WO2022154287A1 - Antibacterial protein efl200 having lytic activity to enterococcus faecium - Google Patents
Antibacterial protein efl200 having lytic activity to enterococcus faecium Download PDFInfo
- Publication number
- WO2022154287A1 WO2022154287A1 PCT/KR2021/019368 KR2021019368W WO2022154287A1 WO 2022154287 A1 WO2022154287 A1 WO 2022154287A1 KR 2021019368 W KR2021019368 W KR 2021019368W WO 2022154287 A1 WO2022154287 A1 WO 2022154287A1
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- WIPO (PCT)
- Prior art keywords
- efl200
- enterococcus
- antibacterial protein
- fasium
- infection
- Prior art date
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Definitions
- the present invention relates to Enterococcus fasium faecium ) relates to an antibacterial protein EFL200 having lytic activity against bacteria, and more particularly, has the ability to specifically lyse Enterococcus fasium bacteria, which can cause diseases by infecting animals, including humans, SEQ ID NO: By Enterococcus fasium bacteria containing the Enterococcus fasium species-specific antibacterial protein EFL200, and the Enterococcus fasium bacteria-specific antibacterial protein EFL200 as an active ingredient, comprising the amino acid sequence represented by 1. It relates to a pharmaceutical composition for treating induced infections and diseases, and a method for producing the antibacterial protein EFL200.
- Enterococcus ( Enterococci ) is a facultative anaerobic Gram-positive coli that resides in the gastrointestinal tract and urogenital system.
- Enterococcus faecalis Enterococcus faecalis
- Enterococcus fasium Enterococcus faecium
- Enterococcus Durans Enterococcus Durans
- Enterococcus casseliflavus including about 19 species (Species) have been reported, and among them, the major bacterial species that actually cause infection are Enterococcus faecalis and Enterococcus fasium. can present
- infection by Enterococcus faecalis was very common, but recently, infection by Enterococcus faecium has been relatively increasing.
- Enterococcus is relatively weak and does not cause disease in normal people, but it causes various opportunistic infections such as urinary tract infections, wound infections, bacteremia, and endocarditis in the elderly, immunocompromised patients, chronic underlying diseases, or hospitalized patients.
- enterococci may induce infectious diseases by acquiring virus factors through gene hybridization from the outside. Among the infections caused by enterococci, urinary tract infection is the most frequent, followed by wound infection. Other major infections caused by enterococci may suggest endocarditis, and 5-20% of bacterial endocarditis is caused by enterococci.
- Enterococci are known to acquire new DNA (plasmid, transposons) or to acquire antibiotic resistance by using mutations.
- Enterococci which have acquired resistance to aminoglycosides, cause endocarditis or severe infections, it is difficult to succeed in treatment because they cannot be sterilized.
- VRE Vancomycin-Resistance Enterococci
- Vancomycin-resistant VRE was first reported in France in 1986, and VRE was first isolated in Korea in 1992. In the United States, since VRE was first isolated in 1988, according to NNIS (National Nosocomial Infection Surveillance) data, 1-15% of Enterococcus isolated between 1990-1997 were VRE, and 25% in 1999 and 2003 It is increasing at 28.5% per year. Recently, the appearance of VRE is increasing worldwide, not only in Europe and the United States, but also in Asia and Oceania including Korea. Accordingly, it is necessary to develop a drug that can be used to prevent or treat antibiotic-resistant enterococcal infection. In particular, it is very urgent to develop a pharmaceutical formulation that can provide a rapid therapeutic effect.
- NNIS National Nosocomial Infection Surveillance
- the present inventors provide an antibacterial protein capable of specifically lysing Enterococcus fasium bacteria, and further It contains as an active ingredient and provides a pharmaceutical composition that can be used to treat Enterococcus fasium infection, and further effectively treats Enterococcus fasium infection or disease using the pharmaceutical composition
- a pharmaceutical composition that can be used to treat Enterococcus fasium infection, and further effectively treats Enterococcus fasium infection or disease using the pharmaceutical composition
- an antibacterial protein EFL200 having an activity capable of specifically lysing Enterococcus fasium and comprising the amino acid sequence represented by SEQ ID NO: 1.
- Another object of the present invention is to provide a method for efficiently producing the antibacterial protein EFL200 having the activity to specifically lyse the Enterococcus fasium bacteria and comprising the amino acid sequence represented by SEQ ID NO: 1.
- Another object of the present invention is to provide a pharmaceutical composition for the treatment of Enterococcus fasium infection comprising the antibacterial protein EFL200 capable of specifically lysing the Enterococcus fasium bacteria as an active ingredient.
- Another object of the present invention is to provide a method for treating Enterococcus fasium infection or disease using a pharmaceutical composition comprising the antibacterial protein EFL200 as an active ingredient.
- Another object of the present invention is to provide a food composition comprising the antibacterial protein EFL200 as an active ingredient and a method for improving infection or disease of Enterococcus fasium using the same.
- the inventors of the present invention prepared various antibacterial protein candidates in the form of recombinant proteins by utilizing various antibacterial protein information known to have antibacterial activity against various bacterial species, and their lysis against Enterococcus fasium bacteria. Antibacterial proteins with excellent lytic activity were selected by examining their activity, and a method for efficiently manufacturing them was developed. The present invention was completed by developing a composition.
- the present invention provides an amino acid sequence of the antibacterial protein EFL200 capable of specifically lysing Enterococcus fasium bacteria.
- EFL200 an antibacterial protein capable of specifically lysing Enterococcus fasium, is composed of 284 amino acid residues and has a molecular weight of about 30.7 kDa.
- amino acid sequence of SEQ ID NO: 1 may be partially modified by those skilled in the art using known techniques. Such modifications include partial substitutions of amino acid sequences, partial additions of amino acid sequences, and partial deletions of amino acid sequences. However, it is most preferable to apply the amino acid sequence of SEQ ID NO: 1 disclosed in the present invention mutatis mutandis.
- the present invention provides a strain TOP10-EFL200 that can be used for the production of the antibacterial protein EFL200 having the amino acid sequence of SEQ ID NO: 1.
- the strain TOP10-EFL200 is a strain prepared to produce the antibacterial protein EFL200 by transforming E. coli using a plasmid containing the gene sequence of SEQ ID NO: 2 prepared by the present inventors.
- This E. coli TOP10-EFL200 was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on November 10, 2020 by the present inventors (accession number KCTC 14364BP).
- the term "gene” has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of genes, are natural nucleotides as well as modified sugars or base sites. Also includes analogs ( Chemical Reviews 90:543-584, 1990).
- the present invention is Enterococcus fasium bacteria characterized by the amino acid sequence of SEQ ID NO: 1 and comprising the antibacterial protein EFL200 having a specific lytic ability against Enterococcus fasium bacteria as an active ingredient.
- a pharmaceutical composition that can be effectively used for treatment of infection or disease.
- the antibacterial protein EFL200 having a specific lytic ability against Enterococcus fasium, characterized by the amino acid sequence of SEQ ID NO: 1 according to the present invention, contained in the pharmaceutical composition of the present invention is Enterococcus fasium as described above. Because it specifically lyses, it shows an effect in the treatment of various diseases caused by Enterococcus fasium bacteria. Therefore, the pharmaceutical composition of the present invention can be utilized for the treatment of diseases caused by Enterococcus fasium bacteria. In addition, the pharmaceutical composition of the present invention is implemented as an antibiotic, disinfectant, bactericide, therapeutic agent, etc., can be utilized for the treatment of various diseases caused by Enterococcus fasium bacteria.
- the present invention provides an antibacterial protein EFL200 specific to Enterococcus fasium, characterized in that it has the amino acid sequence represented by SEQ ID NO: 1, and various types caused by Enterococcus fasium.
- an antibacterial protein EFL200 specific to Enterococcus fasium characterized in that it has the amino acid sequence represented by SEQ ID NO: 1, and various types caused by Enterococcus fasium.
- a method of treating various diseases caused by Enterococcus fasium by administering to a subject having a disease.
- disease caused by Enterococcus fasium refers to a disease caused by Enterococcus fasium infection, including urinary tract infection, wound infection, bacteremia, endocarditis, and the like, but is not limited thereto. does not
- Enterococcus fasium bacteria is not 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.
- prevention refers to (i) prevention of Enterococcus fasium infection; and (ii) inhibiting the development of a disease caused by Enterococcus fasium infection.
- treatment refers to any action that reduces the pathological condition of an infection or disease caused by Enterococcus faecium, and a disease caused by Enterococcus fasium. it means.
- antibacterial substances capable of providing antibacterial activity against other bacterial species may be added to the pharmaceutical composition of the present invention.
- 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, and the like.
- 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.
- the pharmaceutical composition of the present invention may be administered through oral administration or parenteral administration, and in the case of parenteral administration, it may be administered using intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or local administration, It may also be used as a method of application or spraying outside the diseased area, but is not limited thereto.
- the pharmaceutical composition of the present invention is prepared in 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 placing it in a multi-dose container.
- 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.
- suitable application, spraying and dosage of the pharmaceutical composition depend on factors such as formulation method, administration method, age, 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.
- the pharmaceutical composition of the present invention may be embodied as an antibiotic, an antiseptic, a bactericide, a therapeutic agent, and the like.
- the term 'antibiotic' refers to a preservative, a disinfectant, and an antibacterial agent.
- the pharmaceutical composition of the present invention may be implemented in the form of a food composition.
- Enterococcus fasium infection and disease treatment method using a pharmaceutical composition comprising the antibacterial protein EFL200 having the amino acid sequence represented by SEQ ID NO: 1 according to the present invention as an active ingredient, as well as general Enterococcus fasium bacteria, as well as existing antibiotics It can also effectively act on Enterococcus fasium bacteria, which have acquired resistance to bacteria and antibacterial substances.
- the Enterococcus fasium-specific antibacterial protein EFL200 of the present invention does not affect the normal flora in the body other than Enterococcus fasium. can be minimized.
- Existing antibiotics have a disadvantage of causing adverse effects on beneficial bacteria in the body, causing various side effects.
- Lanes 1 to 3 are chromatography flow-throughs during purification, and lanes 4 to 10 are purification fractions.
- FIG. 2 is a result showing the antibacterial activity (lytic activity) of the antibacterial protein EFL200 against Enterococcus fasium (VRE indicated at the bottom), and the transparent part is generated by the antibacterial activity (lytic activity) of the antibacterial protein EFL200.
- a control Buffer control
- a buffer solution not containing the antibacterial protein EFL200 was used, and the presence or absence of the lysis ring indicates the antibacterial activity of the antibacterial protein EFL200.
- Figure 3 is a result showing the experimental results of the turbidity reduction assay (Turbidity reduction assay) performed on Enterococcus fasium bacteria of the antibacterial protein EFL200.
- the negative control group used the buffer itself without antibacterial protein EFL200.
- the horizontal axis is time (minutes) and the vertical axis is absorbance at 600 nm.
- Lane M is a protein size marker, and lanes 1 to 7 are samples obtained every 10 minutes.
- EFL200 Enterococcus fasium-specific antibacterial protein EFL200
- an expression plasmid having the gene sequence of SEQ ID NO: 2 was prepared through a conventional molecular biological method, and transformed by introducing it into E. coli Strain TOP10-EFL200 (Accession No. KCTC 14364BP) was prepared.
- culture was carried out at 37°C.
- anhydrotetracycline was added so that the final concentration was 0.2 ⁇ g/ml to induce the expression of the antibacterial protein EFL200 of the amino acid sequence shown in SEQ ID NO: 1. did.
- culture was further performed for 3 hours.
- the cell culture solution was taken and centrifuged at 7,000 rpm at 4° C. for 15 minutes to recover cell precipitates, and the recovered cell precipitates were suspended in 30 ml of buffer (50 mM K 3 PO 4 , pH 7.0).
- the cell suspension thus prepared was disrupted by sonication, and the sonication method was repeated in an ice bath for a total of 5 minutes under the condition that ultrasonic waves were applied for 3 seconds to break the cells and stopped for 3 seconds.
- the supernatant obtained by centrifuging the cell disruption solution at 7,000 rpm for 15 minutes at 4° C. was purified through a conventional cation-exchange chromatography purification process.
- fractions containing the antimicrobial protein EFL200 at a high concentration were collected, and a buffer (50 mM K 3 PO 4 , pH 7.0) was subjected to dialysis to perform medium exchange. Through this, it was possible to secure an antibacterial protein EFL200 solution having a purity of 90% or more.
- the present inventors investigated the antibacterial activity of the antibacterial protein EFL200 through a conventional spot-on-lawn assay.
- Enterococcus faecium 5 weeks Enterococcus faecium ( Enterococcus ) faecalis ) 2 weeks, Staphylococcus aureus ) 2 weeks, Salmonella 2 weeks, and Escherichia coli 2 weeks were the subjects.
- the bacteria were purchased from an external institution such as The American Type Culture Collection (ATCC) in the United States, or those isolated and identified by the present inventors.
- ATCC American Type Culture Collection
- the antibacterial protein EFL200 had antibacterial activity (dissolution power) only against Enterococcus fasium bacteria, and had no antibacterial activity against other bacterial species. Antibacterial activity against Enterococcus fasium was confirmed for all 5 strains of Enterococcus fasium that were the subject of the experiment. The results of the antimicrobial activity of the antibacterial protein EFL200 against Enterococcus fasium bacteria are presented in FIG. 2 .
- the antibacterial protein EFL200 can provide excellent bactericidal power (antibacterial power) against Enterococcus faecium, and can also be effectively used for preventing or treating infectious diseases caused by Enterococcus faecium. there was.
- Example 3 Investigation of antibacterial activity of antibacterial protein EFL200 through turbidity reduction investigation method
- the antibacterial activity of the antibacterial protein EFL200 was investigated through a turbidity reduction assay for 5 Enterococcus fasium strains tested in Example 2.
- the experimental method of the turbidity reduction investigation method is as follows. After suspending the test bacteria in physiological saline so that the absorbance at 600 nm becomes about 0.7, 0.1 ml of antibacterial protein EFL200 solution is added to 0.9 ml of this suspension (final concentration: 10 ⁇ g/ml), and the absorbance at 600 nm is measured. It was carried out in a manner of measuring for 30 minutes. As a negative control, a buffer solution (50 mM K 3 PO 4 , pH 7.0) not containing the antimicrobial protein EFL200 was used instead of the antimicrobial protein EFL200 solution.
- the Enterococcus fasium-specific antibacterial protein EFL200 of the present invention could eventually be killed by lysing the Enterococcus fasium bacteria.
- a pharmaceutical composition containing the Enterococcus fasium-specific antibacterial protein EFL200 can be utilized for the purpose of killing Enterococcus fasium during Enterococcus fasium infection, and also Enterococcus fasium It shows that it can be used in the same way as conventional antibiotics for the purpose of treating infections.
- the stability of the antibacterial protein EFL200 to the digestive enzymes was investigated by conducting a digestion test by digestive enzymes.
- the experimental method was as follows.
- a SIF-P solution was prepared by dissolving 0.05 g of Pancreatin (Sigma-Aldrich, Cat. No. P1625) in 5 ml of Simulated Intestinal Fluids (SIF, 50 mM KH 2 PO 4 , 0.015 N NaOH, pH 6.8) solution. .
- SIF Simulated Intestinal Fluids
- the negative control group used a buffer solution (50 mM K 3 PO 4 , pH 7.0) not containing the antibacterial protein EFL200 instead of the antibacterial protein EFL200 solution, and the positive control group added the antibacterial protein EFL200 solution to the SIF solution and reacted for 60 minutes. liquid was used.
- Example 5 Enterococcus fasium fungal specific antibacterial protein EFL200's Enterococcus Investigation of the therapeutic effect on Pasium infection
- mice Five-week-old ICR mice [specific pathogen-free (SPF) grade] weighing about 20 g were used as experimental animals. After dividing a total of 20 animals into 2 groups (10 mice per group), 1 ⁇ 10 8 cfu of Enterococcus fasium (ie, 1 ⁇ 10 8 cfu/mouse) was administered to each mouse by intravenous administration to prevent infection. induced. For one group (treatment group), 0.2 ml of antimicrobial protein EFL200 solution (10 mg/ml) was intravenously administered at 30 minutes, 12 hours, and 24 hours after forced bacterial infection. For another group (control group), only buffer (50 mM K 3 PO 4 , pH 7.0) was intravenously administered in the same volume.
- buffer 50 mM K 3 PO 4 , pH 7.0
- Buffer administration was performed at 30 minutes, 12 hours, and 24 hours after forced bacterial infection in the same manner as the administration of the antimicrobial protein EFL200 solution.
- the number of deaths was investigated every day for 5 days after the forced bacterial infection, and the results are presented in Table 1.
- the observation of unusual phenomena was also investigated twice a day, in the morning and in the afternoon.
- the antibacterial protein EFL200 of the present invention was effective in treating Enterococcus fasium infection.
- the pharmaceutical composition containing the antibacterial protein EFL200 of the present invention as an active ingredient can be used for the purpose of treating Enterococcus fasium infection, and also a common antibiotic for the purpose of treating Enterococcus fasium infection. It shows that it can be utilized in the same way.
- this specific technique is only a preferred embodiment, and thus It is clear that the scope is not limited. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
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Abstract
The present invention relates to an antibacterial protein EFL200 having specific antibacterial activity against Enterococcus <i /> faecium, and more specifically, to an antibacterial protein EFL200, a method for producing same, and a pharmaceutical composition for treating infections or diseases caused by Enterococcus faecium, comprising, as an active ingredient, the antibacterial protein CEL200 specific to by Enterococcus faecium, wherein the antibacterial protein EFL200 specific to Enterococcus faecium has the ability to specifically lyse Enterococcus faecium and comprises an amino acid sequence represented by SEQ ID NO: 1.
Description
본 발명은 엔테로코쿠스 패슘(Enterococcus faecium) 균에 대하여 용균활성을 갖는 항균단백질 EFL200에 관한 것으로, 더욱 상세하게는 인간을 포함한 동물에 감염하여 질환을 일으킬 수 있는 엔테로코쿠스 패슘 균을 특이적으로 용균시킬 수 있는 능력을 갖고 서열번호 1로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 엔테로코쿠스 패슘 균종에 특이적인 항균단백질 EFL200, 및 상기 엔테로코쿠스 패슘 균 특이적인 항균단백질 EFL200을 유효성분으로 포함하는 엔테로코쿠스 패슘 균에 의해 유발되는 감염 및 질환 처치용 약학적 조성물, 및 항균단백질 EFL200의 제조방법에 관한 것이다.The present invention relates to Enterococcus fasium faecium ) relates to an antibacterial protein EFL200 having lytic activity against bacteria, and more particularly, has the ability to specifically lyse Enterococcus fasium bacteria, which can cause diseases by infecting animals, including humans, SEQ ID NO: By Enterococcus fasium bacteria containing the Enterococcus fasium species-specific antibacterial protein EFL200, and the Enterococcus fasium bacteria-specific antibacterial protein EFL200 as an active ingredient, comprising the amino acid sequence represented by 1. It relates to a pharmaceutical composition for treating induced infections and diseases, and a method for producing the antibacterial protein EFL200.
장알균(Enterococcus; Enterococci)은 위장관과 비뇨생식계에 상재하는 통성혐기성 그람 양성 알균이다. 장알균 속(Genus)에는 엔테로코쿠스 패칼리스(Enterococcus faecalis), 엔테로코쿠스 패슘(Enterococcus faecium), 엔테로코쿠스 두란스(Enterococcus durans), 엔테로코쿠스 카쎄리프라부스(Enterococcus casseliflavus)를 포함하여 약 19가지 종(Species)이 보고되고 있으며, 이 중에 실제 감염을 일으키는 주요 균종으로는 엔테로코쿠스 패칼리스와 엔테로코쿠스 패슘을 제시할 수 있다. 과거에는 엔테로코쿠스 패칼리스에 의한 감염이 매우 흔하였지만 최근에는 엔테로코쿠스 패슘에 의한 감염이 상대적으로 늘고 있다. Enterococcus ( Enterococci ) is a facultative anaerobic Gram-positive coli that resides in the gastrointestinal tract and urogenital system. Enterococcus faecalis ( Enterococcus faecalis ), Enterococcus fasium ( Enterococcus ) faecium ), Enterococcus Durans ( Enterococcus Durans ), Enterococcus casseliflavus ), including about 19 species (Species) have been reported, and among them, the major bacterial species that actually cause infection are Enterococcus faecalis and Enterococcus fasium. can present In the past, infection by Enterococcus faecalis was very common, but recently, infection by Enterococcus faecium has been relatively increasing.
장알균은 비교적 독성이 약하여 정상인에서는 질병을 일으키지 않으나 노인이나 면역저하 환자, 만성 기저질환자 또는 병원에 입원중인 환자에서는 요로감염, 창상감염, 균혈증, 심내막염 등의 각종 기회감염증을 유발한다. 또한, 장알균은 외부로부터 유전자 교잡을 통하여 병독인자(Virulence factor)를 획득하여 감염성 질환(Infectious disease)을 유발할 수도 있다. 장알균에 의한 감염증 중에 요로감염이 가장 빈번하며, 그 다음으로 창상감염이 흔하다. 장알균에 의한 그 밖의 주요감염으로 심내막염을 제시할 수 있는데, 세균성 심내막염의 5-20%가 장알균에 의한 것이다.Enterococcus is relatively weak and does not cause disease in normal people, but it causes various opportunistic infections such as urinary tract infections, wound infections, bacteremia, and endocarditis in the elderly, immunocompromised patients, chronic underlying diseases, or hospitalized patients. In addition, enterococci may induce infectious diseases by acquiring virus factors through gene hybridization from the outside. Among the infections caused by enterococci, urinary tract infection is the most frequent, followed by wound infection. Other major infections caused by enterococci may suggest endocarditis, and 5-20% of bacterial endocarditis is caused by enterococci.
한편, 일반적으로 장알균 처치에 사용되는 항생제로는 아미노글리코사이드(Aminoglycosides), 세팔로스포린(Cephalosporins), 클린다마이신(Clindamycin) 및 반합성 페니실린 제제들이 사용되나, 이들 항생제에 대한 내성균 발생이 자주 보고 되고 있다. 장알균은 새로운 DNA(플라스미드, Transposons)를 얻거나, 돌연변이를 이용하여 항생제 내성을 획득한다고 알려져 있다. 특히 아미노글리코사이드에 대한 내성을 획득한 장알균이 심내막염이나 중증 감염을 일으킨 경우에는 이 장알균을 살균할 수 없기 때문에 치료에 성공하기 어렵다. 반코마이신 내성 장알균(Vancomycin-Resistance Enterococci, 이하 VRE)에 원인한 균혈증이 있는 경우는 사망률이 67%에 달하는 것으로 보고되고 있다. 1986년 프랑스에서 처음으로 반코마이신에 내성을 보이는 VRE가 보고되었고 국내에서는 1992년에 VRE가 처음으로 분리되었다. 미국의 경우, 1988년 처음으로 VRE가 분리된 이래, NNIS(National Nosocomial Infection Surveillance)의 자료에 따르면 1990-1997년 사이에 분리된 엔테로코쿠스 중 1-15%가 VRE였으며 1999년 25%, 2003년 28.5%로 증가되고 있는 추세이다. 최근에는 유럽, 미국 뿐 아니라 한국을 포함한 아시아 및 오세아니아를 비롯하여 전 세계적으로 VRE의 출현이 증가하고 있다. 이에 따라 항생제 내성 장알균 감염 예방 또는 치료에 활용할 수 있는 약제의 개발이 필요하다. 특히, 신속한 치료 효과를 제공해 줄 수 있는 약학적 제제의 개발이 매우 절실한 형편이다.On the other hand, aminoglycosides, cephalosporins, clindamycin and semi-synthetic penicillin preparations are generally used as antibiotics used to treat Enterococcus, but the occurrence of bacteria resistant to these antibiotics is frequently reported. . Enterococci are known to acquire new DNA (plasmid, transposons) or to acquire antibiotic resistance by using mutations. In particular, if Enterococci, which have acquired resistance to aminoglycosides, cause endocarditis or severe infections, it is difficult to succeed in treatment because they cannot be sterilized. In the case of bacteremia caused by Vancomycin-Resistance Enterococci (VRE), the mortality rate is reported to reach 67%. Vancomycin-resistant VRE was first reported in France in 1986, and VRE was first isolated in Korea in 1992. In the United States, since VRE was first isolated in 1988, according to NNIS (National Nosocomial Infection Surveillance) data, 1-15% of Enterococcus isolated between 1990-1997 were VRE, and 25% in 1999 and 2003 It is increasing at 28.5% per year. Recently, the appearance of VRE is increasing worldwide, not only in Europe and the United States, but also in Asia and Oceania including Korea. Accordingly, it is necessary to develop a drug that can be used to prevent or treat antibiotic-resistant enterococcal infection. In particular, it is very urgent to develop a pharmaceutical formulation that can provide a rapid therapeutic effect.
이에, 유해 병원성 박테리아인 엔테로코쿠스 패슘 균에 대한 기존 항생제 사용에 의해 발생하는 문제점의 해결 방안으로서, 본 발명자들은 엔테로코쿠스 패슘 균을 특이적으로 용균시킬 수 있는 항균단백질을 제공하고, 나아가 이를 유효성분으로 포함하고 엔테로코쿠스 패슘 균의 감염을 처치하는 데에 활용될 수 있는 약학적 조성물을 제공하며, 더 나아가 상기 약학적 조성물을 활용한 엔테로코쿠스 패슘 균의 감염 또는 질환을 효과적으로 처치하는 방법을 제공하고자 한다.Accordingly, as a solution to the problem caused by the use of existing antibiotics for harmful pathogenic bacteria Enterococcus fasium, the present inventors provide an antibacterial protein capable of specifically lysing Enterococcus fasium bacteria, and further It contains as an active ingredient and provides a pharmaceutical composition that can be used to treat Enterococcus fasium infection, and further effectively treats Enterococcus fasium infection or disease using the pharmaceutical composition We want to provide a way
따라서, 본 발명의 목적은 엔테로코쿠스 패슘 균을 특이적으로 용균시킬 수 있는 활성을 갖고 서열번호 1로 표시되는 아미노산 서열을 포함하는 항균단백질 EFL200을 제공하는 것이다.Accordingly, it is an object of the present invention to provide an antibacterial protein EFL200 having an activity capable of specifically lysing Enterococcus fasium and comprising the amino acid sequence represented by SEQ ID NO: 1.
본 발명의 다른 목적은 상기 엔테로코쿠스 패슘 균을 특이적으로 용균시킬 수 있는 활성을 갖고 서열번호 1로 표시되는 아미노산 서열을 포함하는 항균단백질 EFL200을 효율적으로 제조할 수 있는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for efficiently producing the antibacterial protein EFL200 having the activity to specifically lyse the Enterococcus fasium bacteria and comprising the amino acid sequence represented by SEQ ID NO: 1.
본 발명의 또 다른 목적은 상기 엔테로코쿠스 패슘 균을 특이적으로 용균시킬 수 있는 항균단백질 EFL200을 유효성분으로 포함하는 엔테로코쿠스 패슘 감염 처치 목적의 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for the treatment of Enterococcus fasium infection comprising the antibacterial protein EFL200 capable of specifically lysing the Enterococcus fasium bacteria as an active ingredient.
또한, 본 발명의 또 다른 목적은 항균단백질 EFL200을 유효성분으로 포함하는 약학적 조성물을 이용하여 엔테로코쿠스 패슘 균의 감염 또는 질환을 치료하는 방법을 제공하는 것이다.In addition, another object of the present invention is to provide a method for treating Enterococcus fasium infection or disease using a pharmaceutical composition comprising the antibacterial protein EFL200 as an active ingredient.
또한, 본 발명의 또 다른 목적은 항균단백질 EFL200을 유효성분으로 포함하는 식품 조성물 및 이를 이용하여 엔테로코쿠스 패슘 균의 감염 또는 질환을 개선하는 방법을 제공하는 것이다.In addition, another object of the present invention is to provide a food composition comprising the antibacterial protein EFL200 as an active ingredient and a method for improving infection or disease of Enterococcus fasium using the same.
상기 목적들을 달성하고자, 본 발명의 발명자들은 여러 균종들에 대하여 항균활성을 가진다고 알려진 다양한 항균단백질 정보를 활용하여, 여러 항균단백질 후보들을 재조합 단백질 형태로 제조하였고 이들의 엔테로코쿠스 패슘 균에 대한 용균활성을 조사하여 우수한 용균활성을 갖는 항균단백질을 선별하였고, 이를 효율적으로 제조할 수 있는 방법까지 개발하였으며, 마지막으로 이를 유효성분으로 포함하는 엔테로코쿠스 패슘 균 감염 처치 목적으로 활용될 수 있는 약학적 조성물을 개발함으로써 본 발명을 완성하였다.In order to achieve the above objects, the inventors of the present invention prepared various antibacterial protein candidates in the form of recombinant proteins by utilizing various antibacterial protein information known to have antibacterial activity against various bacterial species, and their lysis against Enterococcus fasium bacteria. Antibacterial proteins with excellent lytic activity were selected by examining their activity, and a method for efficiently manufacturing them was developed. The present invention was completed by developing a composition.
따라서, 본 발명의 일 양태에 따르면, 본 발명은 엔테로코쿠스 패슘 균을 특이적으로 용균시킬 수 있는 항균단백질 EFL200의 아미노산 서열을 제공한다. 구체적으로는, 서열번호 1의 아미노산 서열이 이에 해당한다. 엔테로코쿠스 패슘 균을 특이적으로 용균시킬 수 있는 항균단백질 EFL200은 284개의 아미노산 잔기로 구성되며, 분자량은 약 30.7 kDa이다.Therefore, according to one aspect of the present invention, the present invention provides an amino acid sequence of the antibacterial protein EFL200 capable of specifically lysing Enterococcus fasium bacteria. Specifically, the amino acid sequence of SEQ ID NO: 1 corresponds to this. EFL200, an antibacterial protein capable of specifically lysing Enterococcus fasium, is composed of 284 amino acid residues and has a molecular weight of about 30.7 kDa.
서열번호 1의 아미노산 서열은 당업자에 의해 공지의 기술을 이용하여 일부 변형될 수 있음이 자명하다. 상기 변형은 아미노산 서열의 일부 치환, 아미노산 서열의 일부 첨가, 및 아미노산 서열의 일부 결실을 포함한다. 그렇지만 본 발명에서 개시하고 있는 서열번호 1의 아미노산 서열을 준용하는 것이 가장 바람직하다.It is apparent that the amino acid sequence of SEQ ID NO: 1 may be partially modified by those skilled in the art using known techniques. Such modifications include partial substitutions of amino acid sequences, partial additions of amino acid sequences, and partial deletions of amino acid sequences. However, it is most preferable to apply the amino acid sequence of SEQ ID NO: 1 disclosed in the present invention mutatis mutandis.
또한, 본 발명은 서열번호 1의 아미노산 서열을 갖는 항균단백질 EFL200의 생산에 이용될 수 있는 균주 TOP10-EFL200을 제공한다. 상기 균주 TOP10-EFL200은 본 발명자들에 의해 제작된 서열번호 2의 유전자 서열을 포함하는 플라스미드를 사용하여 대장균을 형질전환시켜 항균단백질 EFL200을 생산하도록 제작된 균주이다. 이 대장균 TOP10-EFL200은 본 발명자들에 의해 2020년 11월 10일자로 한국생명공학연구원 생물자원센터에 기탁되었다(수탁번호 KCTC 14364BP). In addition, the present invention provides a strain TOP10-EFL200 that can be used for the production of the antibacterial protein EFL200 having the amino acid sequence of SEQ ID NO: 1. The strain TOP10-EFL200 is a strain prepared to produce the antibacterial protein EFL200 by transforming E. coli using a plasmid containing the gene sequence of SEQ ID NO: 2 prepared by the present inventors. This E. coli TOP10-EFL200 was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on November 10, 2020 by the present inventors (accession number KCTC 14364BP).
본 명세서에서 사용된 "유전자"라는 용어는 DNA(gDNA 및 cDNA) 그리고 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 유전자에서 기본 구성단위인 뉴클레오타이드는 자연의 뉴클레오타이드 뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다(Chemical Reviews 90:543-584, 1990).As used herein, the term "gene" has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of genes, are natural nucleotides as well as modified sugars or base sites. Also includes analogs ( Chemical Reviews 90:543-584, 1990).
본 발명의 다른 일 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 특징지어지고 엔테로코쿠스 패슘 균에 대하여 특이적 용균능을 갖는 항균단백질 EFL200을 유효성분으로 포함하는 엔테로코쿠스 패슘 균의 감염 또는 질환에 대한 치료에 효과적으로 활용될 수 있는 약학적 조성물을 제공한다. According to another aspect of the present invention, the present invention is Enterococcus fasium bacteria characterized by the amino acid sequence of SEQ ID NO: 1 and comprising the antibacterial protein EFL200 having a specific lytic ability against Enterococcus fasium bacteria as an active ingredient. Provided is a pharmaceutical composition that can be effectively used for treatment of infection or disease.
본 발명의 약학적 조성물에 포함되는, 본 발명에 따른 서열번호 1의 아미노산 서열로 특징지어지는 엔테로코쿠스 패슘 균에 대하여 특이적 용균능을 갖는 항균단백질 EFL200은 상술한 바와 같이 엔테로코쿠스 패슘 균을 특이적으로 용균시키므로, 엔테로코쿠스 패슘 균에 의해 유발되는 다양한 질환의 처치에 있어 효과를 나타낸다. 따라서 본 발명의 약학적 조성물은 엔테로코쿠스 패슘 균에 의해 유발되는 질환의 처치에 활용될 수 있다. 또한, 본 발명의 약학적 조성물은 항생제, 소독제, 살균제, 치료제 등으로 구현되어 엔테로코쿠스 패슘 균에 의해 유발되는 다양한 질환들의 처치에 활용될 수 있다.The antibacterial protein EFL200 having a specific lytic ability against Enterococcus fasium, characterized by the amino acid sequence of SEQ ID NO: 1 according to the present invention, contained in the pharmaceutical composition of the present invention is Enterococcus fasium as described above. Because it specifically lyses, it shows an effect in the treatment of various diseases caused by Enterococcus fasium bacteria. Therefore, the pharmaceutical composition of the present invention can be utilized for the treatment of diseases caused by Enterococcus fasium bacteria. In addition, the pharmaceutical composition of the present invention is implemented as an antibiotic, disinfectant, bactericide, therapeutic agent, etc., can be utilized for the treatment of various diseases caused by Enterococcus fasium bacteria.
따라서, 본 발명의 다른 일 양태에 따르면, 본 발명은 서열번호 1로 표시되는 아미노산 서열을 갖는 것을 특징으로 하는 엔테로코쿠스 패슘 균에 특이적인 항균단백질 EFL200을 엔테로코쿠스 패슘 균에 의해 유발되는 다양한 질환을 가진 개체에 투여하여 엔테로코쿠스 패슘 균에 의해 유발되는 다양한 질환들을 치료하는 방법을 제공한다.Therefore, according to another aspect of the present invention, the present invention provides an antibacterial protein EFL200 specific to Enterococcus fasium, characterized in that it has the amino acid sequence represented by SEQ ID NO: 1, and various types caused by Enterococcus fasium. Provided is a method of treating various diseases caused by Enterococcus fasium by administering to a subject having a disease.
본 명세서에서 “엔테로코쿠스 패슘 균에 의해 유발되는 질환”이란 엔테로코쿠스 패슘 균 감염에 의해 유발되는 질환으로서 요로감염, 창상감염, 균혈증, 심내막염 등을 포함하는 질환을 총칭하지만, 이에 제한되지는 않는다.As used herein, the term “disease caused by Enterococcus fasium” refers to a disease caused by Enterococcus fasium infection, including urinary tract infection, wound infection, bacteremia, endocarditis, and the like, but is not limited thereto. does not
본 명세서에서의 엔테로코쿠스 패슘 균은 기존 항생제에 대하여 민감하든지 또는 기존 항생제에 대하여 내성을 가진 내성균이든지 상관이 없다. 즉, 기존 항생제에 대한 내성 획득 여부는 상관이 없다.In the present specification, Enterococcus fasium bacteria is not 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.
본 명세서에서 사용된 "방지" 또는 "예방"이라는 용어는 (i) 엔테로코쿠스 패슘 감염의 방지; 및 (ii) 엔테로코쿠스 패슘 감염에 의한 질병으로의 발전을 억제하는 것을 의미한다.As used herein, the term "prevention" or "prevention" refers to (i) prevention of Enterococcus fasium infection; and (ii) inhibiting the development of a disease caused by Enterococcus fasium infection.
본 명세서에서 사용된 “처치” 또는 “치료”라는 용어는 엔테로코쿠스 패슘 균에 의해 유발된 감염 또는 질환의 억제, 및 엔테로코쿠스 패슘 균에 의해 유발된 질환의 병적상태를 경감시키는 모든 행위를 의미한다.As used herein, the term “treatment” or “treatment” refers to any action that reduces the pathological condition of an infection or disease caused by Enterococcus faecium, and a disease caused by Enterococcus fasium. it means.
이러한 활용 목적에서의 효율성을 높이기 위하여 다른 세균 종에 대하여 항균활성을 제공할 수 있는 항균물질들이 본 발명의 약학적 조성물에 추가될 수 있다. In order to increase the effectiveness for this purpose of utilization, antibacterial substances capable of providing antibacterial activity against other bacterial species may be added to the pharmaceutical composition of the present invention.
본 발명의 약학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.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, and the like. it's 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.
본 발명의 약학적 조성물은 경구 투여 또는 비경구 투여를 통해 투여할 수도 있으며 비경구 투여의 경우 정맥 내 투여, 복강 내 투여, 근육 내 투여, 피하 투여 또는 국부 투여를 이용하여 투여할 수도 있으며, 그 밖에 질환 부위에의 도포 또는 분무하는 방법으로도 이용될 수 있으나 이에 제한되지는 않는다.The pharmaceutical composition of the present invention may be administered through oral administration or parenteral administration, and in the case of parenteral administration, it may be administered using intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or local administration, It may also be used as a method of application or spraying outside the diseased area, but is not limited thereto.
본 발명의 약학적 조성물은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 됨으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수도 있다. 이때 제형은 오일 또는 수성 매질 중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캡슐제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수도 있다.The pharmaceutical composition of the present invention is prepared in 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 placing it in 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.
또한, 상기 약학적 조성물의 적합한 도포, 분무 및 투여량은 제제화 방법, 투여 방식, 연령, 체중, 성, 질병 증상의 정도, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사나 수의사는 소망하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.In addition, suitable application, spraying and dosage of the pharmaceutical composition depend on factors such as formulation method, administration method, age, 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.
본 발명의 약학적 조성물은 항생제, 소독제, 살균제, 치료제 등으로 구현될 수 있다. 본 명세서에 있어서, '항생제’라는 용어는 방부제, 살균제 및 항균제를 총칭한다. 또한, 본 발명의 약학적 조성물은 식품 조성물 형태로도 구현될 수 있다.The pharmaceutical composition of the present invention may be embodied as an antibiotic, an antiseptic, a bactericide, a therapeutic agent, and the like. In the present specification, the term 'antibiotic' refers to a preservative, a disinfectant, and an antibacterial agent. In addition, the pharmaceutical composition of the present invention may be implemented in the form of a food composition.
본 발명에 따른 서열번호 1로 표시되는 아미노산 서열을 갖는 항균단백질 EFL200을 유효성분으로 포함하는 약학적 조성물을 이용한 엔테로코쿠스 패슘 균의 감염 및 질환 치료 방법은 일반적인 엔테로코쿠스 패슘 균뿐만 아니라 기존 항생제들이나 항균물질들에 대하여 내성을 획득한 엔테로코쿠스 패슘 균에도 효과적으로 작용할 수 있다. 한편, 본 발명의 엔테로코쿠스 패슘 균 특이 항균단백질 EFL200은 엔테로코쿠스 패슘 균 외의 다른 체내의 정상 상재균에는 영향을 주지 않아 항균단백질 EFL200을 유효성분으로 포함하고 있는 약학적 조성물의 사용에 따른 부작용을 최소화 시켜 줄 수 있다. 기존 항생제들의 경우에는 체내의 유익균들에도 악영향을 초래하여 여러 가지의 부작용을 유발시키는 단점을 나타내었다.Enterococcus fasium infection and disease treatment method using a pharmaceutical composition comprising the antibacterial protein EFL200 having the amino acid sequence represented by SEQ ID NO: 1 according to the present invention as an active ingredient, as well as general Enterococcus fasium bacteria, as well as existing antibiotics It can also effectively act on Enterococcus fasium bacteria, which have acquired resistance to bacteria and antibacterial substances. On the other hand, the Enterococcus fasium-specific antibacterial protein EFL200 of the present invention does not affect the normal flora in the body other than Enterococcus fasium. can be minimized. Existing antibiotics have a disadvantage of causing adverse effects on beneficial bacteria in the body, causing various side effects.
도 1은 서열번호 1로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200을 재조합 단백질 형태로 제조한 결과를 보여주는 전기영동 사진으로서, 레인 M은 단백질 크기 마커이고, 레인 1부터 레인 3은 정제 시의 크로마토그래피 통과액이고, 레인 4부터 레인 10은 정제 분획이다.1 is an electrophoresis photograph showing the result of preparing Enterococcus fasium-specific antibacterial protein EFL200 in the form of a recombinant protein, characterized in that it includes the amino acid sequence represented by SEQ ID NO: 1, and lane M is a protein size marker. , Lanes 1 to 3 are chromatography flow-throughs during purification, and lanes 4 to 10 are purification fractions.
도 2는 항균단백질 EFL200의 엔테로코쿠스 패슘 균 (하단에 VRE 표시)에 대한 항균활성(용균활성)을 보여주는 결과로서, 투명한 부분은 항균단백질 EFL200의 항균활성(용균활성)에 의해 생성된 것이다. 대조군(Buffer control)은 항균단백질 EFL200을 포함하지 않은 완충액 자체를 사용하였으며, 용균환의 형성 유무가 항균단백질 EFL200의 항균활성을 나타낸다.2 is a result showing the antibacterial activity (lytic activity) of the antibacterial protein EFL200 against Enterococcus fasium (VRE indicated at the bottom), and the transparent part is generated by the antibacterial activity (lytic activity) of the antibacterial protein EFL200. As a control (Buffer control), a buffer solution not containing the antibacterial protein EFL200 was used, and the presence or absence of the lysis ring indicates the antibacterial activity of the antibacterial protein EFL200.
도 3은 항균단백질 EFL200의 엔테로코쿠스 패슘 균을 대상으로 실시한 탁도감소조사법(Turbidity reduction assay)의 실험 결과를 보여주는 결과이다. 음성대조군(Negative control)은 항균단백질 EFL200을 포함하지 않은 완충액 자체를 사용하였으며, 가로축은 시간(분)이고 세로축은 600 nm에서의 흡광도이다.Figure 3 is a result showing the experimental results of the turbidity reduction assay (Turbidity reduction assay) performed on Enterococcus fasium bacteria of the antibacterial protein EFL200. The negative control group used the buffer itself without antibacterial protein EFL200. The horizontal axis is time (minutes) and the vertical axis is absorbance at 600 nm.
도 4는 항균단백질 EFL200의 엔테로코쿠스 패슘 균을 대상으로 실시한 소화효소에 대한 분해 조사법(Digestive enzyme degradation test)의 실험 결과이다. 음성대조군(Negative control)은 항균단백질 EFL200을 포함하지 않은 소화효소만을 사용하였으며, 양성대조군(Positive control)은 항균단백질 EFL200만을 포함한다. 레인 M은 단백질 크기 마커이고, 레인 1부터 레인 7번까지는 10분마다 얻은 시료이다.4 is an experimental result of the digestive enzyme degradation test performed on Enterococcus fasium bacteria of the antibacterial protein EFL200. The negative control group used only digestive enzymes that did not contain the antibacterial protein EFL200, and the positive control group contained only the antibacterial protein EFL200. Lane M is a protein size marker, and lanes 1 to 7 are samples obtained every 10 minutes.
이하, 실시예에 의거하여 본 발명을 보다 구체적으로 설명하지만, 이들 실시예는 본 발명의 예시일 뿐이며 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.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.
실시예 1: 엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200의 제조Example 1: Preparation of Enterococcus fasium-specific antibacterial protein EFL200
엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200의 제조에 대하여 이하에 설명한다. 본 발명자들이 최종 고안한 서열번호 1의 아미노산 서열을 갖는 항균단백질 EFL200의 제조를 위하여 서열번호 2의 유전자 서열을 갖는 발현 플라스미드를 통상의 분자생물학적 방법을 통하여 제작하였고 이를 대장균에 도입시켜 형질전환된 생산균주 TOP10-EFL200(수탁번호 KCTC 14364BP)을 제작하였다. The preparation of Enterococcus fasium-specific antibacterial protein EFL200 will be described below. For the production of the antibacterial protein EFL200 having the amino acid sequence of SEQ ID NO: 1 that the present inventors finally designed, an expression plasmid having the gene sequence of SEQ ID NO: 2 was prepared through a conventional molecular biological method, and transformed by introducing it into E. coli Strain TOP10-EFL200 (Accession No. KCTC 14364BP) was prepared.
이하 생산균주 TOP10-EFL200(수탁번호 KCTC 14364BP)을 사용한 항균단백질 EFL200 제조에 대하여 설명한다. 50 μg/ml 카나마이신이 포함된 LB배지(Tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) 20 ml에 TOP10-EFL200(수탁번호 KCTC 14364BP)을 20 μl 첨가하여 접종한 다음 37℃에서 한밤 동안 진탕 배양하였다. 다음날, 50 μg/ml 카나마이신이 포함된 LB배지 0.5 L에 한밤 배양한 배양액을 1/100 부피비로 첨가하였다. 220 rpm의 교반속도로, 37℃ 온도 조건에서 배양을 실시하였다. 세포 농도가 600 nm에서의 흡광도 기준으로 0.5가 되었을 때, 최종 농도가 0.2 μg/ml이 되도록 언하이드로테트라사이클린(Anhydrotetracycline)을 첨가하여 서열번호 1로 표시되는 아미노산 서열의 항균단백질 EFL200의 발현을 유도하였다. 발현 유도 후에 3시간 배양을 추가 실시하였다.Hereinafter, the production of antibacterial protein EFL200 using the production strain TOP10-EFL200 (Accession No. KCTC 14364BP) will be described. 20 μl of TOP10-EFL200 (Accession No. KCTC 14364BP) was added to 20 ml of LB medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) containing 50 μg/ml kanamycin, and then inoculated Incubated with shaking at 37°C overnight. The next day, the overnight culture solution was added to 0.5 L of LB medium containing 50 μg/ml kanamycin in a 1/100 volume ratio. At a stirring speed of 220 rpm, culture was carried out at 37°C. When the cell concentration reached 0.5 based on the absorbance at 600 nm, anhydrotetracycline was added so that the final concentration was 0.2 μg/ml to induce the expression of the antibacterial protein EFL200 of the amino acid sequence shown in SEQ ID NO: 1. did. After induction of expression, culture was further performed for 3 hours.
배양 종료 후, 세포 배양액을 취하여 7,000 rpm에서 15분간 4℃에서 원심분리하여 세포 침전물을 회수하였고, 회수한 세포 침전물은 30 ml의 완충액(50 mM K3PO4, pH 7.0)에 부유시켰다. 이렇게 준비된 세포 부유액을 초음파 분쇄법을 이용하여 세포를 파쇄하였고, 상기 초음파 분쇄법은 3초간 초음파를 가하여 세포를 깨고, 3초간 멈추는 조건으로 총 5분간 반복하여 얼음조(Ice bath)에서 실시하였다. 세포 파쇄 후에 세포 파쇄액을 7,000 rpm에서 15분간 4℃에서 원심분리하여 얻어진 상등액을 통상의 양이온-교환 크로마토그래피(Cation-exchange chromatography) 정제 공정을 통하여 정제하였다.After completion of the culture, the cell culture solution was taken and centrifuged at 7,000 rpm at 4° C. for 15 minutes to recover cell precipitates, and the recovered cell precipitates were suspended in 30 ml of buffer (50 mM K 3 PO 4 , pH 7.0). The cell suspension thus prepared was disrupted by sonication, and the sonication method was repeated in an ice bath for a total of 5 minutes under the condition that ultrasonic waves were applied for 3 seconds to break the cells and stopped for 3 seconds. After cell disruption, the supernatant obtained by centrifuging the cell disruption solution at 7,000 rpm for 15 minutes at 4° C. was purified through a conventional cation-exchange chromatography purification process.
정제 공정을 간단히 설명하면 다음과 같다. 본 실시예에서는 양이온-교환수지(Cation-exchange resin)로 5 ml의 HiTrapTM SP FF(GE Healthcare)를 사용하였다. 크로마토그래피는 칼럼을 Buffer A(50 mM K3PO4, pH 7.0)로 미리 평형화시킨 후에 실시하였고, 시료를 칼럼에 적하(Loading)한 다음에는 5 ml/min의 유량(Flow rate)으로 상기 buffer A를 10 CV(Column Volume) 흘려주어 세척을 실시하였다. 세척 후에는 5 ml/min의 유량으로 buffer A에서 buffer B(50 mM K3PO4, 1 M NaCl, pH 7.0)로의 농도 기울기(Gradient)가 0%에서 100%가 되게 하는 조건으로 크로마토그래피를 수행하였다. 이 과정에서 목적하는 서열번호 1로 표시되는 아미노산 서열의 항균단백질 EFL200의 용출이 달성되었다. 도 1에 크로마토그래피 정제 공정을 이용하여 정제한 항균단백질 EFL200의 전기영동 분석 결과가 제시되어 있다.A brief description of the purification process is as follows. In this example, 5 ml of HiTrap TM SP FF (GE Healthcare) was used as a cation-exchange resin. Chromatography was performed after pre-equilibrating the column with Buffer A (50 mM K 3 PO 4 , pH 7.0), and after loading the sample into the column, the buffer at a flow rate of 5 ml/min. A was washed by flowing 10 CV (Column Volume). After washing, chromatography was performed under conditions such that the gradient of concentration from buffer A to buffer B (50 mM K 3 PO 4 , 1 M NaCl, pH 7.0) from 0% to 100% at a flow rate of 5 ml/min. carried out. In this process, the elution of the antimicrobial protein EFL200 of the desired amino acid sequence represented by SEQ ID NO: 1 was achieved. 1 shows the results of electrophoresis analysis of the antibacterial protein EFL200 purified using the chromatography purification process.
확보된 정제 분획 중에 항균단백질 EFL200을 고농도로 포함하고 있는 분획들(도 1에서의 레인 4부터 레인 9에 상응하는 정제 분획들)을 모았고, 완충액(50 mM K3PO4, pH 7.0)에 대하여 투석(Dialysis)을 실시하여 매질 교환을 수행하였다. 이를 통하여 90% 이상의 순도를 갖는 항균단백질 EFL200 용액을 확보할 수 있었다. Among the obtained purified fractions, fractions containing the antimicrobial protein EFL200 at a high concentration (purified fractions corresponding to lanes 4 to 9 in FIG. 1) were collected, and a buffer (50 mM K3PO4, pH 7.0) was subjected to dialysis to perform medium exchange. Through this, it was possible to secure an antibacterial protein EFL200 solution having a purity of 90% or more.
실시예 2: 점적 실험을 통한 항균단백질 EFL200의 항균활성 조사Example 2: Investigation of antibacterial activity of antibacterial protein EFL200 through drip experiment
본 발명자들은 항균단백질 EFL200의 항균활성을 통상의 점적 실험(Spot-on-lawn assay)을 통하여 조사해 보았다. 실험에서는 엔테로코쿠스 패슘(Enterococcus faecium) 5주, 엔테로코쿠스 패칼리스(Enterococcus faecalis) 2주, 황색포도상구균(Staphylococcus aureus) 2주, 살모넬라(Salmonella) 2주, 및 대장균 2주를 대상으로 하였다. 상기 박테리아는 미국의 The American Type Culture Collection(ATCC) 등의 외부기관으로부터 분양을 받았거나, 또는 본 발명자들에 의해 분리 및 동정된 것들이었다. The present inventors investigated the antibacterial activity of the antibacterial protein EFL200 through a conventional spot-on-lawn assay. In the experiment, Enterococcus faecium 5 weeks, Enterococcus faecium ( Enterococcus ) faecalis ) 2 weeks, Staphylococcus aureus ) 2 weeks, Salmonella 2 weeks, and Escherichia coli 2 weeks were the subjects. The bacteria were purchased from an external institution such as The American Type Culture Collection (ATCC) in the United States, or those isolated and identified by the present inventors.
실험 방법은 TSA 배지에 37℃ 배양기에서 한밤 진탕 배양한 박테리아 배양액 1 mL을 각각 다른 평판배지에 도말한 후 잘 건조되도록 약 30분간 상온 방치시켰다. 건조된 TSA 배지를 37℃ 배양기에서 5시간 배양하여 꺼낸 후 평판배지에서 박테리아가 자란 것을 확인한 다음에 항균단백질 EFL200 용액(농도: 1 μg/μl) 10 μL씩을 떨어뜨렸다. 대조군(Buffer control)은 EFL200이 포함되지 않은 완충액(50 mM K3PO4 , pH 7.0)을 사용하였다. 점적한 후 건조될 때까지 약 30분간 상온 방치한 다음 각 박테리아의 용균 정도를 관찰하였다. 그 결과, 항균단백질 EFL200은 엔테로코쿠스 패슘 균에 대해서만 항균활성(용균력)이 있었고 다른 균종들에 대해서는 항균활성이 없었다. 엔테로코쿠스 패슘 균에 대한 항균활성은 실험에서 대상이 된 엔테로코쿠스 패슘 5주 모두에 대해서 확인되었다. 항균단백질 EFL200의 엔테로코쿠스 패슘 균에 대한 항균활성 실험 결과를 도 2에 제시하였다.As for the experimental method, 1 mL of a bacterial culture solution cultured overnight at 37 ° C in TSA medium was smeared on different plate media, and then left at room temperature for about 30 minutes to dry well. After incubating the dried TSA medium in an incubator at 37°C for 5 hours and taking it out, it was confirmed that the bacteria grew on the plate medium, and then 10 μL of antibacterial protein EFL200 solution (concentration: 1 μg/μl) was dropped. As a control (Buffer control), a buffer solution without EFL200 (50 mM K 3 PO 4 , pH 7.0) was used. After instillation, it was left at room temperature for about 30 minutes until it dried, and then the degree of lysis of each bacteria was observed. As a result, the antibacterial protein EFL200 had antibacterial activity (dissolution power) only against Enterococcus fasium bacteria, and had no antibacterial activity against other bacterial species. Antibacterial activity against Enterococcus fasium was confirmed for all 5 strains of Enterococcus fasium that were the subject of the experiment. The results of the antimicrobial activity of the antibacterial protein EFL200 against Enterococcus fasium bacteria are presented in FIG. 2 .
이로부터 항균단백질 EFL200은 엔테로코쿠스 패슘 균에 대하여 우수한 용균력(항균력)을 제공할 수 있으며, 또한 엔테로코쿠스 패슘 균에 의해 유발되는 감염성 질환의 예방 또는 치료에 효과적으로 활용될 수 있음을 확인할 수 있었다.From this, it can be confirmed that the antibacterial protein EFL200 can provide excellent bactericidal power (antibacterial power) against Enterococcus faecium, and can also be effectively used for preventing or treating infectious diseases caused by Enterococcus faecium. there was.
실시예 3: 탁도감소조사법을 통한 항균단백질 EFL200의 항균활성 조사Example 3: Investigation of antibacterial activity of antibacterial protein EFL200 through turbidity reduction investigation method
실시예 2에서 시험대상이 된 엔테로코쿠스 패슘 균주 5종에 대하여 탁도감소조사법(Turbidity reduction assay)을 통해 항균단백질 EFL200의 항균활성을 조사하였다. 탁도감소조사법의 실험 방법은 다음과 같다. 생리식염수에 시험대상 박테리아를 600 ㎚에서 흡광도가 0.7 정도가 되도록 부유시킨 다음에 이 부유액 0.9 ml에 항균단백질 EFL200 용액을 0.1 ml을 첨가(최종 농도: 10 μg/ml)한 후에 600 ㎚에서 흡광도를 30분간 측정하는 방식으로 실시하였다. 음성대조로는 항균단백질 EFL200 용액 대신에 항균단백질 EFL200을 포함하지 않은 완충액(50 mM K3PO4, pH 7.0)을 사용하였다. The antibacterial activity of the antibacterial protein EFL200 was investigated through a turbidity reduction assay for 5 Enterococcus fasium strains tested in Example 2. The experimental method of the turbidity reduction investigation method is as follows. After suspending the test bacteria in physiological saline so that the absorbance at 600 nm becomes about 0.7, 0.1 ml of antibacterial protein EFL200 solution is added to 0.9 ml of this suspension (final concentration: 10 μg/ml), and the absorbance at 600 nm is measured. It was carried out in a manner of measuring for 30 minutes. As a negative control, a buffer solution (50 mM K 3 PO 4 , pH 7.0) not containing the antimicrobial protein EFL200 was used instead of the antimicrobial protein EFL200 solution.
실험 결과, 5개 균주에 대하여 유사한 실험 결과를 확인할 수 있었는데, 항균단백질 EFL200은 시험대상 박테리아를 신속히 용균시켜 흡광도를 떨어뜨렸다. 이러한 신속한 용균 효과는 기존의 어떤 항생제들도 제공하지 못했던 본 발명에 따른 항균단백질 EFL200의 특성이라 할 수 있다. 엔테로코쿠스 패슘 균주 5종에 대한 실험 결과를 도 3에 제시하였다.As a result of the experiment, similar experimental results were confirmed for the five strains, and the antibacterial protein EFL200 rapidly lysed the test bacteria and lowered the absorbance. This rapid lytic effect can be said to be a characteristic of the antibacterial protein EFL200 according to the present invention, which was not provided by any existing antibiotics. Experimental results for 5 Enterococcus fasium strains are presented in FIG. 3 .
이상의 결과로 본 발명의 엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200이 엔테로코쿠스 패슘 균을 용균시킴으로 결국 사멸시킬 수 있음을 확인할 수 있었다. 이러한 특성은 엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200을 포함하는 약학적 조성물이 엔테로코쿠스 패슘 균 감염 시에 엔테로코쿠스 패슘 균 사멸 목적으로 활용될 수 있음을 보여 주고, 또한 엔테로코쿠스 패슘 균 감염 처치 목적으로 통상의 항생제와 같은 방식으로 활용될 수 있음을 보여준다.As a result of the above, it was confirmed that the Enterococcus fasium-specific antibacterial protein EFL200 of the present invention could eventually be killed by lysing the Enterococcus fasium bacteria. These characteristics show that a pharmaceutical composition containing the Enterococcus fasium-specific antibacterial protein EFL200 can be utilized for the purpose of killing Enterococcus fasium during Enterococcus fasium infection, and also Enterococcus fasium It shows that it can be used in the same way as conventional antibiotics for the purpose of treating infections.
실시예Example
4: 소화효소에 의한 분해 실험을 통한 4: Through digestion test by digestive enzymes
항균단백질antibacterial protein
EFL200의EFL200's
소화효소에 대한 안정성 조사 Stability study for digestive enzymes
소화효소에 의한 분해 실험을 진행하여 항균단백질 EFL200의 소화효소에 대한 안정성을 조사하였다. 실험 방법은 다음과 같았다. 5 ml의 Simulated Intestinal Fluids(SIF, 50 mM KH2PO4, 0.015 N NaOH, pH 6.8) 용액에 0.05 g의 Pancreatin(Sigma-Aldrich, Cat. No. P1625)을 용해시켜 SIF-P 용액을 제조하였다. 이렇게 준비된 SIF-P 용액 200 μl에 항균단백질 EFL200의 최종 양이 100 μg이 되도록 첨가한 후, 37℃ 조건에서 60분간 정치하였다. 음성대조군은 항균단백질 EFL200 용액 대신에 항균단백질 EFL200을 포함하지 않은 완충액(50 mM K3PO4, pH 7.0)을 사용하였고, 양성대조군은 SIF 용액에 항균단백질 EFL200 용액을 첨가하여 60분간 반응시킨 반응액을 사용하였다. 60 μl의 0.1 N HCl이 분주된 1.5 ml tube에 매 10분마다 회수한 반응액 20 μl를 넣은 후, 그 중 32 μl를 취해 8 μl의 5× sample buffer [10% SDS, 500 mM DTT, 50% glycerol, 0.5% bromophenol blue dye, 250 mM Tris-HCl(pH 6.8)]가 담겨진 1.5 ml tube에 첨가 (EFL200의 양: 1 μg/SDS-PAGE well)하였다. 그리고 위 tube들을 95℃에서 4분간 끓인 후 전기영동을 실시하였다. 실험 결과, 항균단백질 EFL200이 소화효소(Pancreatin)에 대하여 30분 동안 안정적임을 확인할 수 있었다. 실험 결과를 도 4에 제시하였다.The stability of the antibacterial protein EFL200 to the digestive enzymes was investigated by conducting a digestion test by digestive enzymes. The experimental method was as follows. A SIF-P solution was prepared by dissolving 0.05 g of Pancreatin (Sigma-Aldrich, Cat. No. P1625) in 5 ml of Simulated Intestinal Fluids (SIF, 50 mM KH 2 PO 4 , 0.015 N NaOH, pH 6.8) solution. . To 200 μl of the SIF-P solution thus prepared, the final amount of the antimicrobial protein EFL200 was added to 100 μg, and then left at 37° C. for 60 minutes. The negative control group used a buffer solution (50 mM K 3 PO 4 , pH 7.0) not containing the antibacterial protein EFL200 instead of the antibacterial protein EFL200 solution, and the positive control group added the antibacterial protein EFL200 solution to the SIF solution and reacted for 60 minutes. liquid was used. After adding 20 μl of the recovered reaction solution every 10 minutes to a 1.5 ml tube in which 60 μl of 0.1 N HCl is dispensed, 32 μl of it is taken and 8 μl of 5× sample buffer [10% SDS, 500 mM DTT, 50 % glycerol, 0.5% bromophenol blue dye, 250 mM Tris-HCl (pH 6.8)] was added to a 1.5 ml tube (Amount of EFL200: 1 μg/SDS-PAGE well). Then, the above tubes were boiled at 95°C for 4 minutes, and then electrophoresis was performed. As a result of the experiment, it was confirmed that the antibacterial protein EFL200 was stable for 30 minutes with respect to the digestive enzyme (Pancreatin). The experimental results are presented in FIG. 4 .
실시예Example
5: 5:
엔테로코쿠스Enterococcus
패슘fasium
균 특이적 fungal specific
항균단백질antibacterial protein
EFL200의EFL200's
엔테로코쿠스Enterococcus
패슘 균 감염에 대한 치료적 효과 조사 Investigation of the therapeutic effect on Pasium infection
엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200의 엔테로코쿠스 패슘 균 감염에 대한 치료적 효과를 감염 동물 모델을 사용하여 조사하였다. The therapeutic effect of Enterococcus fasium-specific antibacterial protein EFL200 on Enterococcus fasium infection was investigated using an infected animal model.
약 20 g 내외 체중의 5주령 ICR 마우스[specific pathogen-free(SPF) grade]를 실험동물로 사용하였다. 총 20마리를 2개의 군으로 분리(군당 10마리씩)한 다음에 정맥투여 방식으로 각 마우스에 엔테로코쿠스 패슘 균 1× 108 cfu(즉, 1× 108 cfu/mouse)를 투여하여 감염을 유도하였다. 하나의 군(처치군)에 대해서는 균 강제 감염 후에 30분 경과 시점, 12시간 경과 시점, 및 24시간 경과 시점에 항균단백질 EFL200 용액(10 mg/ml) 0.2 ml을 정맥 투여하였다. 또 다른 군(대조군)에 대해서는 완충액(50 mM K3PO4, pH 7.0)만을 동일 부피로 정맥 투여하였다. 완충액 투여는 항균단백질 EFL200 용액 투여와 동일하게 균 강제 감염 후에 30분 경과 시점, 12시간 경과 시점, 및 24시간 경과 시점에 실시하였다. 균 강제 감염 후로부터 5일 동안 매일 사망 개체 수를 조사하였고 그 결과를 표 1에 제시하였다. 또한, 특이 현상의 관찰여부도 매일 오전 및 오후로 하루에 2번씩 조사하였다. Five-week-old ICR mice [specific pathogen-free (SPF) grade] weighing about 20 g were used as experimental animals. After dividing a total of 20 animals into 2 groups (10 mice per group), 1×10 8 cfu of Enterococcus fasium (ie, 1×10 8 cfu/mouse) was administered to each mouse by intravenous administration to prevent infection. induced. For one group (treatment group), 0.2 ml of antimicrobial protein EFL200 solution (10 mg/ml) was intravenously administered at 30 minutes, 12 hours, and 24 hours after forced bacterial infection. For another group (control group), only buffer (50 mM K 3 PO 4 , pH 7.0) was intravenously administered in the same volume. Buffer administration was performed at 30 minutes, 12 hours, and 24 hours after forced bacterial infection in the same manner as the administration of the antimicrobial protein EFL200 solution. The number of deaths was investigated every day for 5 days after the forced bacterial infection, and the results are presented in Table 1. In addition, the observation of unusual phenomena was also investigated twice a day, in the morning and in the afternoon.
실험 결과로서, 확연한 치료 효과가 확인되었다. 본 발명의 항균단백질 EFL200의 투여가 감염 동물의 생존율에서 확연한 개선을 제공하였다. 또한, 대조군에서는 안검염의 발적(Erythema of lid margin), 활동성 감소 등의 다양한 특이 반응들이 관찰됨에 비교하여 항균단백질 EFL200 투여군에서는 그러한 특이 반응이 관찰되지 않았다.As a result of the experiment, a clear therapeutic effect was confirmed. Administration of the antimicrobial protein EFL200 of the present invention provided a marked improvement in the survival rate of infected animals. In addition, in the control group, various specific reactions such as redness of blepharitis (Erythema of lid margin) and decreased activity were observed, whereas in the group administered with the antimicrobial protein EFL200, no such specific reaction was observed.
| 군army | 사망 개체 수number of deaths | 사망 개체 수 /시험 개체 수Number of dead / number of test subjects | 폐사율 (%)Mortality (%) | ||||
| 균 강제 감염 후 경과 일Elapsed days after bacterial forced infection | |||||||
| 1One | 22 | 33 | 44 | 55 | |||
|
대조군 |
00 | 33 | 33 | 1One | 00 | 7/107/10 | 7070 |
|
처치군 |
00 | 00 | 00 | 00 | 00 | 0/100/10 | 00 |
이상의 결과로 본 발명의 항균단백질 EFL200이 엔테로코쿠스 패슘 균의 감염 치료에 효과적임을 확인할 수 있었다. 이러한 특성은 본 발명의 항균단백질 EFL200을 유효성분으로 포함된 약학적 조성물이 엔테로코쿠스 패슘 균 감염 치료 목적으로 활용될 수 있음을 보여주고, 또한 엔테로코쿠스 패슘 균의 감염 치료 목적으로 통상의 항생제와 같은 방식으로 활용될 수 있음을 보여 준다.이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As a result of the above, it was confirmed that the antibacterial protein EFL200 of the present invention was effective in treating Enterococcus fasium infection. These characteristics show that the pharmaceutical composition containing the antibacterial protein EFL200 of the present invention as an active ingredient can be used for the purpose of treating Enterococcus fasium infection, and also a common antibiotic for the purpose of treating Enterococcus fasium infection. It shows that it can be utilized in the same way. As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific technique is only a preferred embodiment, and thus It is clear that the scope is not limited. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (6)
- 엔테로코쿠스 패슘(Enterococcus faecium) 균 특이적 항균활성을 갖고 서열번호 1로 표시되는 아미노산 서열을 포함하는 엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200. Enterococcus fasium faecium ) Enterococcus fasium-specific antibacterial protein EFL200 containing the amino acid sequence represented by SEQ ID NO: 1 having a bacteria-specific antibacterial activity.
- 제1항에 있어서, 상기 항균단백질 EFL200은 284개의 아미노산으로 구성되며 분자량이 30.7 kDa인 것을 특징으로 하는 엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200.According to claim 1, wherein the antibacterial protein EFL200 is Enterococcus fasium specific antibacterial protein EFL200, characterized in that it is composed of 284 amino acids and has a molecular weight of 30.7 kDa.
- 제1항의 엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200을 유효성분으로 포함하는 엔테로코쿠스 패슘 균 감염 또는 질환 처치용 약학적 조성물.A pharmaceutical composition for treating Enterococcus fasium infection or disease comprising the Enterococcus fasium-specific antibacterial protein EFL200 of claim 1 as an active ingredient.
- 제3항에 있어서, 상기 질환은 요로감염, 창상감염, 균혈증 및 심내막염으로 구성된 군으로부터 선택된 어느 하나의 질환인 것을 특징으로 하는 엔테로코쿠스 패슘 균 감염 또는 질환 처치용 약학적 조성물.The pharmaceutical composition for treating Enterococcus fasium infection or disease according to claim 3, wherein the disease is any one disease selected from the group consisting of urinary tract infection, wound infection, bacteremia and endocarditis.
- 제3항에 있어서, 상기 조성물은 식품 조성물, 항생제, 소독제, 살균제 또는 치료제 용도로 사용되는 것을 특징으로 하는 엔테로코쿠스 패슘 균 감염 또는 질환 처치용 약학적 조성물.[Claim 4] The pharmaceutical composition for treating Enterococcus fasium infection or disease according to claim 3, wherein the composition is used as a food composition, antibiotic, disinfectant, disinfectant or therapeutic agent.
- 서열번호 2의 유전자 서열로 표시되는 플라스미드를 사용하여 대장균을 형질전환시켜 제작한 균주 TOP10-EFL200(수탁번호: KCTC 14364BP)을 이용하여 제1항의 엔테로코쿠스 패슘 균 특이적 항균단백질 EFL200을 제조하는 방법.Using the strain TOP10-EFL200 (Accession No.: KCTC 14364BP) prepared by transforming E. coli using the plasmid represented by the gene sequence of SEQ ID NO: 2 to prepare the Enterococcus fasium-specific antibacterial protein EFL200 of claim 1 Way.
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