WO2023113396A1 - Use of recombinant antibacterial protein for effectively killing clostridium difficile bacteria - Google Patents

Abstract

The present invention relates to use of a recombinant antibacterial protein for effectively killing Clostridium difficile bacteria and, specifically, to use of a recombinant protein (CHAP-161^LysSAP26) in which the amino acid sequences of SEQ ID Nos: 162-251 of the C-terminal region of endolysin LysSAP26 having antibacterial activity is deleted. The CHAP-161^LysSAP26 recombinant protein of the present invention exhibits an ability to kill Clostridium difficile, and thus can prevent or treat inflammatory diseases caused by the bacteria. In particular, CHAP-161^LysSAP26 uses peptidoglycan, which is a component of bacterial cell walls, as a substrate so as to exhibit bacteria-killing ability through peptidoglycan decomposition and since peptidoglycan is present only in bacteria and not in humans or animals, CHAP-161^LysSAP26 of the present invention does not affect humans and animals, and thus is safe.

Description

Use of Recombinant Antimicrobial Proteins to Effectively Kill Clostridium difficile Bacteria

The present invention relates to the use of a recombinant antibacterial protein that effectively kills Clostridium difficile bacteria, and in detail, the amino acid sequence at positions 162 to 251 of the C-terminal region of LysSAP26, an endolysin having antibacterial activity, is deleted. It relates to the use of a recombinant protein (CHAP-161 ^LysSAP26 ).

As the multi-drug antimicrobial resistance of pathogenic bacteria increases due to the misuse of antibiotics, the number of cases that cannot be treated with antibiotics increases significantly, becoming a serious public health problem.

Bacteriophage is a bacterial virus, which infects the host bacterium according to its biological life cycle, reproduces a large amount of phage, and makes proteins that break through or decompose the cell wall of the bacterium in the process of being killed by a large number of phages in the cell of the host bacterium. The proteins break down the cell wall made of peptidoglycan, disintegrating the cell wall and killing the bacteria.

Clostridium difficile is an anaerobic bacillus that forms spores as a Gram-positive bacterium. When the intestinal flora is destroyed by the use of antibiotics or drugs, it is colonized by Clostridium difficile and progresses to inflammation by secreted toxin A or B. The more secreted toxin (enterotoxin), the more severe enteritis. In the case of the United States, the incidence of intestinal diseases caused by Clostridium difficile is on the rise, and in particular, the incidence and mortality rates of the elderly over 65 years of age are rapidly increasing. Treatment of intestinal diseases caused by Clostridium difficile is oral metrinidazole or vancomycin treatment, and recently, good effects have been reported with feces transplantation in healthy people, but drug resistance and side effects due to antibiotic treatment, and stool to be transplanted Quality control is emerging as a problem.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating Clostridium difficile infectious diseases comprising CHAP-161 ^LysSAP26 recombinant protein as an active ingredient.

Another object of the present invention is to provide an antibiotic for killing Clostridium difficile containing CHAP-161 ^LysSAP26 recombinant protein as an active ingredient.

In order to achieve the above object, the present invention comprises a CHAP-161 ^LysSAP26 recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient Clostridium difficile ( Clostridium difficile ) A pharmaceutical composition for preventing or treating infectious diseases provides

In addition, the present invention provides an antibiotic for killing Clostridium difficile comprising the CHAP-161 ^LysSAP26 recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

The present invention relates to the use of a recombinant antibacterial protein that effectively kills Clostridium difficile bacteria, and in detail, the amino acid sequence at positions 162 to 251 of the C-terminal region of LysSAP26, an endolysin having antibacterial activity, is deleted. It relates to the use of a recombinant protein (CHAP-161 ^LysSAP26 ). The CHAP-161 ^LysSAP26 recombinant protein of the present invention exhibits the ability to kill Clostridium difficile and can prevent or treat infectious diseases caused by this bacterium. In particular, the CHAP-161 ^LysSAP26 uses peptidoglycan, a component of the cell wall of bacteria, as a substrate to show the ability to kill bacteria by decomposing peptidoglycan, and peptidoglycan exists only in bacteria and does not exist in humans or animals However, CHAP-161 ^LysSAP26 of the present invention is safe because it does not affect humans and animals.

Figure 1 is an SDS-PAGE analysis photograph (A) after purification of LysSAP26 and CHAP-139 ^LysSAP26 proteins, including CHAP-161 ^LysSAP26 , and Western analysis using specific antibodies recognizing the C-terminal 6 histidine amino acid sequences of each protein. to confirm CHAP-161 ^LysSAP26 , LysSAP26, and CHAP-139 ^LysSAP26 (B).

Figure 2 is CHAP-161 ^LysSAP26 , LysSAP26, and CHAP-139 ^LysSAP26 of Acinetobacter baumanii ATCC 17978 (A) and Staphylococcus aureus ATCC 25923 (B) for 20 hours antibacterial activity was determined by measuring the absorbance.

Figure 3 is a photograph of the protein precipitate in the aqueous solution after 15 days of each protein purification in relation to the protein stability of CHAP-161 ^LysSAP26 and LysSAP26.

Figure 4 is the minimum inhibitory concentration (MIC) and minimum killing concentration for 1 week and 3 weeks (PO3654, PO3780, PO3783) of Clostridium difficile standard strain (ATCC 9689) by CHAP-161 ^LysSAP26 and clinical isolates (Minimum bactericidal concentration, MBC) was confirmed as an experimental result.

5 shows a schematic diagram of an in vivo experiment for the treatment effect of CHAP-161 ^LysSAP26 on an animal model infected with Clostridium difficile.

Figure 6 shows the results of the 14-day survival rate change for the Clostridium difficile ATCC 9689 infection test group (groups 4-6).

Figure 7 shows the results of changes in survival rate for 14 days and 14 days for Clostridium difficile CD-M-5 infection test groups (groups 7-9).

Figure 8 shows the results of the 14-day survival rate change for the Clostridium difficile CD-H-7 infection test group (groups 10-12).

Figure 9 shows the results of changes in the body weight of mice for 14 days for Clostridium difficile ATCC 9689 infection test groups (groups 4-6).

Figure 10 shows the results of changes in the body weight of mice for 14 days for Clostridium difficile CD-M-5 infection test groups (groups 7-9).

Figure 11 shows the results of changes in the body weight of mice for 14 days for the test group infected with Clostridium difficile CD-H-7 (groups 10-12).

Accordingly, the inventors of the present invention made a CHAP-161 ^LysSAP26 expression vector by preparing a deletion mutant based on DNA encoding endolysin LysSAP26, and transformed it into E. coli to express the protein. The protein showed the ability to kill Clostridium difficile, in particular, has higher protein productivity than wild type LysSAP26, has a small molecular weight and is stable, and has a better antibacterial effect against Clostridium difficile bacteria, thereby confirming the present invention. completed.

The present invention provides a pharmaceutical composition for the prevention or treatment of Clostridium difficile infectious diseases comprising the CHAP-161 ^LysSAP26 recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

Specifically, the LysSAP26 protein may be derived from bacteriophage SAP26 (KCTC 11665BP) of Siphoviridae, but is not limited thereto. The present inventors induced and isolated a novel bacteriophage from a clinical isolate of Staphylococcus aureus, and deposited the isolated bacteriophage in the gene bank of the Korea Research Institute of Bioscience and Biotechnology on March 11, 2010 (accession number KCTC 11665BP).

Specifically, the gene encoding the CHAP-161 ^LysSAP26 recombinant protein may consist of the nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.

Specifically, the Clostridium difficile infectious disease may be endocarditis, diarrhea, enteritis, inflammatory bowel disease (IBD), pseudomembranous colitis, toxic megacolon, gastrointestinal perforation or sepsis, It is not limited thereto.

The CHAP-161 ^LysSAP26 recombinant protein of the present invention uses peptidoglycan, a bacterial cell wall component, as a substrate to decompose and disintegrate the cell wall to kill bacteria. Since the peptidoglycan exists only in bacteria and not in humans or animals, the CHAP-161 ^LysSAP26 recombinant protein of the present invention does not affect humans and animals, so it is safe and can be applied to the pharmaceutical industry, food industry, and biotechnology. In addition to being possible, there is an advantage in that bacteria can be effectively killed at the target site or target substance without problems with multi-drug antimicrobial resistance.

As used herein, the term 'treatment' refers to the prevention, suppression and alleviation of infectious diseases caused by Clostridium difficile .

When the composition of the present invention is a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described active ingredients for administration. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods. . Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc., in addition to the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It may be prepared by adding various excipients, for example, wetting agents, sweeteners, aromatics, and preservatives, in addition to liquids and liquid paraffin for oral use. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tablets. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, Witepsol, Macrosol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

A suitable dose of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug type, and the time, but can be appropriately selected by a person skilled in the art, and the daily dose of the composition is preferably It is 0.001 mg/kg to 50 mg/kg, and it can be divided and administered once a day to several times as needed.

In addition, the present invention provides an antibiotic for killing Clostridium difficile comprising the CHAP-161 ^LysSAP26 recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

As used herein, the term "antibiotic" is a generic term for preservatives, bactericides and antibacterial agents for medical use.

The CHAP-161 ^LysSAP26 recombinant protein of the present invention has excellent selective bacterial killing ability compared to conventional antibacterial agents. When the protein is used as an antimicrobial agent, unlike conventional antibacterial agents, it has the advantage of not inducing resistance or resistance of bacteria, so it is possible to provide an antibiotic material with a longer life cycle than conventional antibiotic agents. While most antibiotics have a decreasing range of use as they face increased resistance, the antimicrobial agent containing the protein of the present invention as an active ingredient can fundamentally solve the problem of antimicrobial resistance, thereby increasing the product lifespan as an antimicrobial agent. can

Therefore, the antibiotic containing the CHAP-161 ^LysSAP26 recombinant protein of the present invention having a specific killing activity for Clostridium difficile as an active ingredient is useful as an antibiotic with excellent antibacterial, bactericidal and antiseptic effects can be used

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

< Example 1> LysSAP26 Endolysin and LysSAP26 C-terminal deletion mutant recombinant protein CHAP-139 ^LysSAP26 , CHAP-161 ^LysSAP26 production and purification of

Plasmid vectors pLysSAP26 (6.12 Kbp), pCHAP139 (5.78 Kbp), and pCHAP161 (5.89 Kbp) expressing LysSAP26 endolysin and LysSAP26 C-terminal deletion mutant recombinant proteins CHAP-139 ^LysSAP26 and CHAP-161 ^LysSAP26 are prepared as follows.

The genome was extracted from the SAP26 phage and subjected to polymerase chain reaction (PCR) using this as a template, and the primers used at this time are shown in Table 1 below.

Expression vector	Primer name	Primer sequence
pLysSAP26	nde1-SAPlys	GGGAATTCCATATGaaaacatacagtgaagc
	xho1-SAPlys	atccgCTCGAGaaacacttctttcacaatc
pCHAP139	nde1-SAPlys	GGGAATTCCATATGaaaacatacagtgaagc
	xho1-SAPlyschap r	atccgCTCGAGctcacttttatacttagg
pCHAP161	nde1-SAPlys	GGGAATTCCATATGaaaacatacagtgaagc
	xho1-SAPlyschap161-r	atccgCTCGAGtgcagatttaccgactgct

PCR fragments produced using the primers mentioned in Table 1 were obtained by performing electrophoresis on an agarose gel and then eluting the band.

The PCR fragments were digested with restriction enzymes NdeI and XhoI, respectively, and then ligated with the expression vector (pET21a) digested with the same enzymes. After transforming E. coli BL21 (DE3) with the above-constructed vectors, they were cultured in LB liquid medium supplemented with 100 μg/ml ampicillin until the absorbance of the bacteria reached 0.6 (600 nm wavelength). Next, IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.1 mM and incubated at 18 ° C for 16 hours to induce expression of each protein.

Then, after harvesting the bacteria, the bacteria were disrupted using a lysis buffer [50 mM Tris-Cl (pH 8.0), 200 mM NaCl] and an ultrasonicator. The supernatant was collected by centrifugation of the disrupted bacterial lysate, injected into a Ni-NTA column, and C using an elution buffer [500 mM imidazole, 50 mM Tris-Cl (pH 8.0), 200 mM NaCl]. -LysSAP26, CHAP-139 ^LysSAP26 , and CHAP-161 ^LysSAP26 proteins tagged with six histidines at the end were purified.

In order to identify each of the purified proteins, after performing SDS-PAGE (12%), as a result of coomassie blue staining, as shown in FIG. 1, LysSAP26, CHAP-139 ^LysSAP26 , and CHAP-161 ^LysSAP26 proteins It was identified at about 30, 17.4, and 20 kD regions, respectively, and as a result of Western analysis using an anti-6xHis monoclonal antibody, it was confirmed that each 6xHis tagged protein was expressed.

In FIG. 1A, M is a protein size marker, the number of each gel picture is the number of each protein purification fraction, and FIG. 1B is a Western test result using an anti-6×His monoclonal antibody against RKR protein.

The amino acid sequence and number of CHAP-161 ^LysSAP26 is composed of 169 amino acids (SEQ ID NO: 1) including a histidine tag, and the theoretical protein size is 18.6 kDa. The gene coding sequence excluding the histidine tag is 483 bp (SEQ ID NO: 2).

< Example 2> LysSAP26 Endolysin and LysSAP26 C-terminal deletion mutant recombinant protein CHAP-139 ^LysSAP26 , CHAP-161 ^LysSAP26 Antibacterial activity comparison test of

LysSAP26, CHAP-139 ^LysSAP26 , CHAP-161 In order to investigate the antibacterial activity of ^LysSAP26 , Acinetobacter baumani ATCC 17978 and Staphylococcus aureus ATCC 25923 were used as target strains and the antibacterial activity was measured as follows. Each bacteria was cultured on a Blood Agar Plate medium and cultured at 37° C. for 18 hours, and then diluted to about 10 ⁴ CFU/mL using a sterilized Mueller Hinton Broth medium. After mixing 100 μL of the diluted bacterial culture medium and 100 μL of the selected protein (final concentrations of 5, 12.5, 25, 50, and 75 μg/mL) in a 96-well plate, the mixture was incubated at about 37°C for 20 hours, and each antibacterial activity was measured. The absorbance was measured using a 96 well plate reader at Optical Density 600 nm, and the value was expressed as a percentage with Control as the reference value of 100% (FIG. 2). The lower the absorbance value, the higher the antibacterial activity or antibacterial rate.

As a result of the test, CHAP-161 ^LysSAP26 almost completely killed Acinetobacter baumani bacteria (95% or more antibacterial rate) like LysSAP26 at 25 μg/mL and higher concentrations, and at concentrations of 5 and 12.5 μg/mL, CHAP-161 LysSAP26 161 ^LysSAP26 (about 80, 82% antibacterial rate) showed better antibacterial activity than LysSAP26 (about 15, 42% antibacterial rate). CHAP-139 ^LysSAP26 showed lower antimicrobial rates than CHAP-161 ^LysSAP26 at all concentrations tested (about 45, 50, 55, and 65% antibacterial rates at 5, 12.5, 25, and 50 μg/mL, respectively) (FIG. 2A). As a result of the antimicrobial test against Staphylococcus aureus, CHAP-161 ^LysSAP26 (65%, 95%, and 95% or more) in the concentration range of 25, 50, and 75 μg/mL, respectively, LysSAP26 (55%, 95%, and 95% or more, respectively) and CHAP-139 had higher antibacterial rates than ^LysSAP26 (35% and 45%, respectively) (Fig. 2B). CHAP-139 ^LysSAP26 could not be tested at a concentration of 75 μg/mL due to protein yield problems.

As a result of the comparison test of the antibacterial activity of the recombinant protein against Acinetobacter baumani and Staphylococcus aureus, CHAP-161 ^LysSAP26 was found to be the most excellent. In terms of protein stability, after 15 days of protein purification, LysSAP26 precipitated in an aqueous solution and lost its activity, whereas CHAP-161 ^LysSAP26 was slightly precipitated compared to LysSAP26, which was excellent in terms of protein stability (FIG. 3).

When comparing the yield of proteins with antibacterial activity, as shown in Table 2 below, 17.32 mg of CHAP-161 ^LysSAP26 , 2.672 mg of LysSAP26, and 2.032 mg of CHAP-139 ^LysSAP26 could be obtained in 1 liter of the culture medium. 161 ^LysSAP26 showed that the yield of antibacterial enzyme was superior among the three proteins.

Protein purification yield (protein yield showing antimicrobial activity from 1 Liter culture, mg/Liter)
LysSAP26	CHAP-139 LysSAP26	CHAP-161 LysSAP26
2.672	2.032	17.320

< Example 3> CHAP- 161 ^LysSAP26 of Clostridium Difficile Germ ability to kill test

To measure the growth inhibition ability of Clostridium difficile bacteria, CHAP-161 The minimum inhibitory concentration (MIC) of ^LysSAP26 was measured (FIG. 4A).

Each bacterium was prepared to a bacterial count of 5 × 10⁴CFU/well using Mueller Hinton Broth (MHB), and purified CHAP-161 ^LysSAP26 was added thereto to 5, 10, 25, 50, and 75 μg/mL, respectively, to anaerobically After reacting at 37 ° C. for 16 hours under the condition, the degree of bacterial growth was confirmed with the naked eye. As a result, bacterial growth was observed in all control groups (-CHAP-161 ^LysSAP26 ) in the well plate, whereas in the test group (+CHAP-161 ^LysSAP26 ), Clostridium difficile ATCC 9689 and Clostridium difficile PO3645 had a protein amount of 25 μg/mL or more. The growth of bacteria was not observed, and the growth of two other clinical strains was not confirmed at 50 μg/mL or more.

The minimum bactericidal concentration (MBC) of CHAP-161 ^LysSAP26 against Clostridium difficile bacteria was measured as follows (FIG. 4B). After inoculating 20 μL of the mixed solution of each well confirmed in the MIC experiment on a Mueller Hinton Agar (MHA) plate under anaerobic conditions, it was incubated at 37 ° C for 18 to 24 hours and the growth of bacteria was confirmed. As a result, CHAP-161 ^LysSAP26 Clostridium difficile ATCC 9689 and Clostridium difficile PO3645 were killed at 25 μg/mL and higher concentrations, and two other clinical strains were killed at CHAP-161 ^LysSAP26 at 50 μg/mL and higher concentrations (Fig. 4B). Based on the results of MIC and MBC experiments, CHAP-161 ^LysSAP26 It was proved that the MIC and MBC values had the same bactericidal effect.

For reference, LysSAP26 showed 50% or less antibacterial activity compared to CHAP-161 ^LysSAP26 according to the MIC and MBC measurements for Clostrididium difficile.

< Example 4> Clostridium Difficile CHAP- for Infectious Animal Models 161 ^LysSAP26 of Disease treatment effect test

1. Clostridium Difficile Preparation of strains

The expression levels of toxin genes and toxin proteins of Clostridium difficile ATCC 9689 and clinical isolates CD-M-5 and CD-H-7 are shown in Table 3 below.

Clostridium difficile isolate	possessed toxin gene	toxin protein expression	Toxin expression level (ng/mL)
ATCC 9689	tcdA + , tcdB +	A + , B +	A: 1.214, B: 2.224
CD-M-5	tcdA + , tcdB +	A + , B -	A: 1.645, B: 0
CD-H-7	tcdA + , tcdB +	A + , B +	A: 0.954, B: 2.845

2. Clostridium Difficile animal model of infection

Immunodeficiency injections of 5-week-old C57BL/6 mice were administered twice and kanamycin (kanamycin 0.4 mg/mL, Nacalai Tesque), gentamicin (gentamicin 0.035 mg/mL, Nacalai Tesque), and colistin (850 U) were administered in drinking water. /mL, Sigma-Aldrich), metronidazole (0.215 mg/mL, Nacalai), and vancomycin (0.045 mg/mL) were prepared, and mice were then fed the antibiotic cocktail for 5 days, followed by 2 days. fed with sterile water. One day before bacterial infection, a single dose of clindamycin (20 mg/kg) was injected intraperitoneally.

Clostridium difficile ATCC 9689, clinical isolates CD-M-5 and CD-H-7 were orally injected (10 ⁹ CFU/mouse) to induce infection, and after 24 hours, CHAP-161 (150 μg, 300 μg) ), PBS (negative control) was followed up for 14 days after each injection (Fig. 5). The mouse experimental groups are as follows.

- Group 1. Negative control group injected with 100 μL of PBS on days 0 and 7

- Group 2. Safety test group injected with 150 μg of CHAP-161 on days 0 and 7

- Group 3. Safety test group injected with 300 μg of CHAP-161 on days 0 and 7

- Group 4. C. difficile on days 0 and 7 Control group infected with ATCC 9689

- Group 5. C. difficile on days 0 and 7 Infection with ATCC 9689, and injection with 100 μg of CHAP-161 after 3 hours each

- Group 6. Infected with C. difficile ATCC 9689 on days 0 and 7, and injected with 150 μg of CHAP-161 3 hours later and 1 day later, respectively.

-group 7. C. difficile on days 0 and 7 Infection control group infected with clinical strain CD-M-5

-group 8. C. difficile on days 0 and 7 Infection with clinical strain CD-M-5, and injection with 100 μg of CHAP-161 after 3 hours each

- Group 9. Infection with C. difficile clinical strain CD-M-5 on days 0 and 7, and injected with 150 μg of CHAP-161 after 3 hours each

-group 10. C. difficile on days 0 and 7 Infection control group infected with clinical strain CD-H-7

-group 11. C. difficile on days 0 and 7 Infection with clinical strain CD-H-7, and injection with 100 μg of CHAP-161 after 3 hours each

- Group 12. Infection with C. difficile clinical strain CD-H-7 on days 0 and 7, and injection of 150 μg of CHAP-161 after 3 hours each

The survival rate and weight change were recorded every day, and the experiment was terminated after confirmation for a total of 14 days.

As a result of the experiment, groups 1-3 showed a 100% survival rate for 14 days, and group 4 showed a 50% survival rate on the 6th day and a 25% survival rate after the 8th day, but CHAP-161 was injected with 100 μg or 150 μg One group, 5 and 6, had a survival rate of 100%. With similar results, Group 10 showed a survival rate of 66% on the 6th day and 33% after the 9th day, but Groups 11 and 12 injected with 100 μg and 150 μg of CHAP-161 showed a 100% survival rate. looked Group 7 did not die during the test day, unlike groups 4 and 10, which were control groups infected with Clostridium difficile. However, as a result of tracking changes in the body weight of the surviving mice, group 7 showed a significant weight loss from the 4th day of infection and continued until the 14th day, whereas the treatment groups of groups 8 and 9 injected with 100 μg and 150 μg of CHAP-161 showed a steady increase in body weight, similar to normal mouse controls.

C. difficile Changes in body weight of surviving mice in the group infected with ATCC 9689 and clinical strain CD-H-7 also showed a tendency of steady decrease after 4 days of infection in groups 4 and 10, which are infection control groups, until day 14, but groups 5 and 6, which are treatment groups, and groups 11 and 12 showed a steady increase in weight or maintenance of weight up to 14 days.

In all bacterial infection groups, the shape of the stool gradually showed atypical watery diarrhea during the 14 days of infection, but in the treatment group, the shape of the stool showed a typical rugby ball shape, as in the normal control group (this result is not included. did not).

The ATCC 9689 strain expressing both toxin A and B and the clinical isolate CD-H-7 showed similar mouse lethality. showed a reducing effect. Through this, the effect of CHAP-161 on Clostridium difficile-induced enteritis (or diarrhea) for diarrhea and body weight normalization was confirmed. The experimental results are shown in Figures 6 to 11.

On the other hand, examples of preparations using the protein of the present invention are exemplified below, but this is not intended to limit the present invention thereto, but is only intended to be specifically described.

[Formulation Example 1: Preparation of Powder]

Recombinant Protein CHAP-161 ^LysSAP26 300 mg

Lactose 100 mg

Talc 10 mg

A powder is prepared by mixing the above ingredients and filling them in an airtight bag.

[Formulation Example 2: Preparation of tablets]

Recombinant Protein CHAP-161 ^LysSAP26 300 mg

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

[Formulation Example 3: Preparation of capsule formulation]

Recombinant Protein CHAP-161 ^LysSAP26 300 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a conventional capsule preparation method.

[Formulation Example 4: Preparation of Injection]

Recombinant Protein CHAP-161 ^LysSAP26 300 mg

Mannitol 180mg

Sterile Distilled Water for Injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

According to the conventional method for preparing injections, it is prepared with the above ingredient content per 1 ampoule (2).

[Formulation Example 5: Preparation of liquid formulation]

Recombinant Protein CHAP-161 ^LysSAP26 300 mg

Isomerized sugar 10 g

5 g mannitol

Appropriate amount of purified water

According to the conventional liquid preparation method, add and dissolve each component in purified water, add an appropriate amount of lemon flavor, mix the above components, add purified water, adjust the total to 100 by adding purified water, and then fill in a brown bottle to sterilize to prepare a liquid.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and 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 equivalents thereof.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (4)

1. A pharmaceutical composition for the prevention or treatment of Clostridium difficile infectious diseases comprising the CHAP-161 ^LysSAP26 recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
2. The pharmaceutical composition for preventing or treating infectious diseases according to claim 1, wherein the gene encoding the CHAP-161 ^LysSAP26 recombinant protein consists of the nucleotide sequence represented by SEQ ID NO: 2. .
3. The method of claim 1, wherein the Clostridium difficile infectious disease is endocarditis, diarrhea, enteritis, inflammatory bowel disease (inflammatory bowel disease, IBD), pseudomembranous colitis, toxic megacolon, gastrointestinal perforation or sepsis Characterized by Clostridium difficile ( Clostridium difficile ) A pharmaceutical composition for preventing or treating infectious diseases.
4. An antibiotic for killing Clostridium difficile comprising the CHAP-161 ^LysSAP26 recombinant protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

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