KR20200104130A - Bacteriophage of Klebsiella pneumoniae and uses thereof - Google Patents
Bacteriophage of Klebsiella pneumoniae and uses thereof Download PDFInfo
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- KR20200104130A KR20200104130A KR1020190022686A KR20190022686A KR20200104130A KR 20200104130 A KR20200104130 A KR 20200104130A KR 1020190022686 A KR1020190022686 A KR 1020190022686A KR 20190022686 A KR20190022686 A KR 20190022686A KR 20200104130 A KR20200104130 A KR 20200104130A
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- bacteriophage
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- klebsiella pneumoniae
- antibiotics
- klebsiella
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Abstract
Description
[0001] 본원은 박테리오파아지에 관한 것이다.[0001] The present application relates to a bacteriophage.
[0002] 병원균을 제거하기 위해 사용되는 항생제에 대한 내성을 갖는 균의 출현은 전세계적으로 큰 문제를 야기하고 있어, 항생제를 대체할 수 있는 물질의 개발이 시급한 실정이다.[0002] The emergence of bacteria resistant to antibiotics used to remove pathogens is causing a major problem worldwide, and the development of a substance capable of replacing antibiotics is urgent.
[0003] 박테리오파아지는 특정 박테리아를 특이적으로 감염하여, 많은 단백질이 관여하는 복잡한 융해(lysis)과정을 통해 박테리아를 사멸시키는 박테리아 바이러스이다. 박테리오파아지는 숙주 박테리아에 대한 특이성이매우 높아 항생제가 본격적으로 개발되기 전 항생제로서의 가능성이 높이 평가되었으나, 박테리오파아지에 대한부족한 이해 및 본격적인 항생제의 개발과 함께, 그 효용이 줄어드는 추세였다.[0003] Bacteriophage is a bacterial virus that specifically infects certain bacteria and kills bacteria through a complex lysis process involving many proteins. Bacteriophages are highly specific for host bacteria, so their potential as antibiotics was highly evaluated before the full-scale development of antibiotics, but with insufficient understanding of bacteriophages and the development of full-scale antibiotics, its utility tended to decrease.
[0004] 하지만 최근들어 박테리오파아지에 대한 연구결과의 축적과 함께 최근 항생제 남용으로 불거진 문제를 해결하기 위한 대안으로 박테리오파아지를 감염치료 및 방지 등에 사용하는 항생제로서의 개발이 증가하는 추세이다.However, in recent years, along with the accumulation of research results on bacteriophage, the development of antibiotics for use in the treatment and prevention of infections of bacteriophage as an alternative to solve the problem of recent antibiotic abuse is increasing.
[0005] 박테리오파아지를 인간 및 가축에 사용하는 것에 관하여 Merril 등 (J INat Rev Drug Discov 2(6):489-197, 2005)에 기술되어 있다.[0005] It is described in Merril et al. (J INat Rev Drug Discov 2(6):489-197, 2005) with respect to the use of bacteriophage in humans and livestock.
[0006] 미국특허 제7674467호는 살모넬라에 대한 박테리오파아지 및 그 용도에 관하여 개시하고 있다.[0006] US Patent No. 7674467 discloses a bacteriophage for Salmonella and its use.
[0007] 미국특허 제7622293호는 슈도모나스에 대한 박테리오파아지 및 그 용도에 관하여 개시하고 있다.[0007] US Patent No. 76222293 discloses a bacteriophage for Pseudomonas and its use.
[0008] 한국특허 제941892호는 살모넬라 갈리나움에 대한 박테리오파아지 및 그 용도에 관하여 개시하고 있다.[0008] Korean Patent No. 941892 discloses a bacteriophage and its use for Salmonella galinaum.
[0009] 하지만 박테리오파아지의 항균제 등으로서의 용도에 관한 다양한 개시에도 불구하고, 신규한 박테리오파아지에 대한 발굴 및 특정 용도에 적합하게 선별된 박테리오파아지에 대한 개발이 여전히 요구된다.However, despite the various disclosures regarding the use of bacteriophage as an antibacterial agent, etc., there is still a need for the discovery of a new bacteriophage and the development of a bacteriophage selected for a specific use.
[0010] 본원은 상기와 같은 문제점을 해결하기 위해 안출된 것으로 클렙시엘라 뉴모니아에 대한 특이성을 가지는 신규한 박테리오파아지를 제공하고자 한다.The present application is intended to provide a novel bacteriophage having specificity for Klebsiella pneumoniae as conceived to solve the above problems.
[0011] 한 양태에서 본원은 서열번호 1(GenBank accession number MG479124)의 서열을 가지고, 클렙시엘라 뉴모니아에 대한 융해능을 갖는 박테리오파아지를 제공한다. 한 구현예에서 본원에 따른 박테리오파아지는 클렙시엘라 뉴모니아의 융해능을 갖는다.[0011] In one aspect, the present application provides a bacteriophage having a sequence of SEQ ID NO: 1 (GenBank accession number MG479124), and having a melting ability for Klebsiella pneumoniae. In one embodiment, the bacteriophage according to the present invention has a melting ability of Klebsiella pneumoniae.
[0012] 본원은 또한 본원에 따른 박테리오파아지와 동일한 생물학적 특성을 나타내는, 프로지니 박테리오파아지를 제공한다.The present application also provides a progeny bacteriophage, which exhibits the same biological properties as the bacteriophage according to the present application.
[0013] 본원은 또한 한국미생물보존센터에 2018년 8월 7일자로 기탁번호 KFCC11779P로 기탁된 것인, 박테리오파아지를 제공한다.[0013] The present application also provides a bacteriophage that has been deposited with the accession number KFCC11779P as of August 7, 2018 in the Korea Microbial Conservation Center.
[0014] 본원에 따른 박테리오파아지는 기회감염성 균주인 클렙시엘라 뉴모니아(Klebsiella pneumoniae)를 특이적으로 감염하여, 다양한 항생제에 내성을 가져 의료계에서 크게 문제가 병원균인 클렙시엘라 균주를 효과적으로 제거하여 사람은 물론 가축의 항생제로서 사용될 수 있다.[0014] The bacteriophage according to the present application specifically infects the opportunistic strain, Klebsiella pneumoniae, and has resistance to various antibiotics, effectively removing the Klebsiella strain, which is a pathogen that is a major problem in the medical field Therefore, it can be used as an antibiotic for humans as well as livestock.
[0015] 도 1은 본원에서 규명된 박테리오파아지를 우라닐아세테이트로 네가티브 염색을 한 후 관찰 한 전자현미경 사진으로 스케일바는 100나노미터이다.
도 2은 원에서 규명된 박테리오파아지의 단백질 분석결과이다. 레인 1: 본원에 따른 파아지의 분석; 레인 2: 단백질 마커.
도 3는 본원에서 규명된 박테리오파아지의 원스텝 성장 곡선이다. L: 잠복기; B: 방출수.
도 4는 본원에서 규명된 박테리오파아지의 온도에 대한 안정성을 확인한 곡선이다.
도 5는 본원에서 규명된 박테리오파아지의 pH에 대한 안정성을 확인한 곡선이다. 1 is an electron microscope photograph observed after negative staining of the bacteriophage identified in the present application with uranyl acetate, the scale bar is 100 nanometers.
2 is a protein analysis result of the bacteriophage identified in the circle. Lane 1: Analysis of phage according to the present application; Lane 2: protein marker.
Figure 3 is a one-step growth curve of the bacteriophage identified herein. L: incubation period; B: discharge water.
Figure 4 is a curve confirming the stability to the temperature of the bacteriophage identified herein.
Figure 5 is a curve confirming the stability to the pH of the bacteriophage identified herein.
[0016] 클렙시엘라 뉴모니아에는 환경에 흔하게 존재하는 기회 감염 균주로 감염시 주 증상으로 폐렴 및 혈류감염을 일으키는 박테리아로 여러 항생제에 내성을 가진 변종이 발견되어 질병의 치료에 어려움을 가중시킴은 물론 의료장비 등에 생물막(biofilm)을 형성하여 교차감염을 일으켜 이의 해결이 시급한 실정이다.[0016] Klebsiella pneumoniae is an opportunistic strain that is common in the environment. It is a bacterium that causes pneumonia and bloodstream infection as the main symptoms when infected. A strain resistant to various antibiotics was found, which increases the difficulty in treating the disease. Of course, a biofilm is formed on medical equipment, etc., causing cross-infection, which is urgently needed.
[0017] 이에 본원에서 항생제에 비해 특정 박테리아를 표적으로 하여 선택적으로 제거할 수 있고, 숙주내에서 복제를통해 지속적인 효과를 볼 수 있으며, 부작용이 없는 클렙시엘라 뉴모니아에 특이적으로 작용하는 신규한 박테리오파아지를 분리하였다.[0017] Thus, compared to antibiotics herein, specific bacteria can be selectively removed by targeting, and sustained effects can be seen through replication in the host, and acting specifically on Klebsiella pneumoniae without side effects A novel bacteriophage was isolated.
[0018] 따라서 한 양태에서 본원은 서열번호 1의 서열을 갖는 클렙시엘라 뉴모니아에 대한 융해능을 갖는 박테리오파아지, 그 변이체, 유도체 및 프로지니(progeny)에 관한 것이다.Accordingly, in one aspect, the present application relates to a bacteriophage having a dissolving ability for Klebsiella pneumoniae having the sequence of SEQ ID NO: 1, its variants, derivatives and progeny.
[0019] 본원에 포함되는 변이체는 서열번호 1의 서열의 변이 또는 이에 의해 코딩되는 단백질에 변이를 갖는 것으로 본원에 따른 박테리오파아지와 유전적 및 표현형 특징에서 동일한 것이다.[0019] The variant included herein is the same in genetic and phenotypic characteristics as the bacteriophage according to the present application as having a mutation in the sequence of SEQ ID NO: 1 or a mutation in the protein encoded thereby.
[0020] 본원에서 사용된 용어 “프로지니“는 그 것이 유래된 본원의 박테리오파아지와 동일한 생물학적 특성을 나타내는 것으로, 연속적 계대 배양 또는 기타 공지된 다른 방법을 통하여 생성된 본원에 따른 박테리오파아지의 복제산물로, 서열번호 1의 서열과 실질적으로 동일하다. 서열번호 1의 경우 ”실질적으로 동일한“ 두 서열사이의 유사도를 나타내는 것으로 공지된 서열비교 프로그램 또는 수동비교 (visual inspection)을 통해 예를 들면, 90% 이상, 특히 95% 이상, 더욱 특히 99% 이상의 유사도를 의미하는 것으로, 아미노산 변이가 수반되거나 또는 되지 않는 핵산 서열의 변이체를 포함하는 것이다.[0020] The term "progeny" as used herein represents the same biological properties as the bacteriophage of the present application from which it is derived, and is a clone product of the bacteriophage according to the present application produced through continuous passage culture or other known methods As such, it is substantially identical to the sequence of SEQ ID NO: 1. In the case of SEQ ID NO: 1, for example, 90% or more, in particular 95% or more, more particularly 99% or more, through a sequence comparison program known to show the degree of similarity between two sequences that are "substantially identical" or through visual inspection. It refers to the degree of similarity, and includes variants of a nucleic acid sequence with or without amino acid mutations.
[0021] 본원에 사용된 용어 “유도체”는 본원에 따른 박테리오파아지 또는 그 프로지니를 구성하는 서브유니트 또는 발현산물을 의미하는 것으로, 예를 들면 박테리오파아지 유전체, 유전자 전부 또는 일부, 유전자 발현산물, 박테리오파아지를 구성하는 산물을 포함한다.[0021] The term “derivative” as used herein refers to a subunit or expression product constituting a bacteriophage or its progeny according to the present application, for example, a bacteriophage genome, all or part of a gene, a gene expression product, Contains products that make up bacteriophage.
[0022] 본원의 박테리오파아지는 클렙시엘라 뉴모니아를 특이적으로 감염한다. [0022] The bacteriophage of the present application specifically infects Klebsiella pneumoniae.
[0023] 다른 양태에서 본원의 박테리오파아지, 그 유도체, 그 변이체 또는 그 프로지니는 클렙시엘라 뉴모니아의 성장, 분열, 집락화의 조절, 억제 또는 제어에 사용될 수 있으며, 예를 들면 가공 또는 비가공된 식품의 처리, 보관, 각종 기구, 특히 의료장비 처리, 질병의 치료를 위한 항생제를 포함하는 약학적 조성물로서의 용도를 들수 있다.[0023] In another aspect, the bacteriophage of the present application, a derivative thereof, a variant thereof, or a progeny thereof may be used to control, inhibit or control the growth, division, colonization of Klebsiella pneumoniae, for example, processed or unprocessed The use as a pharmaceutical composition containing antibiotics for the treatment, storage, treatment of various devices, particularly medical equipment, and treatment of diseases are mentioned.
[0024] 한 측면에서 본원의 박테리오파아지는 클렙시엘라 뉴모니아로 인한 감염 예방을 위한 가공 비가공 식품 또는 식재료의 처리에 사용될 수 있다. 이 경우, 본원에 따른 박테리오파아지를 대상 식품, 식재료, 또는 장비에스프레이하거나, 또는 박테리오파아지를 포함하는 용액에 침지할 수 있다. 본원의 박테리오파아지는 다양한 식재료에 사용될 수 있으며, 예를 들면 소고기, 특히 분쇄 쇠고기, 분쇄 쇠고기로 만들어진 가공식품 예를 들면햄버거, 노지의 발에서 자라는 각종 야채, 예를 들면 상추, 시금치, 파, 음용수, 1차 감염된 식재료 등에 의해2차 감염의 우려가 있는 식품 들을 들 수 있으나, 이로 제한하는 것은 아니다.In one aspect, the bacteriophage of the present application can be used for processing unprocessed foods or ingredients for preventing infections caused by Klebsiella pneumoniae. In this case, the bacteriophage according to the present application may be sprayed on a target food, food material, or equipment, or immersed in a solution containing the bacteriophage. Bacteriophage of the present application can be used in a variety of ingredients, for example, beef, especially ground beef, processed foods made of ground beef, such as hamburgers, various vegetables grown on the feet of the field, such as lettuce, spinach, green onions, drinking water , Foods that are likely to cause secondary infection due to primary infected food ingredients, but are not limited thereto.
[0025] 본원의 박테리오파아지는 또한 클렙시엘라 뉴모니아에 의한 생물막 형성의 제어가 필요한, 가정, 환경, 의료 및 산업 분야에 걸쳐 사용되는 장비에 폭넓게 사용될 수 있다. 예를 들면, 각종 식재료 처리, 식품 가공 등에 사용되는 장비, 각종 의학기구, 의학장비, 의학시설/설비와 같은 물건은 물론 상피세포, 뼈, 치아, 및 혈관내벽 등을 포함하는 각종 생체 조직/기관과 같은 생체조직 및 생체에 적용되는 각종 인공 삽입 보형물에 사용될 수 있다.[0025] The bacteriophage of the present application may also be widely used in equipment used throughout the home, environment, medical and industrial fields, where control of biofilm formation by Klebsiella pneumoniae is required. For example, various biological tissues/organs including epithelial cells, bones, teeth, and inner walls of blood vessels, as well as items such as equipment used for processing various food materials, food processing, various medical instruments, medical equipment, medical facilities/equipment, etc. It can be used for various artificial implants applied to living tissues and living bodies, such as.
[0026] 본원의 박테리오파아지는 클렙시엘라 뉴모니아의 성장, 분열, 집락화의 조절, 제어, 억제 및/또는 생물막 형성의 방지 또는 저해에 효과적인 양으로 사용될 수 있다. 효과적인 양은 처리하고자 하는 대상의 종류 및 그 면적, 및 목적하는 억제/제어의 정도, 처리 시점 등을 포함하는 조건에 맞추어 결정될 수 있으며, 당업자라면 본원의 기재 및 표적 박테리오파아지에 관한 지식을 근거로 적절한 농도를 선택할 수 있을 것이다. 예를 들면 약 1 × 105 내지 1 × 1015 PFU(Plaque Forming Unit)/ml의 농도 또는 약 10-5 내지 105MOI(Multiplicity of Infection)로 사용될 수 있으며, 하루에 일회 내지 수회 및/또는 수일에 걸쳐 처리될 수 있으며, 초기에 처리하는 것이 유리할 수 있다. 당업자라면 박테리오파아지에 관한 문헌(Adams, M.H (1959), Methods of studybacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd. 443-519)을 참조하여 적절한 농도를 결정할 수 있을 것이다. 예를 들면 처리하고자하는 표면에 대하여 약 1ml/500cm2의 농도로 처리될 수 있다.[0026] The bacteriophage of the present application may be used in an amount effective for controlling, controlling, inhibiting and/or preventing or inhibiting the growth, division, colonization of Klebsiella pneumoniae. The effective amount can be determined according to conditions including the type and area of the object to be treated, the degree of inhibition/control, and the time of treatment, etc., and those skilled in the art may be appropriate based on the knowledge of the description of the present application and the target bacteriophage. You will be able to choose the concentration. For example, it can be used at a concentration of about 1 × 10 5 to 1 × 10 15 Plaque Forming Unit (PFU)/ml or about 10-5 to 105 MOI (Multiplicity of Infection), and once to several times a day and/or several days. It can be treated over, and it may be advantageous to treat it early. Those skilled in the art will be able to determine the appropriate concentration by referring to the literature on bacteriophage (Adams, MH (1959), Methods of studybacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd. 443-519). For example, the surface to be treated may be treated at a concentration of about 1ml/500cm 2 .
[0027] 본원의 박테리오파아지를 포함하는 조성물은 가정, 산업, 의학, 및 환경분야에서의 구체적인 목적에 맞추어 다양한 형태로 제조될 수 있다. 예를 들면 생체는 물론, 각종 장비, 기구의 세척을 위해, 클렙시엘라 뉴모니아의 성장, 집락화, 분열의 예방, 제어, 억제, 및/또는 조절에 유효한 양의 박테리오파아지를 포함하는 액체형태, 분무형태 또는 고체형태로 제조될 수 있으며, 목적에 따라 추가의 성분 예를 들면 이로 제한하는 것은 아니나, 계면활성제, 세정제, 제균제, 살균제, 항생제, 곰팡이제거제, 산도조절제, 염료, 색소와 같은 것을 포함할 수 있다.The composition comprising the bacteriophage of the present application may be prepared in various forms according to specific purposes in the fields of home, industry, medicine, and environment. For example, a liquid form containing bacteriophage in an amount effective to prevent, control, inhibit, and/or regulate the growth, colonization, and division of Klebsiella pneumoniae, for the cleaning of various equipment and devices as well as living organisms. , Spray or solid form, and depending on the purpose, additional ingredients such as, but are not limited to, surfactants, detergents, disinfectants, fungicides, antibiotics, fungicides, acidity regulators, dyes, pigments such as May include.
[0028] 한 구현예에서 본원의 박테리오파아지를 포함하는 클렙시엘라 뉴모니아의 성장, 분열, 집락화의 조절, 제어, 억제 및/또는 생물막 형성의 방지 또는 저해를 위한 조성물의 경우, 이러한 용도에 효과적인 양의 박테리오파아지를 포함하는 수용액 또는 현탁액으로 제조될 수 있다. 다른 구현예에서는 파우더, 코팅제, 스프레이, 분배형타입, 액체에 투입할 수 있는 캡슐/타블릿형태 등으로 제조될 수 있다. 이들 조성물은 음이온성, 비이온성, 양쪽 이온성 및 생물학적 계면활성제 및 이들의 혼합물과 같은 계면활성제를 추가로 포함할 수 있다.[0028] In one embodiment, in the case of a composition for controlling, controlling, inhibiting and/or preventing or inhibiting the growth, division, colonization of Klebsiella pneumoniae comprising the bacteriophage of the present application, for such use It can be prepared as an aqueous solution or suspension containing an effective amount of bacteriophage. In another embodiment, it may be manufactured in powder, coating, spray, dispensing type, capsule/tablet form that can be injected into a liquid. These compositions may further comprise surfactants such as anionic, nonionic, amphoteric and biological surfactants and mixtures thereof.
[0029] 다른 구현예에서는 개인위생용품, 예를 들면, 컨택렌즈 소독약, 비누, 샤워젤, 샴푸, 또는 치아, 인공치아 또는 구강의 세정, 세척을 위한 치실, 치약, 치분 또는 가글링 세정제의 형태로 제조될 수 있다.In another embodiment, in the form of a personal hygiene product, for example, a contact lens disinfectant, soap, shower gel, shampoo, or tooth, artificial tooth or oral cleaning, floss for cleaning, toothpaste, tooth powder or gargling detergent Can be manufactured.
[0030] 다른 구현예에서 의학용 장비/기구/설비의 세정, 세척을 위한 파우더, 코팅제, 스프레이, 물수건(wipe) 형태로 제조될 수 있다.In another embodiment, it may be prepared in the form of cleaning of medical equipment / instruments / facilities, powder for washing, coating, spray, wipe.
[0031] 다른 구현예에서, 본원의 박테리오파아지를 포함하는 생물막 형성 방지 또는 저해제는 식품가공, 수(water)처리장치와 같은 산업환경에서 사용될 수 있으며, 첨가제, 액체, 코팅제의 형태로 제조될 수 있다. 첨가제로 제조될 경우, 수관, 쿨링타워, 열교환기에 사용될 수 있다.[0031] In another embodiment, the biofilm formation prevention or inhibitor comprising the bacteriophage of the present application may be used in industrial environments such as food processing and water treatment devices, and may be prepared in the form of additives, liquids, and coatings. have. When prepared with additives, it can be used in water pipes, cooling towers, and heat exchangers.
[0032] 다른 측면에서 본원은 또한 본원에 따른 박테리오파아지를 포함하는 약학적 조성물에 관한 것이다. 본원에 따른 박테리오파아지는 클렙시엘라 뉴모니아를 특이적으로 사멸시키므로, 이에 의해 유발되는 각종 질환에 대한 치료, 증상의 경감, 예방 및/또는 억제에 유용하게 사용될 수 있다. 질환의 예로는, 클렙시엘라 뉴모니아에 의한 폐렴, 혈류감염 등을 들 수 있다. 구체적으로 폐렴, 혈류감염, 뇌수막염, 요로감염 등을 포함하나 이에 국한되지 않는다.In another aspect, the present application also relates to a pharmaceutical composition comprising the bacteriophage according to the present application. Since the bacteriophage according to the present application specifically kills Klebsiella pneumoniae, it can be usefully used for treatment, symptom relief, prevention and/or inhibition of various diseases caused thereby. Examples of the disease include pneumonia and bloodstream infection caused by Klebsiella pneumoniae. Specifically, it includes, but is not limited to, pneumonia, bloodstream infection, meningitis, urinary tract infection, and the like.
[0033] 본원의 약학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필 히드록시벤조에이트, 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.[0033] A pharmaceutically acceptable carrier included in the pharmaceutical composition of the present application is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, etc. It is not limited thereto. 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.
[0034] 본 발명의 약학적 조성물은 질환 부위에의 도포 또는 분무하는 방법으로 이용할 수 있으며, 그 밖에 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 있다.[0034] The pharmaceutical composition of the present invention may be used as a method of applying or spraying to a diseased area, and may be administered through oral administration or parenteral administration, and in the case of parenteral administration, intravenous administration, intraperitoneal administration Administration, intramuscular administration, subcutaneous administration, or topical administration may be used.
[0035] 본 발명의 약학적 조성물의 적합한 도포, 분무 및 투여량은 제제화 방법, 투여 방식, 대상이 되는 동물 및 환자의 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. 일반적으로, 본 발명의 약학적 조성물은 약 1 × 105 내지 1 × 1015 PFU/ml의 농도로 투여될 수 있다.[0035] Appropriate application, spray and dosage of the pharmaceutical composition of the present invention include formulation method, mode of administration, age, weight, sex, degree of disease symptoms, food, administration time, route of administration, It varies depending on factors such as the rate of excretion and response sensitivity, and usually an experienced physician or veterinarian can easily determine and prescribe the effective dosage for the desired treatment. In general, the pharmaceutical composition of the present invention may be administered at a concentration of about 1 × 10 5 to 1 × 10 15 PFU/ml.
[0036] 본 발명의 약학적 조성물은 본 발명이 속하는 기술분야에서 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 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제[0036] The pharmaceutical composition of the present invention is formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention pertains. It may be manufactured in a form or may be manufactured by placing it in a multi-volume container. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and a dispersant or stabilizer
를 추가적으로 포함할 수도 있다.It may additionally include.
[0037] 다른 측면에서 본원은 또한 박테리오파아지를 유효성분으로 포함하는 항생제에 관한 것이다. 본원에서, '항생제’라는 방부제, 살균제 및 항균제를 포함하는 것이다. 본원의 항생제는 클렙시엘라 뉴모니아에 의해 유발되는 각종 질환의 예방, 치료, 증상의 경감, 억제에 사용될 수 있다.In another aspect, the present application also relates to an antibiotic comprising a bacteriophage as an active ingredient. In the present application, it includes preservatives, fungicides, and antibacterial agents called'antibiotics'. The antibiotics of the present application can be used for the prevention, treatment, alleviation and inhibition of various diseases caused by Klebsiella pneumoniae.
[0038] 본원의 박테리오파아지는 기존 항생제에 비하여 클렙시엘라 뉴모니아에 대한 특이성이 매우 높은 장점을 갖고 있어, 유익한 균은 죽이지 않고 병원균인 클렙시엘라만을 선택적으로 제거할 수 있어 부작용이 없는 항생제로서매우 가치가 높다. 본 발명의 박테리오파아지를 항생 물질로 이용하게 되면 기존의 항생제를 이용하는 것과는 달리 병원균의 내성 내지 저항성(resistance)을 유도하지 않기 때문에 기존의 항생물질에 비하여 제품 수명주기가 긴 신규 항생제로서 이용될 수 있다. 즉 본원의 박테리오파아지를 유효성분으로 포함하는 항생제는 내성 문제를 근본적으로 해결할 수 있기에 그 만큼 항생제로서의 제품수명주기가 길어질 것으로 기대된다.[0038] The bacteriophage of the present application has a very high specificity for Klebsiella pneumoniae compared to existing antibiotics, so it does not kill beneficial bacteria and can selectively remove only the pathogen Klebsiella, so there is no side effect. It is very valuable. When the bacteriophage of the present invention is used as an antibiotic, it can be used as a novel antibiotic that has a longer product life cycle compared to the existing antibiotics because it does not induce resistance or resistance of pathogens unlike using conventional antibiotics. . In other words, antibiotics containing the bacteriophage of the present application as an active ingredient can fundamentally solve the resistance problem, and thus the product life cycle as an antibiotic is expected to be prolonged.
[0039] 다른 측면에서 본원은 또한 본원의 박테리오파아지를 유효성분으로 포함하는 소독제에 관한 것이다.In another aspect, the present application also relates to a disinfectant comprising the bacteriophage of the present application as an active ingredient.
[0040] 본원의 박테리오파아지를 유효성분으로 포함하는 소독제는 특히 식중독 예방 등 식품위생에 그 활용가치가 높다. 식품산업에서 클렙시엘라 오염 방지용 소독제 및 식품첨가제로 활용될 수 있으며 또한 축산업 분야에 활용될 수 있다. 또 생활하수처리장에서 방류수 내 클렙시엘라 균 제거를 위한 살포에도 활용될 수 있으며 조리장소 및 조리 설비, 위생시설의 장비, 의료시설 장비, 어린이집과 같은 공공장소의 시설, 장남감 등의 소독제로도 유용하게 사용될 수 있다.The disinfectant comprising the bacteriophage of the present application as an active ingredient has a high utility value in food hygiene, especially food poisoning prevention. It can be used as a disinfectant and food additive to prevent contamination of Klebsiella in the food industry, and it can also be used in the livestock industry. In addition, it can be used for spraying to remove Klebsiella bacteria in the effluent in domestic sewage treatment plants, and is also useful as a disinfectant for cooking and cooking facilities, sanitary facilities, medical facilities, facilities in public places such as day care centers, and toys. Can be used.
[0041] 또다른 측면에서 본원은 또한 본원의 박테리오파아지를 유효성분으로 포함하는 사료첨가제 및 음용수를 제공한다. 사람보다 가축에 들어가는 항생제가 더 많으며, 축산, 수산업에서 사용되는 사료 첨가용 항생제 사용량은 전체 항생제 판매량의 54%를 차지한다. 특히 예방 목적 항생제 투여는 내성균 발생 가능성을 높이며, 가축에 잔류하는 항생제는 사람에게 전달될 수 있는 문제가 있다. 항생제가 육류를 통해 인체에 흡수되면 항생제 내성을 유발해 질병의 확산을 부를 수도 있으며 다제 내성균 발생 확률이 높아진다. 본원에 따른 항생제는 기존의 항생제를 대체하여 사료첨가제용 항생물질로 사용될 수 있다.In another aspect, the present application also provides a feed additive and drinking water comprising the bacteriophage of the present application as an active ingredient. There are more antibiotics in livestock than humans, and the use of antibiotics for feed addition used in livestock and fisheries accounts for 54% of the total antibiotic sales. In particular, administration of antibiotics for preventive purposes increases the likelihood of developing resistant bacteria, and there is a problem that antibiotics remaining in livestock can be delivered to humans. When antibiotics are absorbed into the human body through meat, antibiotic resistance can be induced, leading to the spread of disease, and the probability of multidrug resistant bacteria increases. The antibiotic according to the present application can be used as an antibiotic for feed additives by replacing the existing antibiotic.
[0042] 본원의 사료첨가제는 건조 또는 액체 상태의 제제 형태일 수 있으며, 하나 또는 그 이상의 효소제제를 첨가할 수도 있다. 첨가되는 효소제제는 건조 또는 액체 상태 모두 가능하며 효소제제로는 리파제(lipase)와 같은 지방 분해효소, 파이트산(phytic acid)를 분해하여 인산염과 이노시톨인산염을 만드는 파이타제(phytase), 녹말과 글리코겐(glycogen) 등에 포함되어 있는 알파-1,4-글리코시드 결합(α-1,4-glycoside bond)을 가수분해 하는 효소인 아밀라제(amylase), 유기인산에스테르를 가수분해하는 효소인 포스파타제(phosphatase), 셀룰로스(cellulose)를 분해하는 카르복시메틸셀룰라제(carboxymethylcellulase), 자일로스(xylose)를 분해하는 자일라나제(xylanase), 말토오스(maltose)를 두 분자의 글루코스(glucose)로 가수분해하는 말타제(maltase), 및 사카로스(saccharose)를 가수분해하여 글루코스-프룩토스(glucose-fructose) 혼합물을 만드는 전환효소(invertase) 등과 같은 당 생성 효소로 구성된 군으로부터 선택되어 사용될 수 있다.The feed additive of the present application may be in the form of a dry or liquid formulation, and one or more enzyme formulations may be added. Enzyme preparations added can be either dry or liquid, and enzyme preparations include lipolytic enzymes such as lipase, phytase, which degrades phytic acid to make phosphate and inositol phosphate, starch and glycogen ( amylase, an enzyme that hydrolyzes alpha-1,4-glycoside bonds contained in glycogen), and phosphatase, an enzyme that hydrolyzes organophosphate esters, Carboxymethylcellulase, which degrades cellulose, xylanase, which degrades xylose, and maltase, which hydrolyzes maltose into two molecules of glucose. ), and sugar-generating enzymes such as invertase to hydrolyze saccharose to form a glucose-fructose mixture.
[0043] 또한, 본원의 박테리오파아지를 포함하는 사료첨가제에는 비병원성의 다른 미생물이 추가로 첨가될 수 있다. 첨가될 수 있는 미생물로는 단백질 분해 효소, 지질 분해효소 및 당 전환 효소를 생산할 수 있는 바실러스 서브틸리스(Bacillus subtilis)와 같은 고초균, 소의 위와 같은 혐기적 조건에서 생리적 활성 및 유기물 분해능이 있는 락토바실러스 균주(Lactobacillus sp.), 가축의 체중을 증가시키며 우유의 산유량을 늘리고 사료의 소화 흡수율을 높이는 효과를 보여주는 예를 들면 아스퍼질러스 오리자에(Aspergillus oryzae)와 같은 사상균 및 사카로미세스 세레비지에(Saccharomyces cerevisiae)와 같은 효모가 사용될 수 있다.In addition, other non-pathogenic microorganisms may be additionally added to the feed additive containing the bacteriophage of the present application. Microorganisms that can be added include Bacillus subtilis, which can produce proteases, lipolytic enzymes, and sugar-converting enzymes, and Lactobacillus, which has physiological activity and ability to degrade organic matter under anaerobic conditions such as the stomach of cattle. Strains (Lactobacillus sp.), which increase the weight of livestock, increase milk production, and increase the digestion and absorption rate of feed, for example, aspergillus oryzae, and Saccharomyces cerevisiae. (Saccharomyces cerevisiae) can be used.
[0044] 또한 각종 곡물 및 대두 단백을 비롯한 땅콩, 완두콩, 사탕무우, 펄프, 곡물 부산물, 동물 내장 가루 및 어분가루 등과 같은 사료원료는 가공되지 않거나 또는 가공된 것을 사용할 수 있다.In addition, feed raw materials such as peanuts, peas, sugar beet, pulp, grain by-products, animal visceral powder and fish meal, including various grains and soy protein, may be unprocessed or processed.
[0045] 또 다른 양태에서 본원에 따른 박테리오파아지를 이용한 클렙시엘라 뉴모니아에 의한 생물막 형성의 방지, 억제 또는 저해 방법에 관한 것이다. 본원의 박테리오파아지를 박테리아가 증식할 수 있는 또는 증식하고 있는 표면에 처리하여, 생물막의 형성을 미리 예방하거나, 또는 저해할 수 있다. 상기 박테리아와의 접촉은 물건의 표면을 본원의 방지 또는 저해제로 처리하는 것을 포함한다. 본원의 생물막 형성 방지 또는 저해제는 상기 언급한In another aspect, it relates to a method for preventing, inhibiting or inhibiting biofilm formation by Klebsiella pneumoniae using the bacteriophage according to the present application. By treating the bacteriophage of the present application on a surface on which bacteria can proliferate or proliferate, formation of a biofilm can be prevented or inhibited in advance. Contact with the bacteria involves treating the surface of the object with an inhibitor or inhibitor herein. The biofilm formation preventing or inhibitor of the present application
바와 같이 목적하는 생물막 형성의 저해에 유효한 양으로 처리 될 수 있다.As such, it can be treated in an amount effective to inhibit the formation of a desired biofilm.
[0046] 상기 표면은 내부 및 외부의 표면을 모두 칭하는 것이며, 단단하거나(고상), 유연한 것을 모두 포함하는것으로, 생체유래의 것은 물론, 가정, 산업, 환경, 의료 분야에서 사용되는 물건을 포함하는 것이다. 단단한 표면은 이로 제한하는 것은 아니나, 예를 들면 배수관, 글레이즈세라믹, 포설린, 유리, 금속, 나무, 크롬, 플라스틱, 비닐 및 포미카를 들 수 있다. 또한 고상 표면은 피부, 치아와 같은 생물체 유래 조직 또는 기관일 수있다. 또한 상기 고상 표면은 의료용 물건 유래일 수 있다. 의료용 물건은 각종 의료용 설비, 장비, 기구, 임시 또는 영구용 인공삽입 보형물 예를 들면 렌즈, 인공판막, 페이스메이커, 수술용 핀, 삽입도관, 카테터 등을 일컫는 것이나, 이로 한정하는 것은 아니다.[0046] The surface refers to both internal and external surfaces, and includes both hard (solid), flexible, and bio-derived, as well as products used in home, industry, environment, and medical fields. will be. Hard surfaces include, but are not limited to, drain pipes, glazed ceramics, porcelain, glass, metal, wood, chrome, plastic, vinyl and formica. In addition, the solid surface may be a tissue or organ derived from an organism such as skin or teeth. In addition, the solid surface may be derived from a medical product. Medical items refer to various medical facilities, equipment, instruments, temporary or permanent prosthetic implants, such as lenses, artificial valves, pacemakers, surgical pins, insertion conduit, catheter, etc., but are not limited thereto.
[0047] 이하, 본 발명을 하기의 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited by the following examples.
[0048] 본 발명은 달리 언급이 없는 한 세포생물학, 세포배양, 분자생물학, 유전자 형질전환 기술, 미생물학,DNA 재조합기술에 관한 당업자의 기술수준 내인 통상의 기술을 사용하여 실시될 수 있다. 또한 일반적인 기술에관한 보다 자세한 설명은 Molecular Biotechnology: (Bernard et al., ASM press 1994); Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley &Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985)을 참고할 수 있다.[0048] The present invention can be practiced using conventional techniques within the skill level of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation technology, microbiology, and DNA recombination technology unless otherwise stated. Also, for a more detailed description of the general technique, see Molecular Biotechnology: (Bernard et al., ASM press 1994); Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985) can be referred.
[0049] [실시예] [0049] [Example]
[0050] 실시예 1 균주의 배양조건[0050] Culture conditions of Example 1 strain
[0051] 클렙시엘라 뉴모니아 (KCTC 2296)을 숙주로 사용하였으며, LB(Luria-Bertani) 배지에서 37℃에서 진탕배양하였다.[0051] Klebsiella pneumoniae (KCTC 2296) was used as a host, and cultured with shaking at 37° C. in LB (Luria-Bertani) medium.
[0052] 실시예 2 박테리오파아지의 선별[0052] Example 2 Screening of bacteriophage
[0053] 클렙시엘라 뉴모니아를 감염하는 파아지를 선별하기 위해서 대한민국 경기도 광주시 오포읍 문형리 332 (하수종말처리장)에서 샘플을 채취하였다. 상기 채취한 샘플을 물에 녹여 그 상층액과 클렙시엘라 뉴모니아를 37℃에서 3 시간진탕배양 한 후, 5000rpm으로 20분간 원심분리하여 상등액을 회수하였다. 이어 상등액을 멸균된 막 필터(0.45um)로 여과하였다. 여과한 시료를 이중 아가층 플라크 분석법(double agar layer plaque assay)을 사용하였다. 요약하면, 5 ml 윗층 (top) 아가에 상기 숙주 박테리아와 파아지 배양액을 0.1 MOI로 섞어 아가 플레이트에 붓고 37℃에서 24시간 동안 배양하여 플라크를 수득하였다.In order to select phages infecting Klebsiella pneumoniae, samples were collected at 332 Munhyeong-ri, Opo-eup, Gwangju-si, Gyeonggi-do, Republic of Korea (sewage terminal treatment plant). The collected sample was dissolved in water, the supernatant and Klebsiella pneumoniae were incubated with shaking at 37° C. for 3 hours, and then centrifuged at 5000 rpm for 20 minutes to recover the supernatant. Then, the supernatant was filtered through a sterilized membrane filter (0.45um). The filtered sample was used as a double agar layer plaque assay. In summary, the host bacteria and phage culture solution were mixed in 5 ml top agar at 0.1 MOI, poured onto an agar plate, and incubated at 37° C. for 24 hours to obtain plaques.
[0054] 이어 상기 박테리오파아지로부터 유전체에 대한 서열분석을 실시하였다. 200 ml LB 배지에 클렙시엘라 뉴모니아를 OD600=0.5까지 배양한 후, 여기에 여과한 박테리오파아지 109PFU/ml 또는 0.1 MOI로 감염시켜 용균하였다. 이 후 여기에 염화나트륨을 최종농도 1M이 되도록 추가한 후 4℃에 1시간 두었다. 이어11000xg 에서 10분간 원심분리 한 후 침전물에 PEG (polyethylene glycol 8000)를 10% (w/v)로 넣고 4℃에 1시간 두었다. 이어 111000xg 에서 10분간 원심분리 한 후, 상층액을 제거하고, 침전물을 SM 완충액 (100mM NaCl, 10mM MgSO4(heptahydrate), 50mM Tris-HCl, pH 7.5)으로 현탁하였다. 여기에 클로로포름을 1:1의 비율로 넣고 볼텍스한 후 3000xg으로 15분간 원심분리하여 상층액을 얻었다. 폴리카보네이트 시험관에 40% 글리세롤 3ml을 넣고, 이어 5% 글리세롤 4ml을 섞이지 않게 넣었다. 여기에 상기 준비한 파아지 시료를 넣고 4℃에서 151000xg로 1시간 원심분리하였다. 이어 상등액을 제거한 후 침전물을 SM 완충액으로 재현탁하였다. 박테리오파아지 유전체 DNA는 phage DNA isolation kit (Norgen biotek corp.)를 제조사의 방법대로 사용하여 분리하였다.Subsequently, a sequence analysis was performed on the genome from the bacteriophage. After culturing Klebsiella pneumoniae in 200 ml LB medium to OD600 = 0.5, the bacteriophage filtered therein was infected with 10 9 PFU/ml or 0.1 MOI to lysis. After that, sodium chloride was added thereto to a final concentration of 1M, and then placed at 4°C for 1 hour. Then, after centrifugation at 11000xg for 10 minutes, PEG (polyethylene glycol 8000) was added to the precipitate in 10% (w/v) and left at 4°C for 1 hour. After centrifugation at 111000xg for 10 minutes, the supernatant was removed, and the precipitate was suspended in SM buffer (100mM NaCl, 10mM MgSO4 (heptahydrate), 50mM Tris-HCl, pH 7.5). Here, chloroform was added in a ratio of 1:1, vortexed, and centrifuged at 3000xg for 15 minutes to obtain a supernatant. 3 ml of 40% glycerol was added to a polycarbonate test tube, followed by 4 ml of 5% glycerol without mixing. Here, the prepared phage sample was put and centrifuged for 1 hour at 151000xg at 4°C. After removing the supernatant, the precipitate was resuspended in SM buffer. Bacteriophage genomic DNA was isolated using a phage DNA isolation kit (Norgen biotek corp.) according to the manufacturer's method.
[0055] 상기와 같이 분리된 총 유전체에 대한 염기서열을 분석하였다. (주) 마크로젠, Roche 454 차세대 유전체 분석기).[0055] The nucleotide sequence of the total genome separated as described above was analyzed. Macrogen Co., Ltd., Roche 454 next-generation genome analyzer).
[0056] 수득한 서열에 대하여 서열비교 프로그램인 BLAST 서치를 한 결과 기존에 알려지지 않은 새로운 파아지임을 확인하였다(비교결과는 나타내지 않음). 서열은 1개의 콘티그로 나타났으며, PBKP05로명명하였다.[0056] As a result of performing a BLAST search, which is a sequence comparison program, for the obtained sequence, it was confirmed that it is a new phage that is not known previously (comparative results are not shown). The sequence appeared as one contig, and was named PBKP05.
[0057] 실시예 3 투과전자현미경을 이용한 바이러스 형태 분석Example 3 Virus morphology analysis using a transmission electron microscope
[0058] 실시예 2에서 수득한 박테리오파아지의 형태를 분석하기 위하여 정제한 박테리오파지를 formvar carbon film (200 mesh copper grids)에 떨어뜨린 후, 2% 우라닐 아세테이트(Uranyl acetate)로 염색(negative staining)하고 건조한 후 투과전자현미경 (LIBRA 120 Energy-Filtering Transmission Electron Microscope (Carl Zeiss))을 통하여 그 형태를 촬영하였다.[0058] In order to analyze the form of the bacteriophage obtained in Example 2, the purified bacteriophage was dropped on formvar carbon film (200 mesh copper grids), and then stained with 2% uranyl acetate (negative staining) After drying, the shape was photographed through a transmission electron microscope (LIBRA 120 Energy-Filtering Transmission Electron Microscope (Carl Zeiss)).
[0059] 결과는 도 1에 있다. 분리된 박테리오파아지는 직경이 약 70nm인 icosahedral head를 가지며 110nm의 긴 꼬리를 가진 형태학적 분류상 시포비리대 과 박테리오파아지 속하는 것으로 판단되었다.The results are in FIG. 1. The isolated bacteriophages had an icosahedral head of about 70 nm in diameter and were considered to belong to the Siphoviridae family and bacteriophages according to their morphological classification with a long tail of 110 nm.
[0060] 실시예 4 박테리오파아지 구조 단백질 분석[0060] Example 4 Bacteriophage structural protein analysis
실시예 2에서 분리된 박테리오파아지 1010 Bacteriophage isolated in Example 2 10 10
[0061] PFU에 시료완충액 (SDS 10% w/v, 10 mM DTT, glycerol 20% v/v, 0.2M tris-HCl, pH 6.8, bromophenol blue 0.05% w/v) 4 ul를 혼합한 후, 3분간 끓이고, 12% SDS-PAGE 젤에 로딩하여 전기영동하고, 분리된 밴드는 쿠마시블루 (coomassie blue) G-250로 염색하여 가시화하였다. 결과는 도 2에 기재되어 있다. 분석 결과 PBKP05은 5개 이상의 단백질로 구성 되어있으며, 분자량은 10에서 150 kDa 범위이며, 가장 주가 되는 단백질의 분자량은 30 kDa 이었다.After mixing 4 ul of sample buffer (
[0062] 실시예 5 박테리오파아지 감염 능력 분석[0062] Example 5 Bacteriophage infection ability analysis
[0063] 실시예 2에서 분리된 박테리오파아지의 감염 능력을 분석하기 위해 원스텝성장 (one-step growth) 실험을 수행하였다.To analyze the infection ability of the bacteriophage isolated in Example 2, a one-step growth (one-step growth) experiment was performed.
[0064] 클렙시엘라 뉴모니아를 OD600=0.5까지 배양한 후 까지 키운 후 0.1 MOI의 실시예 2에서 분리된 박테리오파아지로 감염하였다. 이어 37℃에서 5분간 박테리오파아지 흡착(adsorption)이 일어나도록 한 후 13000rpm으로 5분간 원심분리하여 프리 파아지를 제거하였다. 이 후 20 ml LB 배지로 침전물 현탁하고, 37℃에서 교반하면서 배양 하였다. 이어 배양 중 시료를 5분 간격으로 채취해서 파아지 농도 (phage titer)를 측정하였다. 파아지 농도는 실시예 1에서 기술한 이중 아가층 플라크 분석법을 사용하여 수행하였다.After culturing Klebsiella pneumoniae to OD600 = 0.5, after growing until, it was infected with the bacteriophage isolated in Example 2 of 0.1 MOI. Then, the bacteriophage adsorption (adsorption) took place at 37°C for 5 minutes, and then centrifuged at 13000rpm for 5 minutes to remove the free phage. Thereafter, the precipitate was suspended in 20 ml LB medium and cultured while stirring at 37°C. Subsequently, samples were collected at intervals of 5 minutes during cultivation, and the phage titer was measured. Phage concentration was performed using the double agar plaque assay described in Example 1.
[0065] 원스텝성장 분석을 통해 PBKP05의 잠복기 및 파아지 방출 수(burst size) 를 확인하였다. 결과는 도 3에 기재되어 있다. PBKP05의 잠복기는 20분이었으며, 방출수는 32로 나타났다.Through one-step growth analysis, the incubation period and phage discharge number (burst size) of PBKP05 were confirmed. The results are shown in Figure 3. The incubation period of PBKP05 was 20 minutes, and the number of discharges was 32.
[0066] 실시예 6 박테리오파아지에 대한 온도, pH 안정성 분석Example 6 Temperature, pH stability analysis for bacteriophage
[0067] 박테리아의 성장억제 여부를 확인하기 위해서 먼저 클렙시엘라 뉴모니아를 OD600=0.5까지 배양하였다. 이 후 6.4 × 105 PFU/ml의 실시예 2에서 분리된 박테리오파아지를 4℃,37℃,45℃,55℃,65℃,80℃에서 1시간동안 배양시킨다. 또는 pH3~11까지의 Buffer를 이용하여 1:1 희석하여 1시간동안 상온에서 배양시킨다. 이 후, 연속희석 한 박테리오파아지를 클렙시엘라 뉴모니아에 감염시켜 이중 아가층 플라크 분석법을 사용하여 수행하였다. 결과는 도 4에 기재되어 있다. 도 4에 나타난 바와 같이 PBKP05는 특이적으로 4℃~55℃에서 안정성을 보이며, pH 6~11에서 안정성을 보이는 것으로 나타났다.In order to check whether or not the growth inhibition of bacteria, Klebsiella pneumoniae was first cultured to OD600=0.5. After that, the bacteriophage isolated in Example 2 of 6.4 × 10 5 PFU/ml was incubated at 4°C, 37°C, 45°C, 55°C, 65°C, and 80°C for 1 hour. Alternatively, dilute 1:1 using a buffer of pH 3-11 and incubate at room temperature for 1 hour. Thereafter, the serially diluted bacteriophage was infected with Klebsiella pneumoniae and performed using a double agar plaque assay. The results are shown in Figure 4. As shown in Fig. 4, PBKP05 specifically exhibited stability at 4°C to 55°C, and was found to exhibit stability at
서열번호 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gttaagcgacacctcaagaacctgaattattctg28401cagccgctagggctgtactggagtaccagcaaaagtctccgggcacaggctgggtgtacaagggcggcaaccgctggacc28481tttgactgctctcagtacattaacggtcaacgtaacagagtatgctggggcctatgggagcgcaggcagtggcagagcaa28561agccattggtaatcagtacagcagtgtaaaggaagcactggataaactcacaaaataattggagacagtatggctttaat28641tgagatagtaactgtaggtcccggggacctgacacacgcagagtcctacgaggaccttaagacaaaagtaccgaaagcgg28721ccggtcagcggattcgcttactatcgcacatctccggatgggcggcactggcgcatggtccagtaggcggtggggagttt28801gtggctcgttcaggcacagctgttgatgatggcggttatatctgcgtacccacggggcaggcctcgtactactggcagcg28881tatttcccaaaatccaggtaaggtttgtgccacagagtttggcctatatgatggggctgccctggatagcatactaacga28961aggccatcggatactgcattaaaaactcgttgatgcatctttcaattccagcatatacgcttgccggctcgcagcgcagt29041gcagcgggcttgagtttattaacagtagatctgagtgcatggccctacaaacaagctcatcatcgaaggccctggaatgg29121ctacaaaagggaccagtccggtaataacccacactggggctaatattgcgttaacgtttaagcgcgcgtctcaagtggta29201gataatgttgtagtcagaacgtcacagcgtgggatctggttccactcaaaccctgcatctactgatgatcagacgttgtc29281tttttacggtgccagaattaccaactttggcttccagcacggtatctccggagcctcctacggcatatacgtaggtgacg29361gtgcgcgtgccgataacttatataactgcgacagatggggtggtgggaagttgggggtaacagcacagctctttacgtgg29441cggataaagcgcgagttgatggctttgctaacttccgatatgatggctttgctgcaagcgcaatgacgtctagctcggta29521acccagttaacaatccgtccatggttagccatcgcggaagcagttgccggtgcagctacacctcaccctactctccccgg29601cgagagcatagtaagtgtgccaggaatgaagtgcaagctggtcgggacgttgtttaaggggcagaactcagttatctctg29681ttgtcgggatgcctccgttccatcggtacaaggtcactacccgttgtgggctgtctagtactgcgcagcagcagtacatc29761gttaatatcccgaacggggctaacggtgtacgcaatccaacgttgtgaatgccgagtatcctgtatctatcgagattgag29841gttatagattaacttggcaaccgtgagtccctgcaagtacttgggggctcactagggcttcacccaggggcaagtgtata29921cctgaactcaaaatttgttatactcacgcgaggccctccctcaccctcagcgacgccaattgcccccatagggggtgcct30001agcgtcaatttagggggggggggggggcacccctagggcgcggccagtgcgctggggtagcacctgcagcccgtagtgcg30081cttctattacgcgctagggctatcgctagtgctaccctatggctatgcctgtgcgtgccttgtagggcttcctgtgcgcc30161ctatggggcactagtgctatccctgtgctatccctgtgcgttcactaggcgctccctgtgggccatatgggtgcgccctg30240 Sequence number 1cctcaccctcagcgacgccaattgcccccatagggggtgcctagcgtcaatttagggggggggggggggcacccctaggg81cgcggccagtgcgctggggtagcacctgcagcccgtagtgcgcttctattacgcgctagggctatcgctagtgctaccct161atggctatgcctgtgcgtgccttgtagggcttcctgtgcgccctatggggcactagtgctatccctgtgctatccctgtg241cgttcactaggcgctccctgtgggccatatgggtgcgccctgccttgcttattttgcggagccatagggtgtgtctagtg321ggccagtagtgggccgcctagtgcgctgtagtgcctgcctgtgctatccctagtacgcactaggctatccactggctatc401cttagggctttacattatgccgattatatgctcccactagggcgcacatccactacccggcacactctaccgctatcccg481gcgctatctctgtgctataccttctcccttactttcattcgaaagctaatacgaaatgatgttacaggagtaggagggtt561ttagggcactatacatagtatgtccctgtgcctacgtagtaggctgacatacctacatagtcaccgcatcactccgtgcc641ttcgcttacactacggcactacgctctgctttatgtattattagatagtattggttagtagtggatacagctggtgctgg721cacataggtctatagttcaatcaccgggaggcgctgaggcgataaccggggcagagtcgggagattcagccggattaagg801ctagatagtgtgaagggttagatactcgataaaaagggttgacaccgcgaagaacataagctagattgaatcccagcagt881acggcaggatagttgaagacaggccgctaactaggacacttccacgtggtgagccgccattcaccaaagaggccaggtga961ccgg acacataaaccggacggcttaaaagtaaggtaacttagatagcgaacaaggcaaggataaccaatctagggcacaa1041ggatgtgtgtcccgtcggataggctagacgttaaaaacgacctcttaaaggtttagcagtaagctggtttgcggggaggc1121ccagtaccttgactggtacgttgacgaccaatcctagacaatcagttaaaaagagtaaacgtgccggacgttatcaccgg1201ggtcgctggccgatctgagacaaaccatactgtcgaggcgtgagcagcacgctcctctgaatcgagggaagtgtatcccg1281caaagagcatataacatagcgtagcagtaacaaggctgcgctagattatgttctctaagaggtaagagtatgcgtaaagc1361aaagcgtttagcattaaagcgtaattgggagttatcaaccaacccaactgaagataagaagctgctagttaaccgctgat1441tggtgtcggcggcttttaacccagcaagcaagggctgtgcagtgagcacagggtaaggggaaagcaaaaacagaaaggca1521gcagcaggcagcctaatgggtgactaactccaccaattcaactggtggcagcatccgcaaagcgctgggcgacgtagtgg1601aagcaaagcgtaacatcactatcagtgcgctgtttcatggtctggtaagcagcagggcagctaacctacatcgaatcttt1681gcgtaaaaccaaaggcgagacggcagcagcagatgcggcgacaaaactgtttgtttgcggcggtctggtttcttcttgca1761ggcttcgttttggtaatgagccgttgagtttcggcggttgtgcgacagctgctgatacgatggacaagcgcacgtttcct1841gggctacggatatgcagcgcagcgatgccgctgacttcgatatggtgctgcgtacgctgctgcctatcaacggcaaatat 1921gagttcaacgcgaagaagtgctatgcgtctgctgaaaagctgggcatcgaactggacactatgcgtctggactataaaca2001agttgacaagcagggccgagaagtgattgtagccagcttttatagctcctgtatggccctgtacgctgccgaagcggagc2081aggtgaagaatgacgcactggatgccgatgcagtgcgcttgcaagcgctggggcgcgttaaaaacgccatcacgacgctg2161atttggtgggcatgcttattaatcagggcgtggatgtacgcgctgtactggatgcaactttgaaggtggtggcatgattt2241ataaagttccggttatcaagtggtgggccttgtgtcctgcggccaacgatgctgacgccttcgacagccctatgcgggca2321atcgctcaaaaatgggtgtacgagggattgggcgagtatatctagtagcaagcctatagcgtcctacggggcgctatgtg2401aatgcaactggcgtatgaggtttcatatgaaagcaatactggtttatccggggcatgagctctggtacttagcactgtgc2481tggacgccattaatattggcatcatcaccgattggagacagctttatggttaacgtattcaatatcattgtgaccagcgc2561tatgctggtgctgggcaacgacgtaaacaacccggtgccttattgcacagtgcagatgccggttcagcagccacgccctg2641agtatgaccccgagggtggttgcaaagagcttggcgcacgtatccttgctgcggtgcaagagcagtacccggacgccgca2721gtaagagcaacaacgaaatttaagaggtatcagcggctatgaaagtcccggcgctattaagtgcccgtattgcactggca2801aatacgatccaatgcctgcaagagtcttgtgcgaggaggtcaaagtttccgatgacatagtagctgtgtatg cagagcgc2881cgctatggtagttttgggccttacaccaagctcatgtgtgtacgccttgttagtggaggcggggcatgaagtcttacgga2961aagaatcctgaaacgctgcttatgcgtaagcagcaacaaactattgacgggctggcgcgggagtacaacgcgaaggcagc3041actgcgccagcacttcgggacacaccgtagagttcgtttggactccagcgtatactcctgcgctgagtattacgataaaa3121acaccgagcagtggaaacaatcccttgttcccgctgtcaatttaatcctagggccacataacgtactcgtggcccgcaac3201gtagtgttcaaggacagcatatgctaacagtagacgaaacagcgctgctgatttggcatctactggagacccagggcaag3281tgcgggtgcccgtgggaaacattcaaagaggtccctaatgaactcaagcaaattatcccggtagaacgtagactcctcag3361agttcgtaggcaggactctgcggtggtgctcaccacgtaccgagagtatacagttcagttcgatgtggttacagcactac3441tacggcacggatatcgtggagcctatacaagatttagggcggctgtgcgctcatactataagcaaagacagctcgctgcg3521tggtatgcacgctgaactattaatgcacgatgaaatacaactaattcaggagcaaatgaaaatgcaagagactacaaaag3601aaactaatacagcacctattgaatggaaagtagtgttgccggaaggtgcaaacgcactgccaatgaaatgttcgatgtat3681tccagcggtgattactggaccccgttccaggacttgcaaatgcaaggagaggaccacccccacactgagggtccactgca3761ggcgcttatgggtctacgtactgtcagcgcgtacatcccgggcttagaggtaactataggcgta 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ttggcttccagcacggtatctccggagcctcctacggcatatacgtaggtgacg29361gtgcgcgtgccgataacttatataactgcgacagatggggtggtgggaagttgggggtaacagcacagctctttacgtgg29441cggataaagcgcgagttgatggctttgctaacttccgatatgatggctttgctgcaagcgcaatgacgtctagctcggta29521acccagttaacaatccgtccatggttagccatcgcggaagcagttgccggtgcagctacacctcaccctactctccccgg29601cgagagcatagtaagtgtgccaggaatgaagtgcaagctggtcgggacgttgtttaaggggcagaactcagttatctctg29681ttgtcgggatgcctccgttccatcggtacaaggtcactacccgttgtgggctgtctagtactgcgcagcagcagtacatc29761gttaatatcccgaacggggctaacggtgtacgcaatccaacgttgtgaatgccgagtatcctgtatctatcgagattgag29841gttatagattaacttggcaaccgtgagtccctgcaagtacttgggggctcactagggcttcacccaggggcaagtgtata29921cctgaactcaaaatttgttatactcacgcgaggccctccctcaccctcagcgacgccaattgcccccatagggggtgcct30001agcgtcaatttagggggggggggggggcacccctagggcgcggccagtgcgctggggtagcacctgcagcccgtagtgcg30081cttctattacgcgctagggctatcgctagtgctaccctatggctatgcctgtgcgtgccttgtagggcttcctgtgcgcc30161ctatggggcactagtgctatccctgtgctatccctgtgcgttcactaggcgctccctgtgggccatatgggtgcgccctg30240
Claims (1)
서열번호 1의 서열을 갖는 클렙시엘라 뉴모니아에 대한 융해능을 갖는 박테리오파아지.
청구항 2
제 1 항에 따른 박테리오파아지의 프로지니(progeny)로, 상기 박테리오파아지와 동일한 생물학적 특성을 나타내는, 프로지니 박테리오파아지.
청구항 3
제 1항의 박테리오파아지는 2018년 8월 7일자로 한국미생물보존센터에 기탁번호 KFCC11779P로 기탁된 박테리오파아지.
청구항 4
제 1항의 박테리오파아지를 클렙시엘라 뉴모니아를 융해하기 위한 용도로 사용하는 방법.
Claim 1
Bacteriophage having a melting ability for Klebsiella pneumoniae having the sequence of SEQ ID NO: 1.
Claim 2
With the progeny of the bacteriophage according to claim 1, which exhibits the same biological properties as the bacteriophage, progeny bacteriophage.
Claim 3
The bacteriophage referred to in paragraph 1 is a bacteriophage deposited with the Korea Microbial Conservation Center under the accession number KFCC11779P on August 7, 2018.
Claim 4
A method of using the bacteriophage of claim 1 for melting Klebsiella pneumoniae.
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| KR1020190022686A KR20200104130A (en) | 2019-02-26 | 2019-02-26 | Bacteriophage of Klebsiella pneumoniae and uses thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115029322A (en) * | 2022-05-26 | 2022-09-09 | 山西大学 | Multi-drug-resistant Klebsiella pneumoniae phage and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115029322A (en) * | 2022-05-26 | 2022-09-09 | 山西大学 | Multi-drug-resistant Klebsiella pneumoniae phage and application thereof |
| CN115029322B (en) * | 2022-05-26 | 2023-11-14 | 山西大学 | Klebsiella pneumoniae bacteriophage with multi-drug resistance and application thereof |
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