WO2021241915A1 - Antibacterial substance having lytic activity against actinobacillus pleuropneumoniae and method for producing same - Google Patents

Abstract

The present invention relates to: a proteinaceous antibacterial substance having lytic activity against Actinobacillus pleuropneumoniae, which infects the respiratory system; and a method for producing same. More specifically, the present invention relates to: a proteinaceous antibacterial substance specific to Actinobacillus pleuropneumoniae, the proteinaceous antibacterial substance exhibiting an ability that can specifically lyse Actinobacillus pleuropneumoniae, which infects the porcine respiratory system to cause pleuropneumonia in lung tissue, and being derived from a nucleic acid sequence represented by SEQ ID NO:1; a strain which produces the proteinaceous antibacterial substance and has genetic material encoding the proteinaceous antibacterial substance; a method for producing the proteinaceous antibacterial substance by using the strain; and a pharmaceutical composition for treating respiratory infections and respiratory diseases caused by Actinobacillus pleuropneumoniae, the pharmaceutical composition containing the proteinaceous antibacterial substance as an active ingredient.

Description

Antibacterial substance having lytic activity against Actinobacillus pluropneumoniae, and method for manufacturing the same

The present invention relates to a proteinaceous antibacterial material having lytic activity against the respiratory infection bacterium, Actinobacillus pleuropneumoniae , and a method for manufacturing the same, and more particularly, to a porcine respiratory tract infecting the lung tissue. Actinobacillus pneumoniae, characterized in that it is derived from the nucleic acid sequence represented by SEQ ID NO: 1, and exhibits the ability to specifically lyse the bacteria Actinobacillus pneumoniae that can cause pleural pneumonia. A specific proteinaceous antibacterial material, a strain producing the proteinaceous antibacterial material, characterized in that it has a genetic material encoding the proteinaceous antibacterial material, and the production of the antibacterial material for producing the proteinaceous antibacterial material using the same Method, It relates to a pharmaceutical composition for the treatment of respiratory infections and respiratory diseases caused by Actinobacillus pluropneumoniae bacteria comprising the proteinaceous antibacterial material as an active ingredient.

Swine pleural pneumonia is a worldwide disease, and in Korea, it ranks among the fifth among more than 14 types of bacterial diseases of pigs in 2009 with a high incidence rate. Pig pleural pneumonia occurs in pigs of all ages and tends to occur frequently during the changing seasons. The disease is transmitted mainly through airborne infection, but also through direct contact. The economic loss due to an increase in mortality due to pleural pneumonia in pigs, a decrease in feed efficiency due to the chronic type, and growth delay, etc. is enormous. In particular, pleural pneumonia of pigs is a highly infectious bacterial respiratory disease, and it is a disease with high economic loss accounting for 44.2% of respiratory diseases. Recently, immune-decreasing viral diseases such as swine Ozeski's disease and porcine reproductive and respiratory syndrome (PRRS) are prevalent and the incidence of pleural pneumonia in pigs is continuously increasing as population breeding increases. Swine pleural pneumonia is highly contagious and develops explosively within a short period of time, causing mass death of pigs. In particular, it is a disease that causes enormous economic damage to the pig industry due to a decrease in growth rate and delay in shipment, even if it is chronic, because it often progresses to an acute form in the early stages of infection.

As a major causative agent of porcine pleural pneumonia, Actinobacillus pleuropneumoniae is known. The pathogenic factor of Actinobacillus plur pneumoniae is capsular polysaccharide, lipopolysaccharide, hemolysin, cytotoxin, protein protease, outer cell membrane protein, etc. are known, and the serotype of Actinobacillus pneumoniae is 1 Types 12 through 12 have been identified, and even type 16 is being discussed. In domestic pig farms, pleural pneumonia caused by types 2 and 5 is the most common. Regarding the import of pigs, there have been reports of pleural pneumonia caused by serotypes 1, 3, 4, 6, 7, 10, and 12 in Korea, so it is urgent to establish countermeasures. Therefore, it can be said that there is an urgent need to develop a drug that can be used to effectively treat pleural pneumonia in pigs.

Antibiotics are widely used to treat these infections of Actinobacillus pluropneumoniae, but the treatment efficiency is continuously falling due to the recent increase in antibiotic-resistant bacteria. The development of new antibiotic/antibacterial substances is required to effectively cope with the infection with Actinobacillus pluropneumoniae, which has acquired resistance to these existing antibiotics. In particular, there is an urgent need to develop a pharmaceutical formulation that can provide a rapid therapeutic effect.

On the other hand, bacteriocin (Bacteriocin) is a natural antibacterial material produced by a number of microorganisms, and is mainly divided into protein or peptide series. Most of the bacteriocins have a bactericidal action, and in general, the bacteriocins are not toxic to the human body and do not remain in the body. Pyocin is a kind of bacteriocin, and Pseudomonas strains, especially Pseudomonas aeruginosa ) is a proteinaceous antibacterial substance produced by Pyocin can be generally classified into three types (R-type, F-type, S-type) based on the shape, and R-type is a bacteriophage belonging to the morphologically Myoviridae family. It has a structure similar to that of the tail, and the F-type has a structure similar to that of a bacteriophage belonging to the family Siphoviridae. R-type and F-type exhibit antibacterial activity by forming pores in the membrane of the target strain. On the other hand, S-type is known as a low molecular weight protein that is water-soluble and not a phage-like particle. R-type and F-type have resistance to nuclease and protease, and heat, but S-type has no resistance to protease and heat. Such phyosin can be used as a countermeasure against bacterial infectious diseases.

Accordingly, in order to solve the problem of the existing antibiotics for the treatment of infections caused by the harmful pathogenic bacteria, Actinobacillus pluropneumoniae, the present invention is a harmful pathogenic bacterium, Actinobacillus pneumoniae. Provided are a proteinaceous antibacterial material that can be specifically lysed, a production strain of the proteinaceous antibacterial material, and a method for producing a proteinaceous antibacterial material using the same, comprising the proteinaceous antibacterial material as an active ingredient and using Actinobacillus flu To provide a pharmaceutical composition that can be utilized to treat the infection of pneumoniae, and to provide a method for treating the infection of the bacteria Actinobacillus pneumoniae using this pharmaceutical composition.

Accordingly, an object of the present invention is a proteinaceous antibacterial substance derived from the nucleic acid sequence represented by SEQ ID NO: 1 having the ability to specifically lyse Actinobacillus pluropneumoniae, a respiratory infection bacterium. is to provide

Another object of the present invention is to use the production strain INT-PA05-01 (Accession No. KCTC 14010BP) or the production strain INT-PA05-02 (Accession No. KCTC 14011BP) having a genome comprising the nucleic acid sequence shown in SEQ ID NO: 1 To provide a method for efficiently producing a proteinaceous antibacterial material derived from a nucleic acid sequence represented by SEQ ID NO: 1 that can specifically lyse Actinobacillus pneumoniae pneumoniae, a respiratory infection bacterium.

Another object of the present invention is a solution containing as an active ingredient a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 that can specifically lyse Actinobacillus pluropneumoniae, a respiratory infection bacterium. To provide a pharmaceutical composition for the treatment of Tinobacillus pneumoniae infection and related diseases.

In addition, another object of the present invention is to use a pharmaceutical composition comprising a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 as an active ingredient to infection and related To provide a method for treating a disease.

Therefore, according to one aspect of the present invention, the present invention is derived from the nucleic acid sequence represented by SEQ ID NO: 1 that can specifically lyse Actinobacillus pluropneumoniae, which infects the respiratory tract and causes respiratory diseases. Provides proteinaceous antibacterial substances.

The proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 may be F-type or R-type phyosin, preferably R-type phyosin.

The serotype of the Actinobacillus pneumoniae bacteria may be any one selected from the group consisting of 1, 2, 5 or 11, but is not limited thereto.

It is apparent that the nucleic acid sequence of SEQ ID NO: 1 may be partially modified by natural mutation or artificial mutation. Such modifications include partial substitutions of nucleic acid sequences, partial additions of nucleic acid sequences, and partial deletions of nucleic acid sequences.

According to another aspect of the present invention, the present invention is proteinaceous derived from the nucleic acid sequence represented by SEQ ID NO: 1 that can specifically lyse Actinobacillus p. pneumoniae causing respiratory diseases by infecting the respiratory tract. It provides a method for producing a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 using the production strain of the antibacterial material. The method for producing a proteinaceous antibacterial material derived from the nucleic acid sequence shown in SEQ ID NO: 1 is carried out using the production strain INT-PA05-01 (Accession No. KCTC 14010BP) or the production strain INT-PA05-02 (Accession No. KCTC 14011BP) do.

The production strains of the proteinaceous antimicrobial substance derived from the nucleic acid sequence shown in SEQ ID NO: 1 have a genome comprising the nucleic acid sequence shown in SEQ ID NO: 1 encoding the proteinaceous antimicrobial substance. .

In addition, the nucleic acid sequence represented by SEQ ID NO: 1 encoding the proteinaceous antibacterial material has the same genetic characteristics as shown in FIG. 1 .

The production strain INT-PA05-01 was deposited with the Korea Institute of Bioscience and Biotechnology Biological Resources Center on October 29, 2019 (accession number KCTC 14010BP), and the production strain INT-PA05-02 was also deposited on October 29, 2019, as of October 29, 2019 in Korea Biotechnology It was deposited with the Research Center for Biological Resources (Accession No. KCTC 14011BP).

Specifically, (A) culturing a strain producing a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1; And (B) inducing the production of a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 to prepare a suspension of a proteinaceous antibacterial material containing a proteinaceous antibacterial material; including; Provided is a method for preparing a proteinaceous antibacterial material for the treatment of infections and respiratory diseases. _{When the OD 600} value of the culture medium reaches the level of 0.1 to 0.4, the final concentration is 2 to 4 μg/ml It can be carried out by adding MMC (Mitomycin C) as much as possible and then culturing with shaking at 37°C for 3 to 4 hours.

In addition, according to another aspect of the present invention, the present invention has a specific lytic ability against the respiratory-infecting bacteria, Actinobacillus pneumoniae, and proteinaceous antibacterial derived from the nucleic acid sequence represented by SEQ ID NO: 1 Provided is a pharmaceutical composition that can be effectively used for the treatment of respiratory bacterial infections comprising a substance as an active ingredient.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

Antibacterial substances capable of providing antibacterial activity against other respiratory infection strains may be added to the pharmaceutical composition of the present invention in order to increase the effectiveness for this purpose of application.

Since the proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 according to the present invention, which is included in the pharmaceutical composition of the present invention, specifically lyses Actinobacillus pneumoniae bacteria as described above, It shows an effect in the treatment of various respiratory diseases caused by the bacteria Actinobacillus pluropneumoniae. Therefore, the pharmaceutical composition of the present invention can be utilized for the treatment of diseases caused by Actinobacillus pluropneumoniae bacteria.

In addition, the pharmaceutical composition of the present invention can be implemented as an antibiotic, disinfectant, sterilizer, therapeutic agent, etc., and can be utilized for the treatment of various diseases caused by Actinobacillus pneumoniae bacteria.

In the present invention, “disease caused by the bacteria Actinobacillus pluropneumoniae” means stunted growth, reduced feed efficiency, lethargy, anorexia, fever, cough, Diseases that accompany symptoms such as respiratory problems are collectively referred to.

In the present specification, Actinobacillus pneumoniae bacteria does not matter whether it is sensitive to existing antibiotics or resistant bacteria with resistance to existing antibiotics. That is, it does not matter whether resistance to existing antibiotics is acquired.

The present invention treats diseases caused by the bacteria Actinobacillus pluropneumoniae by administering a pharmaceutical composition comprising a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 as an active ingredient to animals other than humans. method of treatment is provided.

The pharmaceutical composition of the present invention may be administered through oral administration or parenteral administration, and in the case of parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, intranasal administration or local administration may be used. may be, but is not limited thereto.

The pharmaceutical composition of the present invention is prepared in a unit dosage form by being formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. Alternatively, it may be prepared by introducing it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

In addition, suitable application, spraying and dosage of the pharmaceutical composition depend on factors such as formulation method, administration method, age, body weight, sex, degree of disease symptoms, food, administration time, administration route, excretion rate, and reaction sensitivity. , and an ordinarily skilled physician or veterinarian can readily determine and prescribe an effective dosage for the desired treatment.

As used herein, the term "treatment" or "treatment" refers to the suppression of diseases caused by the bacteria Actinobacillus p. means any action that reduces

A proteinaceous antibacterial substance derived from the nucleic acid sequence represented by SEQ ID NO: 1 according to the present invention and a pharmaceutical composition comprising the same as an active ingredient do not affect other normal flora in the body, and the target strain, Actinobacillus pluroni Because it only works against Monaebacteria, the side effects associated with its use are remarkably small compared to treatments based on existing antibiotics. On the other hand, in the case of existing antibiotics, they have a disadvantage of causing adverse effects on beneficial bacteria in the body, causing various side effects. In addition, since the proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 according to the present invention and a pharmaceutical composition comprising the same as an active ingredient still work on strains that have acquired resistance to existing antibiotics, it is also used in the treatment of antibiotic-resistant bacterial infections. can be used effectively. In addition, according to the method for producing a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 of the present invention, it is possible to efficiently produce a high-purity proteinaceous antibacterial material.

1 is a gene analysis result for the nucleic acid sequence represented by SEQ ID NO: 1 of the present invention.

Figure 2 is a result showing the colony formation mode of the production strain of the proteinaceous antibacterial substance derived from the nucleic acid sequence represented by SEQ ID NO: 1 according to the present invention. (A) is a colony of the producing strain INT-PA05-01 (Accession No. KCTC 14010BP), and (B) is a colony of the producing strain INT-PA05-02 (Accession No. KCTC 14011BP).

Figure 3 is a proteinaceous antibacterial substance derived from the nucleic acid sequence represented by SEQ ID NO: 1, prepared using the production strain INT-PA05-01 (Accession No. KCTC 14010BP) according to the present invention, Actinobacillus pluropneumoniae Results showing antibacterial activity against bacteria. Sample 1 is a case in which a buffer solution is instilled, and samples 2 to 5 are a case in which a solution containing a proteinaceous antibacterial material is instilled. Among them, samples 4 and 5 are cases in which a sample of a proteinaceous antibacterial material subjected to heat treatment (60° C., performed for 10 minutes) is instilled. In the case of dripping samples 2 to 5, the transparent part is a transparent ring formed by lysing the test target bacteria with a proteinaceous antibacterial material.

Figure 4 is a proteinaceous antibacterial substance derived from the nucleic acid sequence shown in SEQ ID NO: 1, prepared using the production strain INT-PA05-02 (Accession No. KCTC 14011BP) according to the present invention, Actinobacillus pluropneumoniae Results showing antibacterial activity against bacteria. Sample 1 is a case in which a buffer solution is instilled, and samples 2 to 5 are a case in which a solution containing a proteinaceous antibacterial material is instilled. Among them, samples 4 and 5 are cases in which a sample of a proteinaceous antibacterial material subjected to heat treatment (60° C., performed for 10 minutes) is instilled. In the case of dripping samples 2 to 5, the transparent part is a transparent ring formed by lysing the test target bacteria with a proteinaceous antibacterial material.

5 is an electron micrograph of a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1, prepared using the production strain INT-PA05-01 (Accession No. KCTC 14010BP) according to the present invention.

6 is an electron micrograph of a proteinaceous antibacterial substance derived from the nucleic acid sequence shown in SEQ ID NO: 1, prepared using the production strain INT-PA05-02 (Accession No. KCTC 14011BP) according to the present invention.

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

Example 1: Preparation of proteinaceous antibacterial material

The production of a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 is as follows using the production strain INT-PA05-01 (Accession No. KCTC 14010BP) or the production strain INT-PA05-02 (Accession No. KCTC 14011BP) carried out together. The producing strain INT-PA05-01 (Accession No. KCTC 14010BP) and the producing strain INT-PA05-02 (Accession No. KCTC 14011BP) are both microbiologically Pseudomonas ( Pseudomonas). aeruginosa ), and there was a difference in the colony formation pattern as shown in FIG. 2 . Spread on plate medium (casein digest, 15 g/L; soy bean digest, 5 g/L; NaCl, 5 g/L; agar, 15 g/L) and incubate overnight at 37°C, producing strain INT -PA05-01 (Accession No. KCTC 14010BP) formed white colonies, and the production strain INT-PA05-02 (Accession No. KCTC 14011BP) formed brown colored colonies, and there was a difference in appearance (FIG. 2).

The production of a proteinaceous antibacterial substance derived from the nucleic acid sequence shown in SEQ ID NO: 1 using the production strain INT-PA05-01 (Accession No. KCTC 14010BP) was carried out as follows. TSB (Tryptic Soy Broth) medium (casein digest, 17 g/L; soy bean digest, 3 g/L; dextrose, 2.5 g/ L; NaCl, 5 g / L; dipotassium phosphate, 2.5 g / L) was inoculated, followed by shaking culture at 37 ° C overnight. After overnight incubation, the culture medium (sodium glutamate, 20 g/L; glucose, 5 g/L; sodium hydrogen phosphate, 2.23 g/L; magnesium sulfate, 0.1 g/L; potassium dihydrogen phosphate, 0.25 g/L) ; Yeast extract, 0.5 g/L) was inoculated at a ratio of 1/100, and main culture was performed. When the OD ₆₀₀ value of the culture medium reached the level of 0.2 to 0.3, MMC (Mitomycin C) was added so that the final concentration was 3 μg/ml, and then cultured with shaking at 37° C. for 3 to 4 hours to produce proteinaceous antibacterial substances. was induced (Induction). The solution obtained in this way is referred to as a suspension of proteinaceous antibacterial substances. In the next step, in order to remove the DNA derived from the production strain that may be contained in the suspension of the proteinaceous antibacterial substance, 0.25 μl of DNase I solution is added to 50 ml of the suspension of the proteinaceous antibacterial substance, and then at room temperature for 30 minutes reacted while Then, it was centrifuged at 4,500 rpm for 30 minutes. After centrifugation was completed, the supernatant was recovered and then filtration was performed using a 0.2 μm syringe filter (Sartorius). After filtration, dialysis was performed overnight using a TN50 buffer (50 mM NaCl, 10 mM Tris-Cl, pH 7.5) to prepare a proteinaceous antibacterial material sample.

The production of a proteinaceous antibacterial substance derived from the nucleic acid sequence shown in SEQ ID NO: 1 using the production strain INT-PA05-02 (Accession No. KCTC 14011BP) was carried out as follows. The production strain INT-PA05-02 (Accession No. KCTC 14011BP) was mixed with TSB (Tryptic Soy Broth) medium (casein digest, 17 g/L; soy bean digest, 3 g/L; dextrose, 2.5 g/L) at a ratio of 1/500. L; NaCl, 5 g / L; dipotassium phosphate, 2.5 g / L) was inoculated, and shaking culture was performed overnight at 37 ° C. After overnight incubation, the culture medium was transferred to G medium (sodium glutamate, 20 g/L; glucose, 5 g/L; sodium hydrogen phosphate, 2.23 g/L; magnesium sulfate, 0.1 g/L; potassium dihydrogen phosphate, 0.25 g/L). ; Yeast extract, 0.5 g/L) was inoculated at a ratio of 1/100 and the main culture was performed. OD of the culture medium ₆₀₀When the value reached the level of 0.2-0.3, MMC was added so that the final concentration was 3 μg/ml, and then cultured with shaking at 37°C for 3-4 hours to induce the production of proteinaceous antibacterial substances. The solution obtained in this way is referred to as a suspension of proteinaceous antibacterial substances. As a next step, in order to remove the DNA derived from the production strain that may be contained in the suspension of the proteinaceous antibacterial substance, 2.5 μl of DNase I solution is added to 500 ml of the suspension of the proteinaceous antibacterial substance, and then at room temperature for 30 minutes reacted while It was then centrifuged at 9,800 rpm for 60 minutes. After centrifugation was completed, the supernatant was recovered and then filtered using a 0.2 μm syringe filter (Sartorius). To 400 ml of the filtered sample, PEG (polyethylene glycol) 8,000 and NaCl were added to final concentrations of 10% and 1 M, respectively, and then mixed well and left at 4°C for overnight. After standing overnight, centrifugation was performed at 8,000 rpm for 60 minutes. After centrifugation, the supernatant was discarded, and the precipitate was suspended using 4 ml of EB buffer (50 mM Tris-Cl, pH 7.0). For the suspended sample, CsCl ₂ Density gradient (1.70, 1.50, 1.45 g/cm ³) was subjected to ultracentrifugation (30,000 rpm, 5 hours). After recovering the band formed after ultracentrifugation, dialysis was performed overnight using TN50 buffer (50 mM NaCl, 10 mM Tris-Cl, pH 7.5) to prepare a proteinaceous antibacterial material sample.

Example 2: Investigation of antibacterial activity of proteinaceous antibacterial substances

The antibacterial activity of the proteinaceous antibacterial material prepared according to Example 1 against Actinobacillus pluropneumoniae was investigated. The antibacterial activity was investigated in a manner of examining whether the transparent ring was generated by lysis through a conventional drip test (Spot assay). The Actinobacillus pneumoniae strains used for the investigation of antibacterial activity were isolated by the present inventors and identified as Actinobacillus pneumoniae bacteria, and the serotypes were different from each other for a total of 10 weeks.

The drip experiment was conducted as follows. In TSBY medium (TSB broth, 30 g/L; yeast extract, 0.6%; nicotinamide adenine dinucleotide, 10 μg/mL) at a ratio of 1/1000, Actinobacillus p. pneumoniae was inoculated at 37℃ and Incubated overnight in 5% CO _{2 condition.} BHI-NAD plate medium (BHI-NAD infusion broth, 37 g/L; nicotinamide adenine dinucleotide, 10 μg/mL; agar; 15 g/L) and left to dry. After drying, 37° C., 5% CO ₂ Stationary culture was performed in an incubator for 7 hours. After confirming that Actinobacillus p. pneumoniae was grown on the BHI-NAD plate medium, 10 μl of the proteinaceous antibacterial material sample prepared in Example 1 was instilled. As a negative control, TN50 buffer was instilled. After drying for about 10 minutes on a clean bench, 37° C., 5% CO _{2 After} incubation for 1 hour in an incubator, it was investigated whether a transparent ring was formed at the location where the protein antibacterial material sample was dripped.

The results for the proteinaceous antibacterial material derived from the production strain INT-PA05-01 (Accession No. KCTC 14010BP) are shown in Table 1, and the results for the proteinaceous antibacterial material derived from the producing strain INT-PA05-02 (Accession No. KCTC 14011BP) are shown in Table 1. Table 2 shows.

division	Test strain (serotype)
	Strain 1 (Type 1)	Strain 2 (Type 2)	Strain 3 (Type 3)	Strain 4 (Type 4)	Strain 5 (Type 5)	Strain 6 (Type 7)	Strain 7 (Type 8)	Strain 8 (type 9)	Strain 9 (type 10)	Strain 10 (type 11)
control	-	-	-	-	-	-	-	-	-	-
treatment group	+	+	-	-	+	-	-	-	-	+
+: lytic activity (with antibacterial activity), -: no lytic activity (without antibacterial activity)

division	Test strain (serotype)
	Strain 1 (Type 1)	Strain 2 (Type 2)	Strain 3 (Type 3)	Strain 4 (Type 4)	Strain 5 (Type 5)	Strain 6 (Type 7)	Strain 7 (Type 8)	Strain 8 (type 9)	Strain 9 (type 10)	Strain 10 (type 11)
control	-	-	-	-	-	-	-	-	-	-
treatment group	+	+	-	-	+	-	-	-	-	+
+: lytic activity (with antibacterial activity), -: no lytic activity (without antibacterial activity)

As can be seen from the above results, the proteinaceous antibacterial material derived from the production strain INT-PA05-01 (Accession No. KCTC 14010BP) exhibited antibacterial activity against the Actinobacillus p. pneumoniae strains of serotypes 1, 2, 5 and 11. and proteinaceous antibacterial material derived from the production strain INT-PA05-02 (Accession No. KCTC 14011BP) also exhibited antibacterial activity against the Actinobacillus pluropneumoniae strains of serotypes 1, 2, 5 and 11. Representative experimental results for proteinaceous antibacterial substances derived from the production strain INT-PA05-01 (Accession No. KCTC 14010BP) are presented in FIG. 3 (experimental results for Actinobacillus pneumoniae serotype 2 strain), Representative experimental results for proteinaceous antibacterial substances derived from strain INT-PA05-02 (Accession No. KCTC 14011BP) are presented in FIG. 4 (experimental results for Actinobacillus plur pneumoniae serotype 5 strain). As described above, among the three phyosin types, S-type phyosin is known to lose its antibacterial activity when heat-treated. Antibacterial activity was also shown when phyosin, a proteinaceous antibacterial material secured in the present invention, was heat-treated (at 60° C. for 10 minutes), which indicates that the phyosin of the present invention is F-type or R-type phyosin. result that proves

On the other hand, the antibacterial activity of the proteinaceous antibacterial material derived from the production strain INT-PA05-01 (Accession No. KCTC 14010BP) and the proteinaceous antibacterial material derived from the production strain INT-PA05-02 (Accession No. KCTC 14011BP) of the present invention was investigated by Streptococcus pneumoniae ( Streptococcus pneumoniae ), Salmonella Typhimurium, and Escherichia coli ) was also carried out through the drip experiment described above, as a result, the proteinaceous antibacterial substances of the present invention did not have the killing ability against the above-mentioned strains.

As a result of the above results, it was confirmed that the proteinaceous antibacterial substances of the present invention had excellent killing ability against Actinobacillus pluropneumoniae bacteria. This shows that the proteinaceous antibacterial substances of the present invention can be utilized as active ingredients of the composition for the purpose of treatment for diseases caused by Actinobacillus pneumoniae.

Example 3: Investigation of morphological characteristics of proteinaceous antibacterial substances

The form of the proteinaceous antibacterial material was investigated through electron microscopic analysis using the proteinaceous antibacterial material sample obtained in Example 1. Electron microscopic analysis was performed according to a conventional method. A brief explanation of this is as follows. The proteinaceous antibacterial material sample was buried on a copper grid, and after negative staining and drying with 2% uranyl acetate, the shape was observed through a transmission electron microscope.

The results for the proteinaceous antibacterial material derived from the production strain INT-PA05-01 (Accession No. KCTC 14010BP) are shown in FIG. 5, and the results for the proteinaceous antibacterial material derived from the producing strain INT-PA05-02 (Accession No. KCTC 14011BP) is presented in FIG. 6 . Judging from the morphological characteristics, it was confirmed that the secured proteinaceous antibacterial substances were R-type phyosin.

Example 4: Genome analysis and core gene analysis encoding proteinaceous antibacterial substances

A genome map was prepared by performing genome analysis using the known sequence information (Genbank Accession No. NC_002516.2) of the P. aeruginosa PAO1 strain, which is a test strain. Based on the above-described genome analysis results, a typical polymerase chain reaction (PCR) was performed in the genomes of the production strain INT-PA05-01 (Accession No. KCTC 14010BP) and the production strain INT-PA05-02 (Accession No. KCTC 14011BP). After performing the sequencing of the obtained PCR product, the nucleic acid sequence of the part encoding the information on the proteinaceous antibacterial substance in the genome was obtained. Sequences obtained from each production strain were identical to each other, and are presented in SEQ ID NO: 1. A genome map was prepared by classifying the sequence shown in SEQ ID NO: 1 by function through genome annotation (FIG. 1).

Example 5: actinobacillus Pneumoniae fungal infection treatment example

The therapeutic effect of a composition containing a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 as an active ingredient in pigs induced by a disease caused by Actinobacillus pneumoniae bacteria was investigated. After dividing 20 weaned pigs at the age of 25 days into two groups, the experiment was conducted for 21 days while separately rearing them in an experimental breeding room (1.1m × 1.0m). The surrounding environment was controlled under the thermal insulation facility, the temperature and humidity of the pig room were kept constant, and the floor of the pig room was cleaned every day. From the 8th day (D8) from the start date of the experiment, the suspension of Actinobacillus pneumoniae was sprayed around the pig's nose at a frequency of 3 times a day for 3 days. The amount of one spraying was 10 ml. The suspension of Actinobacillus pneumoniae used for spraying was prepared in the following way. Actinobacillus pneumoniae bacteria using TSBY medium (TSB broth, 30 g / L; yeast extract, 0.6%; nicotinamide adenine dinucleotide, 10 μg / mL) at 37 ° C. and 5% CO ₂ conditions overnight After stationary culture, only the cells were recovered through centrifugation, and then adjusted to 10 ⁹ CFU/ml with physiological saline (pH 7.2) to prepare a suspension of Actinobacillus pneumoniae bacteria. From the next day (D11) after the completion of the Actinobacillus pluropneumoniae spraying, 10 ml of the proteinaceous antibacterial material sample prepared in Example 1 was sprayed around the nose twice daily to the pigs of the experimental group (proteinaceous antibacterial material treatment group). In addition, 1 ml of the proteinaceous antibacterial material sample prepared in Example 1 was intravenously administered once daily. Pigs in the control group (not treated with proteinaceous antibacterial substances) did not receive any treatment after spraying Actinobacillus pneumoniae. Feed and negative water were fed equally to both control and experimental groups. The mortality rate of each group was investigated until the end of the test (D21). The results are presented in Table 3 (cumulative mortality rate).

division	Cumulative mortality (%)
	D11	D12	D13	D14	D15	D16	D17	D18	D19	D20	D21
Control group (untreated group with proteinaceous antibacterial substances)	0	10	10	20	40	60	90	100	100	100	100
Experimental group (protein-type antibacterial substance treatment group)	0	0	0	0	0	0	0	0	0	0	0

From this result, the composition containing the proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 of the present invention as an active ingredient is very effective in the treatment of infectious diseases caused by Actinobacillus pneumoniae bacteria. was able to confirm that

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

1. (A) culturing a strain INT-PA05-02 (Accession No. KCTC 14011BP) producing a proteinaceous antibacterial substance derived from the nucleic acid sequence represented by SEQ ID NO: 1;

   (B) inducing the production of a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1 to prepare a suspension of the proteinaceous antibacterial material containing the proteinaceous antibacterial material;

   (C) removing the DNA derived from the production strain in the suspension and centrifuging to recover the supernatant;

   (D) separating the band layer formed by ultracentrifuging the recovered suspension; and

   (E) recovering a proteinaceous antibacterial material by performing dialysis on the separated band layer;

   A method for producing a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1, comprising a.
2. According to claim 1, wherein the induction of the production of the proteinaceous antibacterial substance derived from the nucleic acid sequence represented by SEQ ID NO: 1 in step (B) is carried out by adding Mitomycin C so that the final concentration is 2 to 4 μg/ml A method for producing a proteinaceous antibacterial material derived from the nucleic acid sequence represented by SEQ ID NO: 1, characterized in that.
3. According to claim 1, wherein the proteinaceous antibacterial material is Actinobacillus pleuropneumoniae ( Actinobacillus pleuropneumoniae ) Bacteria-specific antibacterial activity characterized in that it exhibits, SEQ ID NO: 1 proteinaceous antibacterial derived from the nucleic acid sequence shown A method of making a substance.
4. According to claim 1, wherein the Actinobacillus pleuropneumoniae ( Actinobacillus pleuropneumoniae ) The serotype of the bacteria is characterized in that any one selected from the group consisting of type 1, type 2, type 5 and type 11, SEQ ID NO: A method for producing a proteinaceous antibacterial substance derived from the nucleic acid sequence represented by 1.
5. A proteinaceous antibacterial substance derived from the nucleic acid sequence represented by SEQ ID NO: 1.

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