CN113025582B - 一株肺炎克雷伯菌噬菌体及医用用途 - Google Patents
一株肺炎克雷伯菌噬菌体及医用用途 Download PDFInfo
- Publication number
- CN113025582B CN113025582B CN202110548215.7A CN202110548215A CN113025582B CN 113025582 B CN113025582 B CN 113025582B CN 202110548215 A CN202110548215 A CN 202110548215A CN 113025582 B CN113025582 B CN 113025582B
- Authority
- CN
- China
- Prior art keywords
- phage
- kpnp
- klebsiella pneumoniae
- bacteriophage
- bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000588747 Klebsiella pneumoniae Species 0.000 title claims abstract description 32
- 230000002147 killing effect Effects 0.000 claims abstract description 11
- 241001515965 unidentified phage Species 0.000 claims description 23
- 239000000203 mixture Substances 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 8
- 206010035664 Pneumonia Diseases 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 239000012669 liquid formulation Substances 0.000 claims 1
- 239000012931 lyophilized formulation Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 24
- 239000007788 liquid Substances 0.000 abstract description 14
- 238000000746 purification Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 231100000252 nontoxic Toxicity 0.000 abstract description 3
- 230000003000 nontoxic effect Effects 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 239000007921 spray Substances 0.000 abstract description 2
- 201000008225 Klebsiella pneumonia Diseases 0.000 abstract 1
- 206010035717 Pneumonia klebsiella Diseases 0.000 abstract 1
- 238000004659 sterilization and disinfection Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 9
- 238000012258 culturing Methods 0.000 description 7
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 125000001475 halogen functional group Chemical group 0.000 description 4
- 231100000086 high toxicity Toxicity 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000010865 sewage Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 239000012880 LB liquid culture medium Substances 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 244000039328 opportunistic pathogen Species 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229920002477 rna polymer Polymers 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 206010048282 zoonosis Diseases 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 206010013654 Drug abuse Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010024652 Liver abscess Diseases 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 241000702072 Podoviridae Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 206010046793 Uterine inflammation Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 208000004396 mastitis Diseases 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 208000011117 substance-related disease Diseases 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10211—Podoviridae
- C12N2795/10221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10211—Podoviridae
- C12N2795/10231—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10211—Podoviridae
- C12N2795/10232—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Communicable Diseases (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Oncology (AREA)
- Dentistry (AREA)
- General Chemical & Material Sciences (AREA)
- Environmental Sciences (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一株肺炎克雷伯菌噬菌体vB_KpnP_ZK1,同时还提供了其医用用途,所述的菌株已于2020年11月9日保藏于中国典型培养物保藏中心,保藏名为Klebsiella pneumonia phagevB_KpnP_ZK1,保藏编号为:CCTCC NO:M 2020713。本发明的噬菌体具有较强的特异性,可选择性的杀灭K1型肺炎克雷伯菌,不杀伤其他有益细菌,且清除作用良好;本发明的噬菌体可以单独或与其他物质复配使用,可配置成喷洒液等液体试剂为消毒净化环境提供一种安全、无毒的噬菌体消杀产品。
Description
技术领域
本发明公开了一株肺炎克雷伯菌噬菌体vB_KpnP_ZK1,同时还提供了其医用用途,本发明属于生物工程领域。
背景技术
肺炎克雷伯菌(Klebsiella pneumoniae)是一种普遍存在的机会性致病菌,它不仅是医院和社区主要的获得性机会致病菌,还是一种众所周知的人畜共患病原菌。当免疫力下降或长时间滥用药物导致菌群失调时,肺炎克雷伯菌可引发一系列病理变化,如肺炎、乳腺炎、子宫炎等化脓性炎症甚至败血症。
尤其是以K1型菌株为主的高毒力肺炎克雷伯菌(Hypervirulent K.pneumoniae,hvKP),已证实是导致化脓性肝脓肿的主要原因之一,其对血清补体、中性粒细胞和吞噬细胞等机体清除作用的抵抗力明显强于其他荚膜型菌株,可引起动物和人的多种严重的感染病变,对人类的公共卫生安全和动物养殖业造成巨大威胁。
随着在养殖业和临床大量的和无节制的滥用抗生素,耐药问题日渐严重,各种超级耐药细菌的不断出现,让人们渐渐陷入无药或者少药可选的尴尬境地。肺炎克雷伯菌作为耐药最严重的人畜共患机会性致病菌之一,迫切地需要研发新型药物,以控制愈发严峻的耐药性问题。
噬菌体,是一种能特异性侵袭细菌、支原体、螺旋体、放线菌等微生物的的病毒,是细菌的天然杀手,在自然界广泛存在。裂解性噬菌体可以感染并裂解宿主细菌,并随着宿主细菌的死亡释放到外界,继续感染周围环境中的易感细菌。噬菌体相对于传统抗生素治疗有着高度特异性、高效性、易获得等特点,能在不影响机体正常菌群的情况下,快速杀灭特定病原菌,且对耐药菌株也有良好的杀灭效果。国内外研究均显示噬菌体在防控细菌感染方面表现出巨大潜力。目前,高毒力且具有耐药性的肺炎克雷伯菌已有报道,但关于高毒力肺炎克雷伯菌噬菌体研究较少,开发新型对高毒力肺炎克雷伯菌具有特异性、强效性防治效果的噬菌体具有十分重要的意义。
发明内容
本发明公开一株肺炎克雷伯菌噬菌体,命名为vB_KpnP_ZK1;该噬菌体对K1型肺炎克雷伯菌有很强的特异性裂解能力,可以单独或与其他物质复配使用,为消毒净化环境提供一种安全、无毒的噬菌体消杀产品。
本发明公开的一株肺炎克雷伯菌噬菌体vB_KpnP_ZK1,所述的菌株已于2020年11月9日保藏于中国典型培养物保藏中心,保藏名为Klebsiella pneumonia phagevB_KpnP_ZK1,保藏编号为:CCTCC NO:M 2020713。
本发明所述的噬菌体vB_KpnP_ZK1在制备杀灭空间环境、动物和人中肺炎克雷伯菌药物中的应用。
本发明所述的噬菌体vB_KpnP_ZK1的有效成分在制备用于杀灭人体内、体表、病房、医疗器具的药物添加剂、喷雾添加剂、消毒剂或清洁剂中的用途。
一种用于杀灭肺炎克雷伯菌的组合物,其包含本发明所述的噬菌体vB_KpnP_ZK1作为活性成分;所述组合物为液体制剂、冻干制剂或口服固体制剂等。
本发明的积极效果在于:提供了一株肺炎克雷伯菌噬菌体vB_KpnP_ZK1,并提供了其医用用途;本发明的噬菌体具有较强的特异性,可选择性的杀灭K1型肺炎克雷伯菌,不杀伤其他有益细菌,且清除作用良好;本发明的噬菌体可以单独或与其他物质复配使用,可配置成喷洒液等液体试剂为消毒净化环境提供一种安全、无毒的噬菌体消杀产品。
附图说明
图1. 本发明噬菌体vB_KpnP_ZK1噬菌斑照片;
图2. 本发明噬菌体vB_KpnP_ZK1的透射电镜照片;
图3. 本发明噬菌体vB_KpnP_ZK1的最佳MOI图;
图4.本发明噬菌体vB_KpnP_ZK1的一步生长曲线图。
具体实施方式如下:
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另
有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作和/或它们的组合。
在本发明的实施方案中,提供了对肺炎克雷伯菌具有特异性杀伤活性的新型噬菌体。
噬菌体是能够感染特定细菌并抑制细菌生长的细菌特异性病毒,并且是包含单链或双链脱氧核糖核酸(DNA)或核糖核酸(RNA)作为遗传物质的病毒。
本发明的噬菌体分离自污水的新型噬菌体,该噬菌体具有呈正二十面体的头部和较短的尾部;噬菌体在LB琼脂培养基上可以形成透亮空斑,中心直径0.5-1 mm,周围有直径4-5mm半透明晕环,且半透明晕环随时间的增长而扩大;基因组核酸的酶切图谱显示该噬菌体核酸是双链DNA(dsDNA);已于2020年11月9日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2020713,地址:中国武汉,武汉大学。
实施例1
噬菌体分离及制备
噬菌体的分离过程在下文中详细描述。本发明中的粪液污水样品采自长春市火烧李,宿主菌为肺炎克雷伯菌WKP26。采集污水,用纱布过滤,6000r/min离心10min,取上清,用处理过的污水代替ddH2O配制LB培养基(100mL);培养基内加入1mL过夜培养的宿主细菌WKP26,37℃培养10-12h;取培养物1mL,12000r/min离心5min,上清用0.22μm滤器过滤形成噬菌体原液并保存,将得到的滤液用于空斑测试,以检查是否包括能够裂解肺炎克雷伯菌WKP26的噬菌体。
空斑测试如下:以2%的比例在5mL LB液体培养基中接种肺炎克雷伯菌WKP26,37℃振荡培养过夜。取100μL上述制备的细菌培养液滴于平板正中央,用涂棒将菌液均匀地涂开,室温干燥;待其干燥后,取上述滤液10ul滴于平板中间,平放平板室温干燥;待自然干燥后,置于37℃培养箱培养10h,然后观察滴加滤液区域有无空斑形成。如果滴加滤液区域产生透明区域,则可以判断为滤液中包含能够杀灭肺炎克雷伯菌WKP26的噬菌体。
取上述所得滤液用无菌PBS进行倍比稀释,取10-2、10-4和10-6稀释液各100μL和200μL过夜培养的宿主菌充分混匀,室温孵育5min后,一并加入7mL 、45℃LB半固体培养基中,混匀后迅速倾倒于1.5% 琼脂培养基上制成双层平板,待其凝固后,放置于37℃温箱中孵育6h,观察噬菌斑生长。
实施例2
噬菌体扩增和纯化
在形成噬菌斑的双层平板上,用无菌的移液器的枪头挑取直径较大的、比较圆且透亮的单个噬菌斑,接种于5 mL LB液体培养基中,加入噬菌体宿主菌液200μL,混匀,室温作用15min,37℃培养10~14h,12000rpm,4℃ 离心10min,取上清;重复双层平板实验,如此反复挑取4-5次单个噬菌斑,将噬菌体纯化成大小一样的噬菌斑。
取1mL新鲜培养的宿主菌,加300μL噬菌体裂解液(以单个噬菌体培养物与宿主菌分别按照1:1、1:10和1:100的比例)。37℃温育20min,使噬菌体颗粒吸附于宿主菌;加入800mL LB液体培养基,再加入CaCl 2 母液至终浓度1.25mM,37℃摇振培养6 ~ 8h,12000rpm,4℃ 离心10min,取上清,即为噬菌体裂解液。
PEG纯化:在噬菌体裂解液中加入RNase A、DNaseⅠ至终浓度均为1μg/mL,室温放置30min;加入NaCl至终浓度为1mol/L,混合均匀后,冰浴1-2h;用4℃ 离心机,8000r/min离心15-20min后收集上清;每100ml 混合溶液中加入10g PEG-8000,轻轻搅拌使其充分溶解,冰浴过夜,使噬菌体在PEG-8000作用下形成沉淀;4℃ 离心机,12000r/min离心10-20min,回收沉淀的噬菌体颗粒,加2mL SM液,充分洗涤沉淀,室温作用1h;加入等体积的氯仿抽提,温和振荡30s;4℃ ,5000rpm离心10min以分离有机相和亲水相,回收含有噬菌体颗粒的亲水相,获得纯化的噬菌体。
CsCl等密度梯度离心纯化: 按表制备CsCl梯度液,按照从高密度到低密度的顺序,在5mL半透明聚丙烯酰胺高速离心管中依次加入各梯度液1mL;将噬菌体浓缩液700μL缓慢加入到CsCl梯度液的上面,放到4℃高速离心机中,35000r/min水平离心3h;离心结束后待真空下降为0时,打开仓门,取出样品,关机;样品下端有一层蓝色带,用细针头从带侧面插入,小心吸取;将样品置于透析袋中,用10mM Tris-HCl,PH为7.4,100mM MgCl 2 缓冲液进行透析,2L一次(10-14kd);最终将样品吸出,测定噬菌体效价。
采用双层平板法检测噬菌体效价:将以上纯化的噬菌体液进行10倍梯度稀释,取
相应的几个梯度的噬菌体稀释液各100μL与宿主菌液200μL充分混匀,铺双层琼脂平板,37℃恒温培养6 h左右,对每个琼脂平皿进行噬菌斑计数。选择出现100-200个左右噬菌斑的平皿,根据稀释的倍数计算得到的噬菌体初始浓度即得噬菌体效价。纯化的噬菌体如图1所示,噬菌体在LB琼脂培养基上可以形成透亮空斑,中心直径0.5-1 mm,周围有直径4-5mm半透明晕环,且半透明晕环随时间的增长而扩大。
将纯化的噬菌体自命名为vB_KpnP_ZK1,已于2020年11月9日保藏于中国典型培养物保藏中心,保藏名为Klebsiella pneumonia phagevB_KpnP_ZK1,保藏编号为:CCTCC NO:M2020713。
实施例3
噬菌体vB_KpnP_ZK1透射电镜观察
取实施例2中PEG纯化的噬菌体做电镜观察,具体操作步骤为:加10μL 样本滴在铜网上,待其沉淀15 min, 用滤纸吸去多余的液体,用 2%的磷钨酸(PTA)染色1-2min,干燥后使用透射电镜(日立H-7650)观察;观测结果如图2 所示,头部呈正二十面体,头部直径约为50 ± 5 nm,尾部长约10 ± 5 nm。根据国际病毒分类委员会(ICTV) 2005年发表的《病毒分类—国际病毒分类委员会第八次报告》,vB_KpnP_ZK1属于短尾病毒科(Podoviridae)。
实施例4
噬菌体vB_KpnP_ZK1的最佳MOI测定
最佳MOI测定:将培养至对数期的菌液调到浓度为107cfu/mL,然后按照噬菌体/细菌的比例为0.00000001、0.0000001、0.000001、0.00001、0.0001、0.001、0.01、0.1、1和10将噬菌体与菌进行混合,转接到LB液体培养基中,在37℃振荡培养8h。将培养液在4℃进行10000g离心15min,用0.22μm孔径的一次性滤器对上清液进行过滤得到噬菌体增值液,利用双层平板法对增值液进行滴度测定,低度最高的噬菌体/细菌比例即为最佳MOI,结果如图3所示。
实施例5
噬菌体vB_KpnP_ZK1的一步生长曲线测定
一步生长曲线测定:将培养至对数期的宿主菌与噬菌体按照MOI=0.1的比例进行混合,在37℃孵育2min后,在4℃进行10000g离心10min,用新鲜10mL LB液体培养基将沉淀悬起,将悬液于37℃振荡培养,在培养的0min、1min、2min、3min、4min、5min、10min、15min、20min、25min、30min、40min、50min各取一次样品测定噬菌体的滴度,从而绘制出噬菌体感染细菌的一步生长曲线,结果如图4所示:表明噬菌体感染wkp26的隐蔽期为1min,潜伏期为2min,爆发量约为161PFU每个感染细胞,一个裂解周期大约需要40min。
实施例6
噬菌体vB_KpnP_ZK1宿主谱分析
将实施例2获得的噬菌体vB_KpnP_ZK1效价调整为10 8 pfu/mL备用。试验选择多株肺炎克雷伯菌为对象,对噬菌体vB_KpnP_ZK1的宿主谱进行分析,具体操作如下:
空斑试验测定:分别取待测菌株的过夜培养物100μL,滴加在1.5% LB培养基平板中央,用涂棒分别将它们涂制成均匀的菌苔。取10μL噬菌体vB_KpnP_ZK1滴加在菌苔表面,待液滴干燥后倒置于37℃培养箱培养12 ~ 16h,观察结果,如有空斑产生则记为“+”,否则为“-”,结果如表1所示。
表1. 噬菌体vB_KpnP_ZK1宿主普分析
编号 | 待测细菌名称 | 空斑试验 |
1 | w-kp11 | + |
2 | w-kp13 | + |
3 | w-kp15 | + |
4 | w-kp19 | + |
5 | w-kp25 | + |
6 | w-kp26 | + |
7 | w-kp27 | + |
8 | w-kp28 | + |
9 | w-kp30 | + |
10 | w-kp41 | + |
11 | w-kp44 | + |
12 | w-kp46 | + |
13 | w-kp47 | + |
14 | w-kp55 | + |
15 | w-kp63 | + |
16 | w-kp70 | + |
17 | w-kp76 | + |
18 | w-kp81 | + |
19 | KP10 | + |
20 | KP11 | + |
21 | KP12 | + |
22 | KPP4 | + |
23 | KPP15 | + |
24 | KPP22 | + |
25 | KPP26 | + |
26 | KPP33 | + |
27 | KPP34 | + |
28 | KPP37 | + |
29 | KPP43 | + |
30 | KPP45 | + |
31 | zkp-3 | + |
32 | zkp-5 | + |
33 | zkp-10 | + |
34 | zkp-15 | + |
35 | zkp-18 | + |
36 | zkp-28 | + |
37 | zkp-34 | + |
38 | zkp-36 | + |
39 | zkp-39 | + |
40 | zkp-40 | + |
41 | zkp-44 | + |
42 | zkp-48 | + |
43 | zkp-52 | + |
44 | zkp-54 | + |
45 | zkp-60 | + |
结论:用于测定裂解普的45株菌中,有1株为vB_KpnP_ZK1的宿主菌,44株为K1型肺炎克雷伯菌。由表可知,在空斑试验中,噬菌体vB_KpnP_ZK1裂解液能使全部45株型菌株产生空斑。说明该噬菌体有较宽裂解谱的特性和较强的特异性。
Claims (4)
1.一株噬菌体,命名为:肺炎克雷伯菌噬菌体vB_KpnP_ZK1,已于2020年11月9日保藏于中国典型培养物保藏中心,保藏名为Klebsiella pneumonia phage vB_KpnP_ZK1,保藏编号为:CCTCC NO:M2020713。
2.如权利要求1所述的噬菌体vB_KpnP_ZK1在制备杀灭空间环境、动物和人中肺炎克雷伯菌药物中的应用。
3.一种用于杀灭肺炎克雷伯菌的组合物,其包含权利要求1所述的噬菌体vB_KpnP_ZK1作为活性成分。
4.如权利要求3所述的组合物,其特征在于,所述组合物为液体制剂、冻干制剂或口服固体制剂。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110548215.7A CN113025582B (zh) | 2021-05-19 | 2021-05-19 | 一株肺炎克雷伯菌噬菌体及医用用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110548215.7A CN113025582B (zh) | 2021-05-19 | 2021-05-19 | 一株肺炎克雷伯菌噬菌体及医用用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113025582A CN113025582A (zh) | 2021-06-25 |
CN113025582B true CN113025582B (zh) | 2022-04-15 |
Family
ID=76455437
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110548215.7A Expired - Fee Related CN113025582B (zh) | 2021-05-19 | 2021-05-19 | 一株肺炎克雷伯菌噬菌体及医用用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113025582B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113174372B (zh) * | 2021-06-22 | 2023-04-11 | 吉林大学第一医院 | 一种噬菌体vB_KpnS_ZH01及医用用途 |
CN116004579B (zh) * | 2022-10-31 | 2024-09-10 | 吉林大学 | 一种抑制k2型肺炎克雷伯菌生物被膜的解聚酶及其应用 |
CN116200345A (zh) * | 2023-03-28 | 2023-06-02 | 福建农林大学 | 一种广谱型肺炎克雷伯菌噬菌体及其训化方法和应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017081709A1 (en) * | 2015-11-09 | 2017-05-18 | Universita' Degli Studi Di Siena | Bacteriophage able to lyse klebsiella pneumoniae expressing a cpskkbo-4 capsular polysaccharide and related medical uses thereof |
CN106754745A (zh) * | 2016-11-29 | 2017-05-31 | 江苏省农业科学院 | 一种肺炎克雷伯氏菌噬菌体及其应用 |
CN110438091A (zh) * | 2019-07-12 | 2019-11-12 | 广西大学 | 一株新的肺炎克雷伯氏菌噬菌体及其应用 |
CN112501135A (zh) * | 2020-12-10 | 2021-03-16 | 南京农业大学 | 肺炎克雷伯氏菌噬菌株P560、噬菌体解聚酶Depo43及应用 |
CN112708600A (zh) * | 2020-12-30 | 2021-04-27 | 瑞科盟(青岛)生物工程有限公司 | 一株裂解性肺炎克雷伯菌噬菌体rdp-kp-20005及其应用 |
CN113583973A (zh) * | 2021-08-05 | 2021-11-02 | 瑞科盟(青岛)生物工程有限公司 | 一株高裂解性肺炎克雷伯菌噬菌体rdp-kp-20007及其应用 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TW201130980A (en) * | 2010-03-02 | 2011-09-16 | Univ Ishou | A new lytic phage specific to Klebsiella pneumoniae |
ES2912268T3 (es) * | 2016-12-05 | 2022-05-25 | Technophage Investig E Desenvolvimento Em Biotecnologia Sa | Composiciones de bacteriófagos que comprenden fagos antibacterianos respiratorios y procedimientos de uso de las mismas |
CN109536459B (zh) * | 2018-11-14 | 2021-10-29 | 吉林大学 | 降解肺炎克雷伯氏菌荚膜多糖及生物被膜的噬菌体解聚酶 |
US20210187046A1 (en) * | 2019-12-19 | 2021-06-24 | Technophage, Investigação E Desenvolvimento Em Biotecnologia, Sa | Cocktail compositions comprising respiratory antibacterial phages and methods of use thereof |
CN112159798B (zh) * | 2020-10-20 | 2022-04-15 | 成都医学院 | 一株针对高毒力肺炎克雷伯氏菌的新噬菌体及其应用 |
CN113174372B (zh) * | 2021-06-22 | 2023-04-11 | 吉林大学第一医院 | 一种噬菌体vB_KpnS_ZH01及医用用途 |
-
2021
- 2021-05-19 CN CN202110548215.7A patent/CN113025582B/zh not_active Expired - Fee Related
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017081709A1 (en) * | 2015-11-09 | 2017-05-18 | Universita' Degli Studi Di Siena | Bacteriophage able to lyse klebsiella pneumoniae expressing a cpskkbo-4 capsular polysaccharide and related medical uses thereof |
CN106754745A (zh) * | 2016-11-29 | 2017-05-31 | 江苏省农业科学院 | 一种肺炎克雷伯氏菌噬菌体及其应用 |
CN110438091A (zh) * | 2019-07-12 | 2019-11-12 | 广西大学 | 一株新的肺炎克雷伯氏菌噬菌体及其应用 |
CN112501135A (zh) * | 2020-12-10 | 2021-03-16 | 南京农业大学 | 肺炎克雷伯氏菌噬菌株P560、噬菌体解聚酶Depo43及应用 |
CN112708600A (zh) * | 2020-12-30 | 2021-04-27 | 瑞科盟(青岛)生物工程有限公司 | 一株裂解性肺炎克雷伯菌噬菌体rdp-kp-20005及其应用 |
CN113583973A (zh) * | 2021-08-05 | 2021-11-02 | 瑞科盟(青岛)生物工程有限公司 | 一株高裂解性肺炎克雷伯菌噬菌体rdp-kp-20007及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN113025582A (zh) | 2021-06-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113025582B (zh) | 一株肺炎克雷伯菌噬菌体及医用用途 | |
CN109251898B (zh) | 一株金黄色葡萄球菌噬菌体及其应用 | |
CN112029732B (zh) | 一种耐高温宽裂解谱的沙门氏菌噬菌体及其组合物 | |
CN109207440B (zh) | 弧菌噬菌体及其杀菌组合物制备方法和应用 | |
CN108699531B (zh) | 多杀巴斯德菌噬菌体Pas-MUP-1及其用于抑制多杀巴斯德菌增殖的用途 | |
CN113621584B (zh) | 一株金黄色葡萄球菌噬菌体及其抑菌应用 | |
CN112725287B (zh) | 一株强裂解性金黄色葡萄球菌噬菌体rdp-sr-20001及其应用 | |
CN106929481B (zh) | 一种铜绿假单胞菌噬菌体及其应用 | |
CN113337480B (zh) | 一株广谱的大肠杆菌噬菌体及其应用 | |
CN112143709B (zh) | 一株嗜水气单胞菌噬菌体及其应用 | |
CN113174372B (zh) | 一种噬菌体vB_KpnS_ZH01及医用用途 | |
CN113583966B (zh) | 一株烈性沙门氏菌噬菌体及其应用 | |
CN109266621B (zh) | 一株新浅绿色气球菌噬菌体avp及其用途 | |
CN110747177A (zh) | 一株鲍曼不动杆菌噬菌体及医用用途 | |
CN113913391A (zh) | 一株可跨种裂解的大肠埃希氏菌噬菌体rdp-ec-20128及其应用 | |
Majdani et al. | Isolation and characterization of lytic bacteriophages against Pseudomonas aeruginosa isolates from human infections in the north-west of Iran | |
CN114292822B (zh) | 大肠杆菌噬菌体zjrp5及其应用和杀菌剂、药物 | |
CN112695017B (zh) | 噬菌体vB_Yen_X1及在防治鼠疫杆菌感染中应用 | |
CN113186169B (zh) | 一株粘质沙雷氏菌噬菌体及医用用途 | |
CN114196637A (zh) | 一种沙门氏菌噬菌体(salmonella sp.phage)JNwz02及其用途 | |
CN113215111B (zh) | 一种噬菌体及在防治肉鸡心内膜炎中的医用用途 | |
CN113046328B (zh) | 一株化脓隐秘杆菌噬菌体及其医用用途 | |
CN100363489C (zh) | 一株耐亚胺培南绿脓杆菌噬菌体及其用于治疗耐亚胺培南绿脓杆菌感染的用途 | |
CN115873808B (zh) | 一株弗氏柠檬酸杆菌噬菌体及其用途 | |
CN117603917A (zh) | 一株铜绿假单胞菌单链rna噬菌体及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220415 |