WO2021085834A1 - Novel antibacterial protein cpel-1 having lytic activity against clostridium perfringens - Google Patents

Abstract

The present invention relates to an antibacterial protein CPEL-1 which has the ability to kill Clostridium perfringens and is represented by the amino acid sequence of SEQ ID NO: 2, to a pharmaceutical composition comprising same as an active ingredient, and to a method for preventing or treating diseases caused by Clostridium perfringens using the pharmaceutical composition.

Description

CPEL-1, a novel antibacterial protein that has lytic power against Clostridium perfringens

The present invention relates to a method for preventing or treating Clostridium perfringens infection using an antimicrobial protein having lytic power against Clostridium perfringens and a composition comprising the same as an active ingredient. More specifically, the antimicrobial protein CPEL-1 having the amino acid sequence of SEQ ID NO: 2 having the ability to kill Clostridium perfringens, a pharmaceutical composition comprising the antimicrobial protein CPEL-1 as an active ingredient, and It relates to a method for preventing or treating infection of Clostridium perfringens using this pharmaceutical composition.

Clostridium perfringens (Clostridium perfringens ) is an anaerobic bacterium (bacteria that can hardly grow in the presence of oxygen), and is a causative agent that can cause serious diseases in humans or various animals such as cattle, pigs, and goats, especially food poisoning and necrosis. Necrotizing enteritis, Gas gangrene, Dysentery, Enterotoxemia, Pulpy kidney disease, and Dermatitis are major diseases. Enterotoxin produced by Clostridium perfringens bacteria are usually hemolytic and necrotic poisons, and there are four major toxins: α, β, ε, and i. According to the presence or absence of these toxins, it is classified into six toxin types of AF type. Among them, type A is a representative cause of food poisoning, and type C is known as a causative agent of necrotizing enteritis.

Various antibiotics have been used for the purpose of preventing or treating the infection of Clostridium perfringens bacteria, but as the occurrence of antibiotic-resistant bacteria has recently increased, it is urgent to secure measures other than antibiotics.

Recently, the use of bacteriophage as a countermeasure against bacterial infectious diseases is receiving great attention. In particular, it is getting more attention because of its excellent antibacterial activity against antibiotic-resistant bacteria. Bacteriophages are very small microorganisms that infect bacteria and are usually shortened to phage (Phage). Bacteriophages have the ability to kill bacteria by proliferating inside the bacterial cells after infection with bacteria, and destroying the cell wall of the host bacteria when progeny bacteriophages come out of the bacteria after proliferation. The bacterial infection method of bacteriophage is very specific, so the types of bacteriophage that can infect specific bacteria are limited to some. That is, a specific bacteriophage can infect only a specific category of bacteria, and thus, a specific bacteriophage can provide an antibacterial effect only against specific bacteria.

As a method of overcoming such a relatively narrow antibacterial range, a method of using an antibacterial protein that actually acts when bacteriophage exerts an antibacterial effect against bacteria has been proposed. The antibacterial protein derived from bacteriophage is called by the name of Endolysin or Lysin. In general, the antibacterial protein derived from bacteriophage can provide a wider antibacterial range compared to the bacteriophage that is its parent.

Antibacterial proteins derived from bacteriophages or bacteriophages have high specificity for acting bacterial species compared to conventional antibiotics. That is, it does not affect other bacterial species other than the desired bacterial species. Bacterial specificity of these bacteriophage and bacteriophage-derived antibacterial proteins provides an antibacterial effect (solubility) only for the target bacteria and does not affect the environment or flora in animals. Conventional antibiotics, which have been widely used in the treatment of bacteria, have an effect on several types of bacteria at the same time. This caused problems such as environmental pollution or disturbance of the normal bacterial flora of animals. In contrast, bacteriophage or bacteriophage-derived antibacterial protein works only against specific bacteria, so the use of bacteriophage or bacteriophage-derived antibacterial protein does not cause disturbance of normal flora in the body. Therefore, it can be said that the use of bacteriophage or bacteriophage-derived antibacterial protein is very safe compared to the use of antibiotics, and as such, the possibility of causing side effects by use is relatively low.

Accordingly, the present inventors provide an antimicrobial protein capable of killing the harmful pathogenic bacteria Clostridium perfringens bacteria, and furthermore, the antimicrobial protein developed using this is included as an active ingredient, and the Clostridium perfringens bacteria It is intended to provide a pharmaceutical composition that can be used to prevent or treat an infection of the disease, and to provide a method that can be effectively used for the prevention or treatment of infection of Clostridium perfringens using this pharmaceutical composition. .

Accordingly, an object of the present invention is to provide an antimicrobial protein CPEL-1 having an amino acid sequence represented by SEQ ID NO: 2 having the ability to kill Clostridium perfringens.

In addition, another object of the present invention is to provide a method for efficiently producing an antimicrobial protein CPEL-1 having the ability to kill Clostridium perfringens and having an amino acid sequence represented by SEQ ID NO: 2. .

In addition, another object of the present invention is to provide a pharmaceutical composition for the purpose of preventing or treating Clostridium perfringens infection, comprising the antimicrobial protein CPEL-1 as an active ingredient.

Another object of the present invention is to provide a method for preventing infection of Clostridium perfringens using a pharmaceutical composition containing the antimicrobial protein CPEL-1 as an active ingredient.

Another object of the present invention is to provide a method for treating an infection of Clostridium perfringens using a pharmaceutical composition comprising the antimicrobial protein CPEL-1 as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

The inventors of the present invention use the genetic information of the bacteriophage isolated from nature, which has the ability to kill Clostridium perfringens, which was isolated by the inventors, to achieve the above objects of the present invention, and represented by SEQ ID NO: 2 We developed an antimicrobial protein CPEL-1 that has excellent lytic power against Clostridium perfringens having an amino acid sequence, and further developed a method to efficiently manufacture it, and finally Clostree using it as an active ingredient. The present invention was completed by developing a pharmaceutical composition that can be used for the purpose of preventing or treating against dium perfringens infection.

Accordingly, according to an aspect of the present invention, the present invention can provide an amino acid sequence of an antimicrobial protein CPEL-1 having lytic power against Clostridium perfringens. Specifically, the antimicrobial protein CPEL-1 has an amino acid sequence represented by SEQ ID NO: 2, and the gene encoding it has a nucleotide sequence preferably represented by SEQ ID NO: 1. The antimicrobial protein CPEL-1, which can kill Clostridium perfringens, is composed of 388 amino acids and has a molecular weight of about 44.4 kDa.

It is obvious that the amino acid sequence shown in SEQ ID NO: 2 can be partially modified by a person skilled in the art using known techniques. Such modifications may include partial substitutions of the amino acid sequence, partial additions of the amino acid sequence, and partial deletions of the amino acid sequence. However, it is most preferable to apply mutatis mutandis the amino acid sequence of SEQ ID NO: 2 disclosed in the present invention.

The term "gene" as used herein has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in genes, are not only natural nucleotides, but also sugar or base sites modified. Also includes analogues ( Chemical Reviews 90:543-584, 1990).

In addition, the present invention can provide E. coli pBAD-CPEL-1-TOP10, which is a transformed E. coli strain that can be used for the production of the antimicrobial protein CPEL-1 having the amino acid sequence of SEQ ID NO: 2. This E. coli pBAD-CPEL-1-TOP10 was developed by the present inventors and deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on October 23, 2019 (accession number KCTC 14003BP).

In addition, according to another aspect of the present invention, the present invention comprises as an active ingredient the antimicrobial protein CPEL-1 having the amino acid sequence of SEQ ID NO: 2 and having lytic power against Clostridium perfringens. It is possible to provide a pharmaceutical composition that can be effectively used for the prevention of infection of Gens or treatment after infection. As an example of the pharmaceutical composition, a disinfectant or an antibiotic may be presented, but it is obvious that the present invention is not limited thereto.

The antimicrobial protein CPEL-1 having the amino acid sequence of SEQ ID NO: 2 of the present invention contained in the pharmaceutical composition containing the antimicrobial protein CPEL-1 of the present invention as an active ingredient has lytic power against Clostridium perfringens. Therefore, Food poisoning, Necrotizing enteritis, Gas gangrene, Dysentery, Enterotoxemia, and Pulpy kidney caused by Clostridium perfringens disease), Dermatitis, and other diseases (prevention of infection) or treatment (infection treatment). Therefore, the pharmaceutical composition of the present invention can be used for the purpose of preventing or treating diseases caused by Clostridium perfringens. Diseases caused by Clostridium perfringens in the present specification are food poisoning, necrotizing enteritis, gas gangrene, dysentery, enterotoxemia, and renal It is a generic term for pulpy kidney disease and dermatitis.

Clostridium perfringens in the present specification does not matter whether it is sensitive to existing antibiotics or resistant bacteria resistant to existing antibiotics. In other words, it is irrelevant whether or not resistance to existing antibiotics is acquired.

The term “prevention” or “prevention” as used herein means (i) prevention of Clostridium perfringens infection; And (ii) inhibiting the development of a disease caused by Clostridium perfringens infection.

The term “treatment” or “treatment” as used herein refers to (i) inhibition of a disease caused by Clostridium perfringens; And (ii) all actions that alleviate the pathological condition of a disease caused by Clostridium perfringens.

Pharmaceutically acceptable carriers included in the pharmaceutical composition containing the antimicrobial protein CPEL-1 of the present invention as an active ingredient are commonly used in preparation, 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, stearic acid It includes, but is not limited to, magnesium and mineral oil. The pharmaceutical composition comprising the antimicrobial protein CPEL-1 of the present invention as an active ingredient may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components, but is not limited thereto. .

The pharmaceutical composition containing the antimicrobial protein CPEL-1 of the present invention as an active ingredient can be used as a method of applying or spraying to a diseased area, and may be administered through oral or parenteral administration, and parenteral administration. In the case of, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration may be used, but the present invention is not limited thereto.

Appropriate application, spray, and dosage of the pharmaceutical composition containing the antimicrobial protein CPEL-1 of the present invention as an active ingredient is a formulation method, a mode of administration, the age, weight, sex, degree of disease symptoms of the target animal and patient, It varies depending on factors such as food, time of administration, route of administration, rate of excretion and sensitivity to reaction, and usually a skilled physician or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.

The pharmaceutical composition comprising the antimicrobial protein CPEL-1 of the present invention as an active ingredient contains 0.0001 to 10% (w/v or w/w) of the antimicrobial protein CPEL-1 of the present invention as an active ingredient, preferably 0.001 It includes ~ 1% (w/v or w/w), and most preferably 0.1% (w/v or w/w), but is not limited thereto.

The pharmaceutical composition containing the antimicrobial protein CPEL-1 of the present invention as an active ingredient is a pharmaceutically acceptable carrier and/or according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention belongs. By being formulated using an excipient, it may be prepared in a unit dosage form, or may be prepared by incorporating it into a multi-dose container. At this time, the formulation may be any one formulation selected from the group consisting of a solution, suspension or emulsion in an oil or aqueous medium, a liquid solution, a pill, an extract, a powder, a granule, a tablet or a capsule, and a diluent, a dispersant, or It may further include a stabilizer, but is not limited thereto.

The pharmaceutical composition comprising the antimicrobial protein CPEL-1 of the present invention as an active ingredient is not limited thereto, depending on the method of use, but may be implemented as a disinfectant or antibiotic. In the present specification, the term'antibiotic' refers to preservatives, fungicides and antibacterial agents.

Antibacterial substances capable of providing antibacterial activity against other bacterial species may be added to the pharmaceutical composition of the present invention in order to increase the efficiency for this purpose of use. In addition, other types of bacteriophage-derived antibacterial proteins (endolysin) having antibacterial activity against Clostridium perfringens may also be added. Even with antibacterial proteins derived from bacteriophages having antibacterial activity against Clostridium perfringens, there are differences in the strength and exertion of antibacterial activity, so an appropriate combination of them can maximize the effect.

Clostridium perfringens infection prevention or treatment method using a pharmaceutical composition comprising the antimicrobial protein CPEL-1 having the amino acid sequence represented by SEQ ID NO: 2 according to the present invention as an active ingredient is based on conventional antibiotics, etc. Compared to the method, it can provide an advantage that the specificity for Clostridium perfringens is very high. This means that it can be used for the purpose of preventing or treating infection of Clostridium perfringens without affecting other useful flora, and it means that the side effects of its use are very few. In general, when antibiotics are used, common organisms are also damaged, resulting in various side effects.

1 is an electrophoresis photograph showing the separation and purification process of the antimicrobial protein CPEL-1, lane M is a protein size marker, lane 1 is a sample before purification, lane 2 is a chromatographic pass-through solution during purification, and from lane 3 Lane 10 is the purified fraction.

2 is a result showing the antibacterial activity (lytic activity) of the antimicrobial protein CPEL-1 against Clostridium perfringens, and the transparent part is generated by the antibacterial activity (lytic activity) of the antimicrobial protein CPEL-1.

3 is an experimental result confirming the antimicrobial activity of the antimicrobial protein CPEL-1 against Clostridium perfringens through a turbidity reduction assay. As a negative control, a buffer solution that did not contain the antimicrobial protein CPEL-1 was used. The horizontal axis is time (minutes) and the vertical axis is the absorbance at 600 nm.

Hereinafter, the present invention will be described in more detail based on examples, but these examples are only examples of the present invention, and the scope of the present invention is not limited to these examples.

Example 1: derived from bacteriophage Antibacterial protein Securing sequence

Example 1-1: Preparation of bacteriophage suspension

A suspension containing pure bacteriophage (accession number KCTC 12664BP) having a killing ability against Clostridium perfringens bacteria was prepared through the following purification process. Clostridium perfringens bacteria culture solution was added to a volume of 1/50 of the total volume of the solution including pure bacteriophage, and then cultured again for 12 hours. After incubation, centrifugation was performed at 8,000 rpm for 20 minutes to obtain a supernatant. This process was repeated a total of 5 times to obtain a solution containing a sufficient number of bacteriophage. The supernatant obtained by the final centrifugation was filtered using a 0.45 μm filter, and then a conventional polyethylene glycol (PEG) precipitation process was performed. Specifically, PEG and NaCl were added to 100 mL of the filtrate to become 10% PEG 8000/0.5 M NaCl, and then allowed to stand at 4° C. for 2 to 3 hours, followed by centrifugation at 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate. The thus obtained bacteriophage precipitate was suspended in 5 mL of a buffer (buffer; 10 mM Tris-HCl, 10 mM MgSO ₄ , 0.1% Gelatin, pH 8.0). This is referred to as bacteriophage suspension or bacteriophage liquid.

Example 1-2: Securing the genome of bacteriophage

The genome of the bacteriophage was separated from the suspension of the bacteriophage prepared according to Example 1-1 as follows. First, in order to remove DNA and RNA of Clostridium perfringens that may be contained in the suspension, 200 units of DNase I and RNase A were added to 10 mL of the bacteriophage suspension, and then left at 37°C for 30 minutes. In order to remove the activities of DNase I and RNase A after standing for 30 minutes, 500 μL of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added and then allowed to stand for 10 minutes. Then, after allowing it to stand at 65°C for an additional 10 minutes, 100 μL of proteinase K (20 mg/mL) was added to disintegrate the outer wall of the bacteriophage, and then reacted at 37°C for 20 minutes. Thereafter, 500 μL of 10% sodium dodecyl sulfate (SDS) was added, and then reacted again at 65° C. for 1 hour. After 1 hour reaction, 10 mL of a mixture of phenol: chloroform: isoamylalcohol having a composition ratio of 25:24:1 was added to the reaction solution, and then mixed well. Then, this was centrifuged at 13,000 rpm for 15 minutes to separate the layers, and then the upper layer was taken out of the separated layers, and 1.5 volume ratio of isopropyl alcohol was added thereto, followed by centrifugation at 13,000 rpm for 10 minutes. The dielectric was precipitated. After recovering the precipitate, 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, dried in vacuum, and then dissolved in 100 μL of water. By repeating the above process, a large amount of the genome of the bacteriophage was secured.

Example 1-3: Analysis of genome sequence of bacteriophage and Antibacterial protein Securing sequence

Using the genome obtained in Example 1-2, bacteriophage by performing a next generation sequencing analysis using an illumina Mi-Seq device at the National Instrumentation Center for Environmental Management, Seoul National University. The genome sequence information of was obtained.

From the bacteriophage genome sequence, the gene sequence corresponding to the antibacterial protein of the bacteriophage could be estimated using NCBI GLIMMER and BLAST. The estimated gene sequence (1,167 bp) of the antimicrobial protein was used to develop recombinant production technology for the antibacterial protein derived from bacteriophage. The gene sequence of the used antimicrobial protein is shown in SEQ ID NO: 1. For reference, the amino acid sequence (consisting of 388 amino acid residues) of the antimicrobial protein corresponding to the gene sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2.

The antimicrobial protein represented by the amino acid sequence of SEQ ID NO: 2 thus obtained was named CPEL-1.

Along with this fact, from the fact that the antibacterial properties that can be provided are different if the types of antibacterial proteins derived from bacteriophage are different, the antibacterial protein CPEL-1 represented by the amino acid sequence of SEQ ID NO: 2 is different from other previously reported antibacterial proteins derived from bacteriophage. It was judged that it could provide other antibacterial effects.

Example 2: Antibacterial protein CPEL -1 expression plasmid and Production strain making

For the production of the antimicrobial protein CPEL-1, an expression plasmid of the antimicrobial protein CPEL-1 was constructed. The gene of the antimicrobial protein CPEL-1 identified in Example 1-3 was cloned into a pBAD-TOPO vector (Invitrogen) using Nco I and Not I restriction enzyme sites. To this end, the Enterokinase cleavage site that was present in the pBAD-TOPO vector before cloning was removed and Not After making the one in which the I restriction enzyme site was inserted, it was used for PCR cloning. In addition, a site-directed mutagenesis kit (iNtRON Biotechnology) was used to match the start codon after cloning, and through this process, a plasmid for the expression of the antimicrobial protein CPEL-1 was finally produced. . The thus prepared expression plasmid of the antimicrobial protein CPEL-1 was named pBAD-CPEL-1. The base sequence of pBAD-CPEL-1 is shown in SEQ ID NO: 3.

This pBAD-CPEL-1 was used to transform E. coli TOP10 to produce a strain producing the antimicrobial protein CPEL-1, and this production strain was named pBAD-CPEL-1-TOP10 (accession number KCTC 14003BP).

Example 3: Antibacterial protein CPEL Preparation of -1

Preparation of the antimicrobial protein CPEL-1 having the amino acid sequence represented by SEQ ID NO: 2 will be described below. In this example, E. coli pBAD-CPEL-1-TOP10 produced by the present inventors was used as a production strain. However, those skilled in the art can use the expression plasmid prepared based on SEQ ID NO: 2 presented in the present invention or the expression plasmid prepared based on SEQ ID NO: 1 presented in the present invention to create a production strain different from pBAD-CPEL-1-TOP10. It will be obvious that it can be produced and utilized.

Inoculate E. coli pBAD-CPEL-1-TOP10 to 20 mL of LB medium (trypton, 10 g/L; yeast extract, 5 g/L; sodium chloride, 10 g/L) containing kanamycin to be 50 μg/mL ( 10 μL) and then incubated with shaking at 37° C. overnight (200 rpm). _{The next day, an OD 600} (absorbance at 600 nm) of OD 600 (absorbance at 600 nm) was added in a volume ratio of 1/100 in an incubator containing 1 L of LB medium containing kanamycin to be 50 μg/mL. Culture was performed under the conditions of 9 L aeration, 200 rpm/min, and 37°C. When the cell concentration reached 0.5 based on the absorbance at 600 nm, L-arabinose was added so that the final concentration was 0.2%, thereby inducing the expression of the antimicrobial protein CPEL-1 having the amino acid sequence represented by SEQ ID NO: 2. After induction of expression, culture was performed at 37°C for 4 hours.

After completion of the culture, the cell culture solution was collected and centrifuged at 13,000 rpm and 4° C. for 5 minutes to recover the cell precipitate. The recovered cell precipitate was suspended in a manner using 20 mL of a buffer solution (20 mM K ₃ PO _{4, pH 6.0) per 1 g of the cell precipitate.} The cell suspension thus prepared was subjected to cell disruption by applying an ultrasonic grinding method. The application conditions of the ultrasonic grinding method were repeated for a total of 30 minutes to break the cells by applying ultrasonic waves for 3 seconds and stop for 3 seconds. At this time, it was conducted in an ice bath state. After cell disruption, the cell disruption solution was centrifuged at 4,500 rpm and 4° C. for 20 minutes to recover the supernatant. The obtained supernatant was filtered using a 0.2 μm filter, and then purified through a conventional cation-exchange chromatography purification process. A brief description of the purification process is as follows. As a cation-exchange resin, 5 mL of HiTrap ^TM SP FF (GE Healthcare) was used. Chromatography was performed after equilibrating the column with Buffer A (20 mM K ₃ PO ₄ , pH 6.0) in advance, and after dropping the sample onto the column, Buffer A was added to 10 CV at a flow rate of 5 mL/min. (Column volume) was flushed and washed. After washing, chromatography was performed under conditions such that the gradient from Buffer A to Buffer B (20 mM K ₃ PO ₄ , 1 M NaCl, pH 6.0) is 0% to 100% at a flow rate of 5 mL/min. Performed. In this process, elution of the antimicrobial protein CPEL-1 having the amino acid sequence represented by the desired SEQ ID NO: 2 was achieved. The results of analyzing the purified antimicrobial protein CPEL-1 through electrophoresis are shown in FIG. 2.

Fractions containing a high concentration of the antimicrobial protein CPEL-1 in the obtained purified fractions (purified fractions corresponding to lanes 4 to 10 in FIG. 1) were collected, and this was obtained in a buffer solution (50 mM Tris-HCl, pH 7.5). Dialysis was carried out for medium exchange. Through this, a solution of the antimicrobial protein CPEL-1 having a purity of 90% or more could be obtained.

Example 4: Drip Through experiment Antibacterial protein CPEL Investigation of antimicrobial activity of -1

The present inventors investigated the antibacterial activity of the antimicrobial protein CPEL-1 through a usual drop experiment. In the experiment, Clostridium perfringens 2 weeks, Enterococcus faecalis 2 weeks, Staphylococcus aureus) 2 weeks, Salmonella (Salmonella) were enrolled in two weeks, and E. coli two weeks. The bacteria were pre-sale from external organizations such as The American Type Culture Collection (ATCC) in the United States, or were isolated and identified by the present inventors.

In the experimental method, 2 mL of each bacterial culture solution having an absorbance of about 1 at 600 nm in TSA medium was plated on a different plate medium, and then dried. In the case of Clostridium perfringens, the experimental bacterial culture was obtained by performing midnight static culture in an anaerobic incubator at 37°C, and other strains were obtained by performing midnight shaking culture in an incubator at 37°C. After confirming that the bacteria were grown on the plate medium, 10 μL of the antibacterial protein CPEL-1 solution (concentration: 0.5, 1.0, 1.5 μM) was added dropwise. As a negative control, a buffer solution (50 mM Tris-HCl, pH 7.5) containing no CPEL-1 was dropped. After instillation, the culture was further cultured for about 1 hour, and the degree of lysis of each bacteria was observed. As a result, the antimicrobial protein CPEL-1 had antimicrobial activity (lytic activity) only against Clostridium perfringens and no antibacterial activity against other strains. Antimicrobial activity against Clostridium perfringens was confirmed for all two weeks of Clostridium perfringens, which were the subjects in the experiment. Figure 2 shows the results of the antimicrobial activity of the antimicrobial protein CPEL-1 against Clostridium perfringens.

From this, the antimicrobial protein CPEL-1 can provide excellent lytic power (antibacterial power) against Clostridium perfringens, and can also be effectively used for the prevention or treatment of infectious diseases caused by Clostridium perfringens. It could be confirmed that there is.

Example 5: Through the turbidity reduction investigation method Antibacterial protein CPEL Investigation of antimicrobial activity of -1

The antimicrobial activity of the antimicrobial protein CPEL-1 was investigated by using the antibacterial protein CPEL-1 solution through a turbidity reduction assay. The bacteria to be tested were the same as in Example 4.

The experimental method of the turbidity reduction investigation method was as follows. After suspending the bacteria to be tested in physiological saline so that the absorbance at 600 nm is about 0.5, add the antibacterial protein CPEL-1 solution to 2.48 μM, 0.248 μM, and 0.0248 μM to 270 μL of this suspension, and then at 600 nm. The absorbance was measured for 30 minutes. As a negative control, a buffer solution (50 mM Tris-HCl, pH 7.5) that did not contain the antimicrobial protein CPEL-1 was used.

As a result of the experiment, the antibacterial protein CPEL-1 showed lytic activity only against Clostridium perfringens, but did not have lytic activity against other test bacteria. The experimental results for Clostridium perfringens bacteria are presented in FIG. 3. In the investigation of the antimicrobial activity of the antibacterial protein CPEL-1 through the turbidity reduction method, it was confirmed that the antimicrobial activity of the antimicrobial protein CPEL-1 was very fast. This rapid antimicrobial activity can be said to be a characteristic that no existing antibiotics have been able to provide.

Example 6: Antibacterial protein CPEL -1 of Clostridium Perfringens For infection prevention Application example

In one tube containing 9 mL of nutrient broth (3 g/L of beef extract, 5 g/L of peptone), 100 μL of about 1 mg/mL of the antibacterial protein CPEL-1 solution was added. Instead of the antibacterial protein CPEL-1 solution, 100 μL of nutrient medium was additionally added to a tube containing 9 mL of the same composition. Finally, each Clostridium perfringens culture solution was added so that the absorbance at 600 nm was about 0.5, and then transferred to an anaerobic incubator at 37° C. and stationary cultured to observe the growth state of Clostridium perfringens. As can be seen from the results of Table 1, in the tube to which the antibacterial protein CPEL-1 solution was not added, Clostridium perfringens grew very well so that the absorbance at 600 nm became about 1.3 after 60 minutes. In the tube to which the antibacterial protein CPEL-1 solution was added, it was observed that the absorbance at 600 nm gradually decreased to about 0.1 level after 10 minutes and to 0.05 level after 60 minutes.

division	0 hours incubation	10 minutes after incubation	60 minutes after incubation
Control (no treatment)	0.5	0.7	1.3
Experimental group (antibacterial protein CPEL-1 solution added)	0.5	0.1	0.05

From this result, it was confirmed that the antimicrobial protein CPEL-1 of the present invention has the ability to inhibit the growth of Clostridium perfringens as well as kill it, and from this, the antimicrobial protein CPEL-1 is Clostridium perfringens. It could be concluded that it could be used as an active ingredient in the composition for the purpose of preventing infection.

Example 7: Antibacterial protein CPEL -1 of Clostridium Perfringens Therapeutic effect on infection Research

The therapeutic effect of the antimicrobial protein CPEL-1 on Clostridium perfringens infection was investigated using an infected animal model.

Specifically, a 5-week-old BALB/c mouse [specific pathogen-free (SPF) grade] of about 20 g of body weight was used as an experimental animal. After separating a total of 24 animals into 2 groups (12 animals per group), induce a wound on the skin epidermis by a tape-stripping method, and Clostridium perfringens bacteria 1×10 ⁸ cfu (i.e., 1×10) on the wound site. ⁸ cfu/mouse) was applied to induce infection. For one group (treatment group), 0.1 mL of an antibacterial protein CPEL-1 solution (10 mg/mL) was treated twice a day for 5 days at 24 hours after the forced infection. For another group (control group), only Phosphate buffered saline was treated in the same volume. The buffer solution was administered at the same interval from the time point 24 hours after the forced infection of the bacteria, similar to the administration of the antibacterial protein CPEL-1 solution. The degree of wound recovery was confirmed by observing the wound site on days D0, D4, D7, and D14 after the forced infection of the fungus, and the number of colonies was investigated by extracting the tissues of the infected site in the same individual, and the antimicrobial protein CPEL- The effectiveness of 1 was investigated.

The degree of wound recovery was conducted by measuring the clinical score (Clinical Scores; 0, no symptoms; 1, mild; 2, intermediate; 3, severe) according to the severity, and the number of colonies in the tissue sample was 0.1. After adding g of tissue sample to physiological saline and crushing, the sample was spread on a Clostridium perfringens selection medium (TSC agar plate; OXOID) and incubated for 18-24 hours at 37℃ under anaerobic conditions. Then, among the colonies formed, colonies presumed to be Clostridium perfringens bacteria were selected, and the selected colonies were each sampled to perform a Clostridium perfringens bacteria specific polymerase chain reaction (PCR). It was carried out in a manner that finally confirms whether the colony is Clostridium perfringens bacteria.

The results are shown in Tables 2 and 3, and administration of the antimicrobial protein CPEL-1 provided a distinct effect in healing wounds at the infected site and reducing the number of Clostridium perfringens bacteria remaining in the infected tissue.

group	Clinical Scores (average)
	D0	D4	D7	D14
Control	0.75	2.5	2.75	2.5
Treatment group	One	0.75	0.5	0

group	Number of colonies of Clostridium perfringens detected per plate medium (average)
	D0	D4	D7	D14
Control	4.75	5.25	5.5	5.75
Treatment group	5	0.75	0	0

As a result of the above, it can be confirmed that the antimicrobial protein CPEL-1 of the present invention is effective in treating an infection of Clostridium perfringens bacteria. These characteristics show that a pharmaceutical composition containing the antimicrobial protein CPEL-1 as an active ingredient can be used for the purpose of treating Clostridium perfringens infection, and also for the purpose of treating infection of Clostridium perfringens bacteria. It shows that it can be used in the same way as conventional antibiotics.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

[Accession number]

Depositary Institution Name: KCTC

Accession number: KCTC 14003BP

Consignment Date: 20191023

Claims (8)

1. Clostridium perfringens (Clostridium perfringens ) having lytic power, antimicrobial protein CPEL-1 represented by the amino acid sequence of SEQ ID NO: 2.
2. A method for producing the antimicrobial protein CPEL-1 of claim 1 using a production strain produced by transforming using an expression plasmid into which the gene represented by SEQ ID NO: 1 has been introduced.
3. A pharmaceutical composition for preventing or treating diseases caused by Clostridium perfringens , comprising the antimicrobial protein CPEL-1 of claim 1 as an active ingredient.
4. The pharmaceutical composition for preventing or treating diseases caused by Clostridium perfringens according to claim 3, wherein the pharmaceutical composition is used in the form of a pharmaceutical composition including antibiotics. .
5. The method of any one of claims 3 and 4, wherein the disease is food poisoning, necrotizing enteritis, gas gangrene, dysentery, enterotoxemia, Seen leukomalacia (Pulpy kidney disease) and dermatitis (dermatitis), characterized in that one selected from a group consisting of Clostridium perfringens (Clostridium perfringens )-induced disease prevention or treatment pharmaceutical composition
6. Claim 3 and claim 4, wherein any one of the antimicrobial protein CPEL-1 for containing a pharmaceutical composition comprising, as an active ingredient, the step of administering a treatment subject non-human, Clostridium perfringens (Clostridium according to one wherein perfringens ) to prevent or treat diseases caused by.
7. 7. The method of claim 6, for preventing or treating diseases caused by, Clostridium perfringens (Clostridium perfringens), characterized in that the said pharmaceutical composition is administered to the treatment target by the pharmaceutical composition form, including antibiotics .
8. The method of claim 6 and 7, wherein the disease is food poisoning, necrotizing enteritis, gas gangrene, dysentery, enterotoxemia, and nephropathy kidney disease) and dermatitis (Dermatitis), characterized in that any one selected from the group consisting of, Clostridium perfringens (Clostridium perfringens) a method for preventing or treating a disease caused by.

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