WO2014084644A1 - Method for detecting bacillus subtilis through fluorescent imaging of yqjl gene expression products - Google Patents

Method for detecting bacillus subtilis through fluorescent imaging of yqjl gene expression products Download PDF

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WO2014084644A1
WO2014084644A1 PCT/KR2013/010934 KR2013010934W WO2014084644A1 WO 2014084644 A1 WO2014084644 A1 WO 2014084644A1 KR 2013010934 W KR2013010934 W KR 2013010934W WO 2014084644 A1 WO2014084644 A1 WO 2014084644A1
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bacillus
bacillus subtilis
yqjl
subtilis
gene
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French (fr)
Korean (ko)
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이준석
한원석
이진각
윤창노
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한국과학기술연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)

Definitions

  • the present invention relates to a Bacillus subtilis detection kit comprising a fluorescent compound specifically binding to a hydrolase expressed from the yqjL gene of Bacillus subtilis and a Bacillus subtilis detection method using the same.
  • Bacterial identification is one of the most important processes of infectious disease diagnosis.
  • Conventional bacterial identification methods require a large number of test items and special media or reagents, and commercially available identification kits can produce simple and relatively fast results.
  • the kits are expensive and may not be identified correctly depending on the product. have.
  • Bacillus spp. Cause meningitis, endocarditis, osteomyelitis, bacteremia, pneumonia, and most of these strains are reported as Bacillus subtilis and Bacillus cereus. There is a need to distinguish between them.
  • CMFDA 5-Chloromethylfluorescein diacetate
  • the present invention aims to provide a simple, fast and accurate method for distinguishing Bacillus subtilis and Bacillus cereus by detecting yqjL gene expression products with fluorescent compounds.
  • One embodiment relates to a method of distinguishing Bacillus subtilis from Bacillus sp. Strains using fluorescent compounds.
  • Another embodiment is to provide a Bacillus subtilis detection kit comprising a fluorescent compound that specifically binds to a hydrolase expressed from the yqjL gene of Bacillus subtilis.
  • One aspect includes obtaining a bacterial extract from at least two Bacillus genus strains, including Bacillus subtilis;
  • Protein labeled with 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one from the bacterial extract It provides a method for distinguishing Bacillus subtilis from Bacillus sp. Strain, comprising the step of detecting.
  • the present inventors have studied the method of easily distinguishing Bacillus subtilis from among the strains of Bacillus, and confirmed that the specific fluorescent compound specifically binds to the hydrolase expressed from the yqjL gene of Bacillus subtilis. By completing the present invention.
  • the method may first comprise obtaining a bacterial extract from at least two Bacillus strains, including Bacillus subtilis.
  • the strain includes Bacillus cereus, Bacillus anthracis (in addition to Bacillus subtilis) Bacillus anthracis ), Bacillus brevis ( Bacillus brevis ), Bacillus thuringiensis ( Bacillus thuringiensis ), And Bacillus family ( Bacillus pumilus It may comprise one or more strains of the genus Bacillus selected from the group consisting of).
  • the strain may comprise Bacillus cereus in addition to Bacillus subtilis.
  • bacterial extract may be interpreted to mean the entire protein group contained in the strain of the genus Bacillus.
  • the bacterial extract may be obtained by using a kit capable of dissolving the bacteria, or by pulverizing the bacteria using ultrasonic waves and obtaining only protein in the bacteria by centrifugation according to methods well known in the art.
  • the present invention relates to a 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-), a fluorescent compound that specifically binds to a hydrolase expressed from the yqjL gene of Bacillus subtilis.
  • a fluorescent compound that specifically binds to a hydrolase expressed from the yqjL gene of Bacillus subtilis.
  • 1 (3H), 9 '-(9H) xanthene) -3-one it is possible to distinguish Bacillus subtilis from various kinds of Bacillus strains.
  • the step of obtaining may be to obtain a bacterial extract from Bacillus subtilis and Bacillus cereus.
  • the fluorescent compound used in the present invention Is a compound sold under the name of Cell Tracker TM Green CMFDA (Invitrogen, inc. Cat # C2925), which may be represented by the formula of Formula I below.
  • the fluorescent compound after directly contacting the fluorescent compound with the obtained bacterial extract, it may further comprise the step of confirming the protein pattern of the bacterial extract by electrophoresis of the contacted result.
  • the electrophoresis may be performed through the SDS-PAGE method well known in the art.
  • the method is a 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'- (9H) xanthene) -3 from the bacterial extract
  • the -on step is followed by a detection of the labeled protein.
  • the protein may be a hydrolase expressed from the yqjL gene of Bacillus subtilis, the hydrolase expressed from the yqjL gene is shown in SEQ ID NO: 1.
  • the fluorescent compound may be detected by using a scanner capable of detecting the fluorescent compound after electrophoresis of the contacted product, by comparing the fluorescent compound bound protein or the pattern of the protein, strains of the genus Bacillus Bacillus subtilis can be distinguished from.
  • Another aspect provides a biomarker for detecting Bacillus subtilis comprising a hydrolase expressed from the yqjL gene of Bacillus subtilis represented by the amino acid sequence of SEQ ID NO: 1.
  • Another aspect is a 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 () that specifically binds to a hydrolase expressed from the yqjL gene of Bacillus subtilis.
  • a kit for detecting Bacillus subtilis comprising 3H), 9 '-(9H) xanthene) -3-one is provided.
  • Bacillus subtilis detection kit is 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'- (9H) xanthene ) -3-one, a buffer capable of stabilizing the same, and the like, and may be used as a method for detecting a hydrolase expressed from the yqjL gene of Bacillus subtilis.
  • the kit may be used to distinguish Bacillus subtilis from strains of the genus Bacillus, but most preferably, the kit may be used to distinguish Bacillus subtilis and Bacillus cereus.
  • the methods and kits of the invention comprise 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene Hydrolase expressed from the yqjL gene of Bacillus subtilis that specifically binds to) -3-one can be detected to distinguish Bacillus subtilis from Bacillus cereus, thereby providing an easy, fast and accurate identification result.
  • 1 is a result of comparing the protein pattern of Bacillus subtilis ATCC 6633 and Bacillus cereus ATCC 27348 by a method according to an embodiment.
  • Bacillus subtilis ATCC 6633 and Bacillus cereus ATCC 2734 were purchased from ATCC to prepare proteins. Bacillus subtilis and Bacillus cereus were inoculated in 1 ml of LB broth (Difco TM LB Agar, Becton, Dickinson and Company, Lot #: 0049528) and incubated for 2 hours at 37 ° C. TM LB Agar, Becton, Dickinson and Company, Lot #: 0137418) to obtain a single colony and then incubated in 100 ml of LB medium.
  • the bacterial protein mixture solution in 20 mM Tris-HCl, pH 7.5 was treated with 20 uM of the fluorescent compound contained in 1% of DMSO and incubated at 25 ° C. for 1 hour. After completion of the reaction, the Laemmli sample buffer was added, heated at 95 ° C. for 2 minutes, loaded onto a polyacrylamide gel containing 15% SDS, followed by electrophoresis (120 V 20 minutes, followed by 180 V 60 minutes). Patterns of the proteins of Bacillus subtilis and Bacillus cereus were confirmed with a fluorescent SDS-PAGE Scanner (Typhoon TM 9400, GE Health Science). At this time, the label protein image of the Cell Tracker TM Green CMFDA was obtained under ex: 488 nm, em: 526-SP of the Typhoon TM 9400 instrument.
  • the SDS-PAGE showed that the protein pattern of Bacillus cereus did not appear, but the 28 kDa protein shown in the protein pattern of Bacillus subtilis interacted with Cell Tracker TM Green. Then, the following bioinformatics method was used to identify the 28 kDa protein.
  • Each three-dimensional structure is required to predict the interaction of the fluorescent compound with the 28 kDa protein.
  • a protein structure modeling program was used.
  • the 28 kDa protein 3D structure was predicted using a homology modeling program 'MODELLER' (University of California San Francisco).
  • a docking program was used to predict the interaction based on the three-dimensional structure of the protein and the compound.
  • the binding of the 28 kDa protein and the fluorescent compound was predicted using the 'AutoDock' (The Scripps Research Institute) program.
  • the 28 kDa protein was confirmed to be a hydrolase (Hydrolase) expressed from the yqjL gene of Bacillus subtilis. From the above results, it was confirmed that Cell Tracker TM Green interacted with the hydrolase expressed from the yqjL gene. Therefore, it was confirmed that the Bacillus subtilis and Bacillus cereus can be distinguished by checking the presence of the hydrolase expressed from the yqjL gene using the Cell Tracker TM Green.
  • Hydrolase Hydrolase

Abstract

The present invention relates to a Bacillus subtilis detection kit comprising a fluorescent compound which specifically binds to the hydrolase expressed from yqjL gene of the Bacillus subtilis and to a method for detecting Bacillus subtilis using said kit. The method and the kit according to one embodiment of the present invention detect the hydrolase expressed from yqjL gene of the Bacillus subtilis which specifically binds to 3',6'-bis(acetyloxy)-5-(chloromethyl)-spiro(isobenzofuran-1(3H),9'-(9H)xanthene)-3-on so as to distinguish between Bacillus subtilis and Bacillus cereus, thus providing an easy, quick and accurate identification result.

Description

yqjL 유전자 발현 산물의 형광 영상화를 통한 바실러스 서브틸리스 검출 방법Bacillus subtilis detection method through fluorescence imaging of yqjL gene expression product
본 발명은 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제와 특이적으로 결합하는 형광 화합물을 포함하는 바실러스 서브틸리스 검출 키트 및 이를 이용한 바실러스 서브틸리스 검출 방법에 관한 것이다.The present invention relates to a Bacillus subtilis detection kit comprising a fluorescent compound specifically binding to a hydrolase expressed from the yqjL gene of Bacillus subtilis and a Bacillus subtilis detection method using the same.
세균 동정은 감염성 질병 진단의 가장 중요한 과정 중의 하나이다. 기존의 세균 동정 방법은 많은 검사 종목과 특수 배지나 시약을 필요로 하고, 상용화된 동정 키트는 간단하고 비교적 빠른 결과를 얻을 수 있으나, 키트의 가격이 비싸며 제품에 따라서는 정확한 동정이 이루어지지 않을 수도 있다. Bacterial identification is one of the most important processes of infectious disease diagnosis. Conventional bacterial identification methods require a large number of test items and special media or reagents, and commercially available identification kits can produce simple and relatively fast results. However, the kits are expensive and may not be identified correctly depending on the product. have.
바실러스 종은 뇌막염, 심내막염, 골수염, 균혈증, 폐렴등을 일으키며, 이와 연관된 균주는 대부분이 바실러스 서브틸리스 (Bacillus subtilis)와 바실러스 세레우스 (Bacillus cereus)로 보고되고 있으므로 상기 두 균주의 개별 항생제 처방을 위해 구별해야 될 필요성이 있다.Bacillus spp. Cause meningitis, endocarditis, osteomyelitis, bacteremia, pneumonia, and most of these strains are reported as Bacillus subtilis and Bacillus cereus. There is a need to distinguish between them.
바실러스 속의 여러 균주로부터 바실러스 서브틸리스를 구별하는데 있어, 유전자 서열 분석기와 같은 거대 장비를 이용하지 않고, 균주 사이의 단백체 차이를 실험실 수준에서 확인하는 방법은 기존에 알려져 있지 않다. In distinguishing Bacillus subtilis from several strains of Bacillus, it is not known how to identify protein differences between strains at the laboratory level without using large equipment such as gene sequence analyzers.
5-클로로메틸플루오레세인 디아세테이트 (5-chloromethylfluorescein diacetate, CMFDA)는 세포막을 통과하는 형광 화합물로서, 표지된 세포의 이동 분석 등에 사용될 수 있는 세포 추적체 (cell tracker)이다. 5-Chloromethylfluorescein diacetate (CMFDA) is a fluorescent compound that crosses the cell membrane and is a cell tracker that can be used for analyzing the movement of labeled cells.
본 발명에서는 yqjL 유전자 발현 산물을 형광 화합물로 검출하여 바실러스 서브틸리스와 바실러스 세레우스를 간단하고, 빠르며, 정확하게 구별하는 방법을 제공하고자 한다.The present invention aims to provide a simple, fast and accurate method for distinguishing Bacillus subtilis and Bacillus cereus by detecting yqjL gene expression products with fluorescent compounds.
일 구체예는 형광 화합물을 이용하여 바실러스 속 균주로부터 바실러스 서브틸리스를 구별하는 방법에 관한 것이다. One embodiment relates to a method of distinguishing Bacillus subtilis from Bacillus sp. Strains using fluorescent compounds.
다른 구체예는 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제와 특이적으로 결합하는 형광 화합물을 포함하는 바실러스 서브틸리스 검출 키트를 제공하는 것이다.Another embodiment is to provide a Bacillus subtilis detection kit comprising a fluorescent compound that specifically binds to a hydrolase expressed from the yqjL gene of Bacillus subtilis.
일 양상은 바실러스 서브틸리스를 포함한 2 이상의 바실러스 속 균주로부터 세균 추출물을 수득하는 단계;One aspect includes obtaining a bacterial extract from at least two Bacillus genus strains, including Bacillus subtilis;
상기 세균 추출물과 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 접촉시키는 단계; 및 Contacting the bacterial extract with 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one ; And
상기 세균 추출물로부터 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온이 표지된 단백질을 검출하는 단계를 포함하는, 바실러스 속 균주로부터 바실러스 서브틸리스를 구별하는 방법을 제공한다. Protein labeled with 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one from the bacterial extract It provides a method for distinguishing Bacillus subtilis from Bacillus sp. Strain, comprising the step of detecting.
본 발명자들은 바실러스 속 균주들 중에서 바실러스 서브틸리스를 쉽게 구별할 수 있는 방법을 연구한 결과, 특정 형광 화합물이 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제와 특이적으로 결합한다는 사실을 확인함으로서 본 발명을 완성하였다. The present inventors have studied the method of easily distinguishing Bacillus subtilis from among the strains of Bacillus, and confirmed that the specific fluorescent compound specifically binds to the hydrolase expressed from the yqjL gene of Bacillus subtilis. By completing the present invention.
상기 방법은 먼저, 바실러스 서브틸리스를 포함한 2 이상의 바실러스 속 균주로부터 세균 추출물을 수득하는 단계를 포함할 수 있다. 상기 균주는 바실러스 서브틸리스 외에 바실러스 세레우스, 바실러스 안트라시스 (Bacillus anthracis), 바실러스 브레비스 (Bacillus brevis), 바실러스 튜린겐시스 (Bacillus thuringiensis), 및 바실러스 프밀리스 (Bacillus pumilus)로 구성된 군으로부터 선택되는 하나 이상의 바실러스 속 균주를 포함할 수 있다. 일 구체예에 따르면, 상기 균주는 바실러스 서브틸리스 외에 바실러스 세레우스를 포함할 수 있다.The method may first comprise obtaining a bacterial extract from at least two Bacillus strains, including Bacillus subtilis. The strain includes Bacillus cereus, Bacillus anthracis (in addition to Bacillus subtilis)Bacillus anthracis), Bacillus brevis (Bacillus brevis), Bacillus thuringiensis (Bacillus thuringiensis), And Bacillus family (Bacillus pumilusIt may comprise one or more strains of the genus Bacillus selected from the group consisting of). According to one embodiment, the strain may comprise Bacillus cereus in addition to Bacillus subtilis.
본 명세서에서 용어, "세균 추출물"은 구별하고자 하는 대상의 바실러스 속 균주 내에 포함되어 있는 단백체 전체 집단을 의미하는 것으로 해석될 수 있다. 세균 추출물은 당업계에 널리 알려진 방법에 따라, 세균을 용해할 수 있는 키트를 이용하거나, 초음파를 이용하여 세균을 분쇄하고 원심분리를 통해 세균 내 단백체만 얻는 방법 등을 통해 수득할 수 있다. As used herein, the term "bacterial extract" may be interpreted to mean the entire protein group contained in the strain of the genus Bacillus. The bacterial extract may be obtained by using a kit capable of dissolving the bacteria, or by pulverizing the bacteria using ultrasonic waves and obtaining only protein in the bacteria by centrifugation according to methods well known in the art.
본 발명은 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제에 특이적으로 결합하는 형광 화합물인 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 이용하는 것으로, 여러 종류의 바실러스 속 균주로부터 바실러스 서브틸리스를 구별할 수 있다. 일 구체예에 따르면, 상기 수득하는 단계는 바실러스 서브틸리스 및 바실러스 세레우스로부터 세균 추출물을 수득하는 것일 수 있다. The present invention relates to a 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-), a fluorescent compound that specifically binds to a hydrolase expressed from the yqjL gene of Bacillus subtilis. By using 1 (3H), 9 '-(9H) xanthene) -3-one, it is possible to distinguish Bacillus subtilis from various kinds of Bacillus strains. According to one embodiment, the step of obtaining may be to obtain a bacterial extract from Bacillus subtilis and Bacillus cereus.
다음으로, 상기 세균 추출물과 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 접촉시키는 단계를 거치게 된다. Next, the bacterial extract and 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one The contacting step is performed.
본 발명에서 사용되는 형광 화합물인 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온은 Cell TrackerTM Green CMFDA (Invitrogen, inc. Cat# C2925)의 이름으로 판매되는 화합물로서, 하기 화학식 I의 화학식으로 나타낼 수 있다. 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one, the fluorescent compound used in the present invention Is a compound sold under the name of Cell Tracker ™ Green CMFDA (Invitrogen, inc. Cat # C2925), which may be represented by the formula of Formula I below.
화학식 I Formula I
Figure PCTKR2013010934-appb-I000001
Figure PCTKR2013010934-appb-I000001
일 구체예에 따르면, 상기 형광 화합물을 상기 수득한 세균 추출물에 직접 접촉시킨 이후, 상기 접촉된 결과물을 전기영동하여 상기 세균 추출물의 단백질 패턴을 확인하는 단계를 더 포함할 수 있다. 상기 전기영동은 당업계에 잘 알려진 SDS-PAGE 방법을 통해 이루어질 수 있다. According to one embodiment, after directly contacting the fluorescent compound with the obtained bacterial extract, it may further comprise the step of confirming the protein pattern of the bacterial extract by electrophoresis of the contacted result. The electrophoresis may be performed through the SDS-PAGE method well known in the art.
마지막으로, 상기 방법은 상기 세균 추출물로부터 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온이 표지된 단백질을 검출하는 단계를 거치게 된다. Finally, the method is a 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'- (9H) xanthene) -3 from the bacterial extract The -on step is followed by a detection of the labeled protein.
일 구체예에 따르면, 상기 단백질은 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제일 수 있으며, 상기 yqjL 유전자로부터 발현되는 하이드롤라아제는 서열번호 1에 나타내었다. According to one embodiment, the protein may be a hydrolase expressed from the yqjL gene of Bacillus subtilis, the hydrolase expressed from the yqjL gene is shown in SEQ ID NO: 1.
상기 형광 화합물은 상기 접촉된 결과물을 전기영동한 후, 상기 형광 화합물을 검출할 수 있는 스캐너를 이용하여 검출할 수 있으며, 상기 형광 화합물이 결합된 단백질 또는 상기 단백질의 패턴을 비교함으로써, 바실러스 속 균주로부터 바실러스 서브틸리스를 구별할 수 있다. The fluorescent compound may be detected by using a scanner capable of detecting the fluorescent compound after electrophoresis of the contacted product, by comparing the fluorescent compound bound protein or the pattern of the protein, strains of the genus Bacillus Bacillus subtilis can be distinguished from.
다른 양상은 서열번호 1의 아미노산 서열로 표시되는 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제를 포함하는 바실러스 서브틸리스 검출용 바이오 마커를 제공한다. Another aspect provides a biomarker for detecting Bacillus subtilis comprising a hydrolase expressed from the yqjL gene of Bacillus subtilis represented by the amino acid sequence of SEQ ID NO: 1.
또 다른 양상은 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제와 특이적으로 결합하는 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 포함하는 바실러스 서브틸리스 검출용 키트를 제공한다. Another aspect is a 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 () that specifically binds to a hydrolase expressed from the yqjL gene of Bacillus subtilis. A kit for detecting Bacillus subtilis comprising 3H), 9 '-(9H) xanthene) -3-one is provided.
일 구체예에 따른 바실러스 서브틸리스 검출용 키트는 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온 및 이를 안정화시킬 수 있는 버퍼 등을 포함할 수 있으며, 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제를 검출하는 용도로서 사용될 수 있다. Bacillus subtilis detection kit according to one embodiment is 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'- (9H) xanthene ) -3-one, a buffer capable of stabilizing the same, and the like, and may be used as a method for detecting a hydrolase expressed from the yqjL gene of Bacillus subtilis.
한편, 상기 키트는 바실러스 속 균주들로부터 바실러스 서브틸리스를 구별하는 데 사용될 수 있으나, 가장 바람직하게는, 상기 키트는 바실러스 서브틸리스 및 바실러스 세레우스를 구별하기 위한 것일 수 있다.Meanwhile, the kit may be used to distinguish Bacillus subtilis from strains of the genus Bacillus, but most preferably, the kit may be used to distinguish Bacillus subtilis and Bacillus cereus.
일 구체예에 따르면, 본 발명의 방법 및 키트는 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온에 특이적으로 결합하는 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제를 검출하여 바실러스 서브틸리스와 바실러스 세레우스를 구별하므로, 쉽고 빠르며 정확한 동정 결과를 제공할 수 있다.According to one embodiment, the methods and kits of the invention comprise 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene Hydrolase expressed from the yqjL gene of Bacillus subtilis that specifically binds to) -3-one can be detected to distinguish Bacillus subtilis from Bacillus cereus, thereby providing an easy, fast and accurate identification result.
도 1은 일 구체예에 따른 방법에 의해 바실러스 서브틸리스 ATCC 6633와 바실러스 세레우스 ATCC 27348의 단백질 패턴을 비교한 결과이다.1 is a result of comparing the protein pattern of Bacillus subtilis ATCC 6633 and Bacillus cereus ATCC 27348 by a method according to an embodiment.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more embodiments will be described in more detail with reference to Examples. However, these examples are provided to illustrate one or more embodiments illustratively and the scope of the present invention is not limited to these examples.
1. 실험 방법 1. Experiment Method
(1) 세균의 배양 및 단백체의 준비 (1) Cultivation of bacteria and preparation of proteins
바실러스 서브틸리스 ATCC 6633와 바실러스 세레우스 ATCC 2734를 ATCC로부터 구입하여 단백체를 준비하였다. Bacillus subtilis 및 Bacillus cereus를 각각 1 ml의 LB broth (DifcoTM LB Agar, Becton, Dickinson and Company, Lot#: 0049528)에 접종하여 37℃에서 2시간 동안 배양한 후, 20 ul를 LB Agar plate (DifcoTM LB Agar, Becton, Dickinson and Company, Lot#: 0137418)에서 배양하여, 단일 콜로니를 얻은 다음, 100 ml의 LB 배지에 배양하였다. 배양한 세균을 원심분리 하여 상층액을 버리고, PBS 용액(pH 7.4)으로 2번 씻어 준 후, Genlantis 사의 SoluLyse kit(Cat #: L200125)를 이용하여 최종적으로 20 mM Tris-HCl, pH7.5에 용해된 상기 세균들의 단백질 혼합물 용액을 얻었다. Bacillus subtilis ATCC 6633 and Bacillus cereus ATCC 2734 were purchased from ATCC to prepare proteins. Bacillus subtilis and Bacillus cereus were inoculated in 1 ml of LB broth (Difco TM LB Agar, Becton, Dickinson and Company, Lot #: 0049528) and incubated for 2 hours at 37 ° C. TM LB Agar, Becton, Dickinson and Company, Lot #: 0137418) to obtain a single colony and then incubated in 100 ml of LB medium. Centrifuge the cultured bacteria, discard the supernatant, wash twice with PBS solution (pH 7.4), and finally, using SolanLyse kit (Cat #: L200125) of Genlantis in 20 mM Tris-HCl, pH7.5. A solution of the protein mixture of the above dissolved bacteria was obtained.
(2) 형광 화합물 처리 및 SDS-PAGE 이미지 분석 (2) Fluorescent Compound Treatment and SDS-PAGE Image Analysis
20 mM Tris-HCl, pH 7.5 내 상기 세균 단백질 혼합물 용액에, DMSO 1%에 포함된 20 uM의 상기 형광 화합물을 처리하고, 25℃에서 1시간 동안 인큐베이션 하였다. 반응을 마친 후, Laemmli 샘플 버퍼를 넣고, 95℃에서 2분 동안 가열한 다음, 15% SDS가 포함된 폴리아크릴아미드 겔에 로딩하여 전기영동(120V 20분, 이어서 180V 60분)한 후, 상기 Bacillus subtilis 및 Bacillus cereus의 단백질의 패턴을 형광 SDS-PAGE Scanner (TyphoonTM 9400, GE Health Science)로 확인하였다. 이때, Cell TrackerTM Green CMFDA의 표지 단백질 이미지는 TyphoonTM 9400 기기의 ex: 488 nm, em: 526-SP 조건에서 얻었다. The bacterial protein mixture solution in 20 mM Tris-HCl, pH 7.5 was treated with 20 uM of the fluorescent compound contained in 1% of DMSO and incubated at 25 ° C. for 1 hour. After completion of the reaction, the Laemmli sample buffer was added, heated at 95 ° C. for 2 minutes, loaded onto a polyacrylamide gel containing 15% SDS, followed by electrophoresis (120 V 20 minutes, followed by 180 V 60 minutes). Patterns of the proteins of Bacillus subtilis and Bacillus cereus were confirmed with a fluorescent SDS-PAGE Scanner (Typhoon ™ 9400, GE Health Science). At this time, the label protein image of the Cell Tracker ™ Green CMFDA was obtained under ex: 488 nm, em: 526-SP of the Typhoon ™ 9400 instrument.
2. 실험 결과 2. Experimental Results
도 1에서 나타낸 바와 같이, SDS-PAGE 결과로부터 Bacillus cereus의 단백질 패턴에서는 나타나지 않지만, Bacillus subtilis의 단백질 패턴에서 보이는 28 kDa 정도의 단백질이 Cell TrackerTM Green과 상호 작용함을 알 수 있었다. 이후, 상기 28 kDa의 단백질을 확인하기 위하여 다음과 같은 생물정보학적 방법을 사용하였다. As shown in FIG. 1, the SDS-PAGE showed that the protein pattern of Bacillus cereus did not appear, but the 28 kDa protein shown in the protein pattern of Bacillus subtilis interacted with Cell Tracker TM Green. Then, the following bioinformatics method was used to identify the 28 kDa protein.
상기 형광 화합물과 28 kDa 단백질의 상호 작용을 예측하기 위해서는 각각의 3차원 구조가 필요하다. 이를 위해, 단백질 구조 모델링 프로그램을 사용하였으며, 본 실시예에서는 호몰로지 모델링(homology modeling) 프로그램인 'MODELLER' (University of California San Francisco)를 이용하여 상기 28 kDa 단백질 3차원 구조를 예측하였다. 또한, 상기 28 kDa 단백질과 Cell TrackerTM Green과의 상호작용을 예상하기 위하여 단백질과 화합물의 3차원 구조를 바탕으로 상호작용을 예측할 수 있는 도킹(docking) 프로그램을 사용하였다. 본 실시예에서는 'AutoDock' (The Scripps Research Institute) 프로그램을 이용하여 상기 28 kDa 단백질과 상기 형광 화합물의 결합을 예측하였다. 그 결과, 상기 28 kDa 단백질은 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제(Hydrolase)임을 확인할 수 있었다. 상기 결과로부터 Cell TrackerTM Green은 yqjL 유전자로부터 발현되는 하이드롤라아제와 상호 작용함을 확인할수 있었다. 따라서, Cell TrackerTM Green을 사용하여 yqjL 유전자로부터 발현되는 하이드롤라아제의 존재 여부를 확인함으로써, 바실러스 서브틸리스와 바실러스 세레우스를 구별할 수 있음을 확인할 수 있었다.Each three-dimensional structure is required to predict the interaction of the fluorescent compound with the 28 kDa protein. To this end, a protein structure modeling program was used. In this example, the 28 kDa protein 3D structure was predicted using a homology modeling program 'MODELLER' (University of California San Francisco). In addition, in order to predict the interaction between the 28 kDa protein and Cell Tracker ™ Green, a docking program was used to predict the interaction based on the three-dimensional structure of the protein and the compound. In this example, the binding of the 28 kDa protein and the fluorescent compound was predicted using the 'AutoDock' (The Scripps Research Institute) program. As a result, the 28 kDa protein was confirmed to be a hydrolase (Hydrolase) expressed from the yqjL gene of Bacillus subtilis. From the above results, it was confirmed that Cell Tracker TM Green interacted with the hydrolase expressed from the yqjL gene. Therefore, it was confirmed that the Bacillus subtilis and Bacillus cereus can be distinguished by checking the presence of the hydrolase expressed from the yqjL gene using the Cell Tracker TM Green.

Claims (9)

  1. 바실러스 서브틸리스(Bacillus subtilis)를 포함하는 2 이상의 바실러스 속 균주로부터 세균 추출물을 수득하는 단계; Obtaining a bacterial extract from at least two Bacillus genus strains including Bacillus subtilis ;
    상기 세균 추출물과 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 접촉시키는 단계; 및 Contacting the bacterial extract with 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one ; And
    상기 세균 추출물로부터 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온이 표지된 단백질을 검출하는 단계를 포함하는, 세균으로부터 바실러스 서브틸리스를 구별하는 방법.Protein labeled with 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one from the bacterial extract Detecting the Bacillus subtilis from the bacterium.
  2. 청구항 1에 있어서, 상기 균주는 바실러스 서브틸리스 외에 바실러스 안트라시스 (Bacillus anthracis), 바실러스 세레우스(Bacillus cereus), 바실러스 브레비스 (Bacillus brevis), 바실러스 튜린겐시스 (Bacillus thuringiensis), 및 바실러스 프밀리스 (Bacillus pumilus)로 구성된 군으로부터 선택되는 하나 이상의 바실러스 속 균을 포함하는 것인 방법.The method according to claim 1, wherein the strain is Bacillus subtilis in addition to the Bacillus anthraquinone system (Bacillus anthracis), Bacillus cereus (Bacillus cereus), Bacillus brevis (Bacillus brevis), Bacillus tube ringen sheath (Bacillus thuringiensis), and Bacillus program mm's ( Bacillus pumilus ) comprising one or more Bacillus genus bacteria selected from the group consisting of.
  3. 청구항 2에 있어서, 상기 균주는 바실러스 서브틸리스 외에 바실러스 세레우스를 포함하는 것인 방법.The method of claim 2, wherein the strain comprises Bacillus cereus in addition to Bacillus subtilis.
  4. 청구항 1에 있어서, 상기 접촉시키는 단계 이후, 상기 접촉된 결과물을 전기영동하여 상기 세균 추출물의 단백질 패턴을 확인하는 단계를 더 포함하는 것인 방법.The method of claim 1, further comprising, after the step of contacting, confirming the protein pattern of the bacterial extract by electrophoresis of the contacted result.
  5. 청구항 1에 있어서, 상기 단백질은 하이드롤라아제인 것인 방법.The method of claim 1, wherein the protein is hydrolase.
  6. 청구항 5에 있어서, 상기 하이드롤라아제는 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 것인 방법.The method of claim 5, wherein the hydrolase is expressed from the yqjL gene of Bacillus subtilis.
  7. 서열번호 1의 아미노산 서열로 표시되는 바실러스 서브틸리스의 yqjL 유전자로부터 발현되는 하이드롤라아제를 포함하는 바실러스 서브틸리스 검출용 바이오 마커.A biomarker for detecting Bacillus subtilis comprising a hydrolase expressed from the yqjL gene of Bacillus subtilis represented by the amino acid sequence of SEQ ID NO: 1.
  8. 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 포함하는 바실러스 서브틸리스 검출용 키트.Bacillus subtilis detection, including 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one Kit.
  9. 청구항 8에 있어서, 상기 키트는 바실러스 서브틸리스 및 바실러스 세레우스를 구별하기 위한 것인 키트.The kit of claim 8, wherein the kit is for distinguishing Bacillus subtilis and Bacillus cereus.
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