. In this process, elution of Bacillus-specific lysate protein BPS13 of the amino acid sequence represented by SEQ ID NO: 1 was achieved. The result of analyzing the purified protein BPS13 through electrophoresis is shown in FIG. Through this process, more than 90% purity could be obtained.

Example  2: Bacillus  Bacillus specific lytic protein BPS13 has  Preparation of Included Pharmaceutical Compositions

In this example, a pharmaceutical 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.

Various buffer conditions, various types of stabilizers, and various types of additives applicable to pharmaceutical 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. In selecting buffer conditions, stabilizer types, and additive types, the pharmacologically acceptable components (ie, drug limits) and 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).

Furtherance	Detail ingredients
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

Using the pharmaceutical composition (1 mg / ml) to which the composition 2 prepared in Example 2 was applied, the lytic activity of Bacillus specific lysate protein BPS13 against Bacillus bacteria was investigated. 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. On the other hand, for evaluation of lytic activity against Bacillus and other species, three strains of Salmonella, two strains of E. coli, two strains of Streptococcus mutans , and Enterococcus faecalis ) 3 weeks and 2 weeks of Staphylococcus aureus were also included as the test bacteria.

Species	Strain	Source
Bacillus cereus	ATCC  4342	ATCC
Bacillus licheniformis	KCOM  1491	Korea Oral Microbial Bank
Bacillus circulans	KCTC  3004 [ ATCC  21783]	Microbial Resource Center
Bacillus megaterium	KCTC  2178 [ ATCC  10778]	Microbial Resource Center
Bacillus pumilus	KCTC  3713	Microbial Resource Center

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 μg / ml). After the addition of ml, the absorbance was performed at 600 nm in a manner of measuring 20 minutes.

As a result, 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.

As a result, it can be seen that the Bacillus specific bacterium protein BPS13 of the present invention can be killed by lysing Bacillus bacteria. These properties show that 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

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.

In this study, 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. For another group (control), only excipients (10 mM L-Histidine, 0.1 % Polysobate 20, 1% Sucrose, pH 6.0) were administered to each animal with excipients corresponding to the average of the test group doses. 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.

As a result, a marked therapeutic effect was confirmed. The number of deaths was as shown in Table 3 below, and administration of the pharmaceutical composition containing Bacillus specific lysate protein of the present invention showed a marked improvement in survival rate. In addition, in the control group, there were almost no specific reactions observed in the treatment group compared with various reactions such as Erthema of lid margin, Ptosis, and decreased activity.

group	Death population					Death population / test population	Mortality (%)
	Days after fungal forced infection
	One	2	3	4	5
Control	0	2	2	One	0	5/10	50
Aid group	0	0	0	0	0	0/10	0

As a result, it can be confirmed that Bacillus specific lysate protein BPS13 of the present invention is effective in treating Bacillus bacteria infection. These properties show that 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. .

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Depositary Name: KCTC

Accession number: KCTC 12892BP

Deposited Date: 20150903

Claims (5)

1. Bacillus (Bacillus) bacteria specific lysis having activity, specific lysis proteins BPS13 Bacillus bacterium comprising the amino acid sequence shown in SEQ ID NO: 1.
2. The Bacillus specific lysate protein BPS13 according to claim 1, wherein the Bacillus specific lysate protein BPS13 is composed of 277 amino acids and has a molecular weight of 31 kDa.
3. A pharmaceutical composition for treating Bacillus infection, comprising the Bacillus specific lysate protein BPS13 of claim 1 as an active ingredient.
4. The pharmaceutical composition of claim 3, wherein the composition is used as a food additive, an antibiotic, a disinfectant, a bactericide, or a therapeutic agent.
5. Esherichia A method for producing Bacillus bacteria specific lysate protein BPS13 of claim 1 using coli BL21-pBAD-BPS13 (Accession No. KCTC 12892BP).

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