JP2011239749A - Detection method and identification method of spore using differential response of spore surface antigen protein and antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish a technology for determining the position of spore coat protein in spore-forming bacteria, and to provide a method and means for searching the protein that exists on spore surface layer.SOLUTION: There is provided a method for estimating whether a candidate protein exists on the spore surface in spore-forming bacteria or not includes steps of: (a) measuring the diameter of the spore of the spore-forming bacteria; (b) measuring the diameter of the shell comprising the candidate protein; (c) obtaining the diameter ratio of the shell comprising the candidate protein when the diameter of the spore is 1, subtracting the diameter ratio from 1 divided by 2 to gain the value as an absolute position of the candidate protein, and estimating the candidate protein which exists on the spore surface when the absolute position is ≤0.06.

Description

  The present invention relates to a technique for searching for proteins present in the spore surface of spore-forming bacteria. The present invention also relates to development and improvement of a method for detecting spore-forming bacteria based on this technology, a method for removing spore-forming bacteria, and applied technology such as pharmaceutical production.

  A spore-forming bacterium (endospore-forming bacterium) is a series of biological cells: vegetative cell division / proliferation (logarithmic growth phase), spore formation, spore (dormant), germination, post-emergence growth, and logarithmic growth of vegetative cells. It has a life cycle with change. Vegetative cells undergo unequal division due to changes in environmental conditions such as depletion of nutrients, and differentiate into two cells: mother cells and Prespore. The prespore is taken into the mother cell and becomes a forespore (Forespore). When the forespore matures and becomes a spore, the mother cell self-lyses.

  Since spores have high durability (heat resistance, pressure resistance, chemical resistance) and long-term dormancy, strict sterilization operations that require spore contamination are required in the fields of food, beverages, and pharmaceuticals. In addition, since the strength of durability of the spore varies depending on the type (bacterial species and strains), when it is mixed in raw materials or products, the type must be identified quickly and accurately and a response corresponding to the type must be taken. For general bacteria and vegetative cells, the polymerase chain reaction (PCR) method can be used for rapid testing. However, in the case of spores, a strong outer shell structure makes it difficult to extract DNA and RNA efficiently. In addition, it is difficult to efficiently extract DNA and RNA from spore present in a small amount in a sample, and the PCR method is not very effective. In addition, examination methods such as immunostaining method based on antigen-antibody reaction, enzyme-linked immunosorbent assay (ELISA) method, flow cytometry (FCM) method are also conceivable. Such an inspection method has not been carried out because few antibodies are known to react with the above.

  Since spore-forming bacteria are rich in diversity and antigens common to all spores are not known, to develop a test method based on an antigen-antibody reaction, obtain an antigen for each spore to be detected, Antibodies need to be prepared. If an antibody is obtained by a method in which the type and nature of the antigen are not specified, it is necessary to search for an antibody having reactivity suitable for the purpose from as many candidate samples as possible, and the antibody reacts with the target spore. Can not be put to practical use with antibodies that react with cells other than the desired spores because of their low specificity. Therefore, even if an antibody is produced without specifying an antigen, a specific method for detecting a target spore cannot be developed while requiring a great amount of labor and cost until practical use. Furthermore, if the specified antigen cannot be stably obtained, it is difficult to stably prepare a polyclonal antibody exhibiting the same reactivity and cannot be put into practical use.

  Regarding the method of detecting spores of Bacillus anthracis (Bacillus anthracis), which is known as a spore-forming bacterium causing serious health damage, there are sporadic prior art techniques, and protective antigen (PA) is known as a surface antigen. It has been. Although it has already been disclosed that this antigen is present on the cell surface (Non-Patent Document 1), the determination of the site where the antigen is present has been made as a comprehensive judgment using various methods, and the determination method thereof. Is hard to say.

  Moreover, although patent document 1 and 2 are producing and using the antibody which reacts with anthrax and its spore, since the antibody is acquired by the method in which an antigenic substance is not specified, between the strains of the same bacterial species Differences in antibody reactivity and the presence or absence of antibody cross-reactivity with other spore-forming bacteria cannot be predicted from the base sequence of the gene, and a reactivity test is required for each bacterial species or strain. Therefore, although it is considered to be an effective means when the application range is limited to Bacillus anthracis or a specific strain of Bacillus anthracis, it does not contribute to the development of a detection method for other spore-forming bacteria.

  Furthermore, Non-Patent Document 2 discloses a two-color FCM method using a PA antigen-antibody assay, and it is stated that a surface antigen for distinguishing Bacillus bacteria is being searched using this method. ing. However, since a specific method for searching for a surface antigen is not shown, it is not possible to carry out the method, and no success report has been made.

  The spore of the genus Bacillus is, in order from the inside, the core, the inner spore membrane, the germ cell wall, the cortex, the outer spore membrane, and the spore. It consists of a shell (Spore Coat). Such a basic structure is common to almost all spores of Firmicutes bacteria. By observation of thin sections of spores using a transmission electron microscope, the spore shell is roughly divided into two structures (inner coat and outer coat), each of which has a multilayer structure (multi-shell structure). Furthermore, in B. cereus and Bacillus anthracis, Exosporium exists outside the spore shell. Exosporium is a thin film that covers spores and is an atypical bag-like structure.

  The spore shell and exosporium are considered to be composed of proteins and the like, and the protein constituting the spore shell is called a coat protein (spore shell protein). It is presumed that single or plural types of coat proteins accumulate in layers along the shape of the spore and form each shell-like structure so as to wrap the spore. To date, research using mainly Bacillus subtilis (Bacillus subtilis) has identified a wide variety of coat proteins (spore shell proteins), localization of these proteins in the spore, network, spore Much research has been done on its role in formation. Based on some experimental data, it is believed that the inner coat and outer coat described above are composed of different proteins. Many of the coat proteins (spore shell proteins) were originally identified by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Edman analysis. Later, with the progress of Bacillus subtilis genome analysis, it was identified by a new research method. For example, the present inventors previously separated a protein sample obtained by treatment of Bacillus subtilis spores with SDS and mercaptoethanol by SDS-PAGE in Non-Patent Document 3, and then mass spectrometer (LC-MS / MS) is used to identify the type of protein contained. Furthermore, the expression time of the gene encoding each protein is analyzed, and the type of protein present in the spore shell is estimated. However, at this point in time, the idea and technique for specifying the protein present in the spore surface as shown in the present invention has not been obtained.

  Non-Patent Document 4 examines the localization site in spores by labeling Bacillus subtilis spore shell protein with green fluorescent protein (GFP). The main purpose of this paper is to know whether or not the studied protein exists in the spore shell, and does not mention the relative positional relationship of each protein or the existence of spore surface proteins.

  Non-Patent Document 5 discloses a new detection method using an atomic force microscope for spore surface proteins of Bacillus subtilis using an anti-CotA antibody, but is not accompanied by comparison experiment data using a negative control. There is doubt about authenticity. In addition, only one type of anti-CotA antibody is tested from the few existing antibodies, and other spore shell proteins have not been studied. Therefore, the validity of selecting an anti-CotA antibody could not be confirmed. Cannot be evaluated. Furthermore, the atomic force microscope requires a long time for data acquisition and cannot be used for daily inspection.

  Non-Patent Document 6 is a report that the anti-CotB antibody was presumed to be a protein present in the spore surface, reacted with wild type spores of Bacillus subtilis, and detected by the FCM method. This report does not disclose comparison experiment data using a negative control, so there is doubt about its authenticity. Further, according to the disclosed data, the reactivity between the anti-CotB antibody and CotB is weak, and there is almost no utility value as a spore detection technique.

  According to Non-Patent Document 7, the present inventors have also shown that the spore shell protein labeled with GFP or red fluorescent protein (RFP) exists in the forespore (prespore) in the fluorescence microscope observation of the forspore (prespore). . As a result, the localization site of the protein in the mature spore can be estimated and the relative positional relationship between the two types of spore shell proteins can be grasped. However, even at this time, the absolute position determination method of these proteins in the spore shell has not been conceived, and the spore surface protein has not been specified.

  None of the above reports provide estimations or hypotheses about the proteins present on the spore surface, but none have been proved, and the proteins present on the spore surface were not clear. Therefore, it has been difficult to provide a method or means that can detect spores with high accuracy.

Special Table 2007-537277 Publication JP 2003-238599 A

Redmond, C. et al., Microbiol. 150 (2): 355-363, 2004 Schumacher, W.C. et al., Appl.Environ.Microbiol. 74 (16): 5220-5223, 2008 Kuwana, R. et al., Microbiol. 148 (12): 3971-3982, 2002 Kim, H. et al., Mol. Microbiol. 59 (2): 487-502, 2006 Tang, J. et al., J. Mol. Recognit. 20 (6): 483-489, 2007 Isticato, R. et al., J. Bacteriol. 183 (21): 6294-6301, 2001 Takamatsu, H. et al., J. Bacteriol. 191 (4): 1220-1229, 2009

  As described above, it has been generally accepted as a known fact that the spore shell is composed of a wide variety of proteins and that some of them may be present on the surface layer of the spore.

  By the way, Bacillus subtilis is a non-pathogenic bacterium that can be easily genetically modified, so it can be used as a genetically modified bioreactor by fusing spore shell protein and any useful enzyme, or spore shell protein and any pathogenic microorganism. A technique for use as a genetically modified vaccine is attempted by fusing these antigens, and a typical spore shell protein is used as a support. However, the position of the spore shell protein as a support in the spore is not clear, and therefore it has not been confirmed to be on the surface layer, and no evidence has been given as to whether the selection is optimal.

  For Bacillus anthracis, spore detection methods using antibodies were developed because of their extremely strong pathogenicity, and their usefulness was confirmed, but for other spore-forming bacteria, active development of detection antibodies It wasn't done. On the other hand, most of these spore-forming bacteria are harmless to humans and livestock or have low pathogenicity, but have strong resistance and long-term dormancy. Remains as it is now. In this field, development of a technique capable of detecting a specific spore quickly and easily using an optimal antibody is desired.

  As described above, in the bioreactor, vaccine production field, food hygiene field, medical hygiene field, etc., as a basic technology for use as a support or antibody preparation, a technique for determining the position of spore shell protein in mature spores Establishment is desired.

  As a result of intensive studies to solve the above problems, the present inventors have labeled the candidate protein and expressed the diameter in the shell formed by the candidate protein identified based on the label when expressed in the spore-forming bacteria, We compared the spore length of the spore-forming bacteria and found that the position of the candidate protein in the spore can be identified based on the difference. In addition, the present inventors have confirmed that the antibody against the protein presumed to be present on the surface layer of the spore actually reacts with the spore-forming bacteria. It has been found that spore-forming bacteria can be detected by using it. The present inventors have completed the present invention based on the above findings.

That is, the present invention is as follows.
[1] A method for estimating whether or not a candidate protein is present on a spore surface in a spore-forming bacterium,
(A) measuring the spore length of spore-forming bacteria;
(B) measuring a diameter of a shell formed by the candidate protein;
(C) The ratio of the diameter of the shell formed by the candidate protein when the diameter of the spore is 1 is obtained, and the value obtained by subtracting the diameter ratio from 1 and dividing by 2 is defined as the absolute position of the candidate protein. Estimating the candidate protein in the spore surface when the target position is 0.06 or less.
[2] The spore length is obtained as the distance between the minimum or maximum values of the brightness of the observed image when the brightness on a straight line passing through the center of the spore and connecting the two poles is evaluated using an optical microscope. ] Method.
[3] The maximum value of the labeled signal of the image obtained when the diameter of the shell constituted by the candidate protein is evaluated using a microscope for the candidate protein labeled while maintaining the shape and location of the spore. The method according to [1] or [2], obtained as a distance between. [4] The method according to [3], wherein the label is a fluorescent protein.
[5] For the candidate protein presumed to be present on the spore surface in step (c), the method further comprises a step of verifying that the candidate protein is present on the spore surface by immunostaining. The method according to any one.
[6] A spore-forming bacterium belongs to the genus Bacillus, the bacterium belonging to the genus Clostridium, the bacterium belonging to the genus Sporolactobacillus, the bacterium belonging to the genus Geobacillus, the bacterium belonging to the genus Thermonerobacter, or the genus Thermonerobacterium The method according to any one of [1] to [5], which is selected from bacteria, bacteria belonging to the genus desulphotomacrum, bacteria belonging to the genus Morella, bacteria belonging to the genus Alicyclobacillus, and bacteria belonging to the genus Ricinibacillus .
[7] The method according to [6], wherein the spore-forming bacterium is Bacillus subtilis.

  The present invention provides a technique for efficiently and reliably searching for proteins present in the spore surface of spore-forming bacteria. The present invention also contributes to the development and improvement of a spore detection method based on this technology, the removal method of spores, and the development of applied technologies such as pharmaceutical production using spores. Therefore, the method for detecting and removing spore-forming bacteria provided by the present invention is useful for hygiene management in the field of production of food and beverages and pharmaceuticals, and basic research techniques for bacteria. In addition, the identification of spore surface proteins can contribute to the development of genetically modified vaccines and the development and improvement of genetically modified bioreactors in the field of bacterial applied technology.

The schematic diagram of the spore structure of a spore formation bacterium is shown. It is a schematic diagram which shows the relationship between the spore length measured in the estimation method of the spore surface protein which concerns on this invention, and the diameter of the shell which comprises protein. The result of having measured the spore length and the diameter of the shell which comprises protein is shown. B is a phase contrast microscope image, C is a fluorescence microscope image, and A is an image obtained by superimposing B and C. E is a graph showing the relationship between length and brightness in a phase contrast microscope image. F is a graph showing the relationship between length and brightness in a fluorescence microscope image. D is a graph in which E and F are superimposed. It is a graph which shows the average of the position of the calculated spore shell protein in a pixel unit by making a spore periphery into a reference | standard (0). One pixel corresponds to 64.5 nanometers. A phase contrast microscopic image (center), a fluorescent microscopic image (right) and a superimposed image (left) of spores of Bacillus subtilis 168 strain immunofluorescently stained with an anti-GFP antibody are shown. A phase-contrast microscopic image (center), a fluorescent microscopic image (right) and a superimposed image (left) of the spores of Bacillus subtilis 168 strain immunofluorescently stained with an anti-CgeA antibody are shown.

Hereinafter, the present invention will be described in detail.
1. TECHNICAL FIELD The present invention relates to a method for estimating whether or not a protein is present on a spore surface in a spore-forming bacterium.

  The spore-forming bacterium used in the method according to the present invention refers to a bacterium that forms a bacterial spore and is also called a spore bacterium. Spore-forming bacteria include, but are not limited to, bacteria belonging to the genus Bacillus, such as Bacillus anthracis, Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis Bacillus brevis, Bacillus coagulans, Bacillus amyloliquefaciens, Bacillus pumilis, Bacillus licheniformlen, Bacillus licheniformis, coillus ); Bacteria belonging to the genus Clostridium, such as Clostridium tetani, Clostridium botulinum, Clostridium perfringens; bacteria belonging to the genus Sporolactocillus, such as Sporola Bacteria belonging to the genus Geobacillus, for example, Geobacillus stearothermophilus; Bacteria belonging to the genus Thermonerobacter, for example, Thermoanaerobacter mathranii, Thermoneerobacter aceto erobic Thermonererobacter brockii subsp. Brockii, Thermoanaerobacter brockii subsp. Finni, Thermonererobacter brockii subsp. Thermoanaerobacter cellulolyticus, Thermoanaerobacter ethanolicus, Thermonererobacter itacus Cass (Thermoanaerobacter italicus), Thermoanaerobacter kivui, Thermoanaerobacter siderophilus, Thermoanaerobacter sulfurophile (Thermoanaerobacter sulfurophile) Thermoanaerobacter thermohydrosulfuricus, Thermoanaerobacter wiegelii; bacteria belonging to the genus Thermonerobacterium, such as Thermoanaerobacterium thermosaccharolyticum, Thermonerobacterium thermosaccharolyticum Acidi Tolerance (Thermoanaerobacterium aciditolerans), Thermoanaerobacterium aotearoens (Thermoanaerobacter ium aotearoense), Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosulfurigenes, Thermonererobacterium thermosulfurigenes・ Xylanolyticum, Thermoanaerobacterium zeae; bacteria belonging to the genus Morella, such as Moorella thermoacetica, Moorella glycerini, Moorella ella mulderi), Moorella thermoautotrophica; bacteria belonging to the genus Alicyclobacillus, eg Alicyclobacillus acid telestris (Alicyc) lobacillus acidoterrestris); bacteria belonging to the genus Desulfotomaculum, such as Desulfotomaculum nigrificans, Desulfotomaculum thermoacetoxidans, Desulfotomaculum thermoacetoxidans, subsp. Thermobenzoicum (Desulfotomaculum thermobenzoicum subsp. Thermobenzoicum), Desulfotomaculum thermobenzoicum subsp. Thermosyntrophicum, Desulfotomaculum thermobenzoicum subsp. Thermobenzoicum, Desulfotomaculum thermocisternum, Desulfotomaculum thermocisternum -Thermosapovorans (Desulfotomaculum thermosapovorans), Desulfotomaculum thermosubterraneum; Bacteria belonging to the genus Ricinibacillus, Rishinibachirusu sphaericus (Lysinibacillus sphaericus) and the like if example. As is well known to those skilled in the art, the names and classifications of the bacteria exemplified above may be changed in the future.

  In the present invention, any species and strain can be used as long as they are the spore-forming bacteria described above. Since the spore structure and constituent proteins may change during the life cycle of the spore-forming bacterium, the spore-forming bacterium used in the present method is preferably a mature spore having a surface layer structure (dormant spore). . The mature spores (dormant spores) refer to spores that are not germinating spores and are not prespores or forespores present in the mother cells of spore-forming bacteria. It is desirable that the mature spore is not damaged by germination or artificial treatment (treatment with acid, alkali, organic solvent, surfactant, etc.).

  Bacterial culture methods for spore formation (preferably mature spore formation) are known and not particularly limited. For example, it can be prepared by culturing under appropriate conditions using a medium usually used for culturing bacteria. Specifically, carbon sources (lactose, glucose, sucrose, fructose, galactose, waste molasses, etc.), nitrogen sources (peptone, casein hydrolyzate, whey protein hydrolysate, soy protein hydrolyzate, etc.) Product), inorganic salts (phosphate, sodium, potassium, magnesium, etc.) and any medium that can efficiently culture spore-forming bacteria (natural medium or synthetic medium, liquid medium or solid medium) Available). The spore-forming bacteria are cultured at 20 ° C. to 70 ° C., preferably 30 ° C. to 60 ° C. under aerobic conditions or anaerobic conditions. The culture format is stationary culture, shake culture, tank culture, or the like. The culture time can be 12 hours to 20 days, preferably 12 hours to 10 days. It is preferable to maintain the pH of the medium at the start of the culture at 5 to 9, preferably 6 to 8. A person skilled in the art can select appropriate culture conditions according to the type of spore-forming bacteria to be used.

  Bacterial spores have high durability (heat resistance, pressure resistance, chemical resistance), and a schematic diagram of the structure is shown in FIG. Although the detailed structure differs depending on the bacterial species, the bacterial spore is generally composed of a core, a cortex, and a spore coat (spoor coat) in order from the inside. The spore shell is further divided into two layers, the inner and outer layers, each of which has been shown to have a multilayer structure. Furthermore, in Bacillus cereus and Bacillus anthracis, exosporium, which is a thin, atypical bag-like structure covering the spores, exists outside the spore shell.

  In the present invention, it is estimated whether or not the candidate protein to be evaluated exists on the surface layer of the bacterial spore. The “surface layer” is a layer composed of proteins or the like formed at the periphery of the spore and is a region where the spore is in contact with the outside world. Although details of the structure and constituent factors of the spore surface layer have not been clarified, it is considered that low molecules such as water, ions, amino acids, and monosaccharides permeate, and that macromolecules such as proteins do not. The surface layer means a layer that allows a protein present on the surface layer to bind to the antibody when an antibody against the protein present on the surface layer is reacted with a spore-forming bacterium, for example.

  In the method of the present invention, specifically, (a) a step of measuring the spore length of spore-forming bacteria, and (b) a step of measuring the diameter of the shell formed by the candidate protein are performed. These steps (a) and (b) may be performed simultaneously or before and after, and the order thereof is not particularly limited. Preferably, step (a) is performed first and step (b) is performed later.

  When evaluating one type of candidate protein, the number of spore-forming bacteria to be subjected to the measurement is not limited, but an increase in accuracy can be expected as the number increases. Therefore, the number of spores to be subjected to the above step is 1 or more, preferably 2 or more, for example, 20 to 100, preferably about 40 to 60.

  The “spore length” in step (a) means the diameter of the spore. Some of the spore-forming bacteria include an oval shape, so the straight line that defines the diameter may be set in any direction, but the point connecting the two poles (ie, from end to end in the long axis direction of the spore) ) And through the center of the spore. This is because the longer the measurement distance, the more clearly the positional relationship between the spore periphery and the shell formed by the candidate protein can be identified. In addition, when measuring a plurality of spores, the measurement direction is always constant, so there is less error between spores and the accuracy is improved. Accordingly, the “spore length” preferably refers to a distance of a straight line passing through the center of the spore and connecting the two poles when the long-axis end of the spore periphery is defined as a pole, and is represented by ab in FIG. .

  The spore length can be measured using any method that can measure the distance of a straight line that passes through the center of the spore and connects the two poles as described above. For example, the spore length can be measured by observation with an optical microscope. Preferably, observation is performed with an optical microscope equipped with a phase difference device, an objective lens, a camera adapter lens, a CCD camera, and the like to obtain a phase difference image. In place of the phase contrast image, a microscopic image obtained by an optical microscope using a spore that has been appropriately stained, or a microscopic image obtained by a differential interference microscope should also be used for the same purpose as the phase contrast image (specific identification of the spore periphery). Can do.

  When observing with a phase contrast microscope, the objective lens can be a commonly used 100 × oil immersion lens for phase contrast microscopes, but in order to obtain accurate data, an objective lens with excellent optical performance is desirable. Camera adapter lenses are limited by the type of CCD camera used. The CCD camera may be used for black-and-white photography or color photography, but a camera with higher sensitivity and a larger number of pixels is desirable. Since the size of the spore is generally about 1 to several micrometers, the area of the object projected per pixel of the CCD camera can be less than 64.5nm (nanometer) by combining the objective lens and camera adapter lens. It is desirable to become.

  In the phase contrast image (positive contrast), the body of the spores released from the forespores and mother cells are observed as bright parts, and the vegetative cells and prespores are observed in dark black, so that the vegetative cells and prespores can be distinguished from the forespores and spores. . Furthermore, since the spore is observed as a dark part, the spore is distinguished from the forespore where the dark part is not observed.

  In a phase contrast microscope, a phase contrast image is obtained by irradiating normal light. It is preferable to photograph this, capture it as image data, and calculate the shading using image analysis software. In the phase contrast image, the darkest part of the spore outline can be regarded as the spore periphery (for example, B in FIG. 3). Therefore, the spore length is the distance between the minimum values of the brightness of the observed image, or in some cases the distance between the maximum values when the lightness on a straight line passing through the center of the spore and connecting the two poles is evaluated using an optical microscope. Obtained (for example, the length between a and b in E of FIG. 3).

  The “shell formed by the protein” (also referred to as “protein shell” in the present specification) in step (b) represents a position or region in the spore where the candidate protein exists, and is schematically illustrated in FIG. Shown as an ellipse. “The diameter of the shell formed by the protein” refers to the distance of a straight line connecting both ends of the long axis direction of the protein shell through the center of the spore, and is represented by cd in FIG.

  Proteins (candidate proteins) whose location is to be examined are selected in consideration of conventional knowledge. For example, if it is Bacillus subtilis, the spore shell protein will be considered, and if it is, for example, Bacillus cereus, the exosporium constituent protein or spore shell protein will be considered. It has not been confirmed at present whether the proteins contained in the spore shell and exosporium are strictly different or in common. Examples of such spore shell proteins and exosporium constituent proteins include those described in Non-Patent Documents 3-7.

  The diameter of the shell of the candidate protein (protein shell) can be determined by using a method that can measure the distance of the straight line connecting the long ends of the protein shell through the center of the spore as described above. Can be measured. For example, measuring the diameter of the protein shell by labeling a candidate protein in the spore while maintaining the structure of the spore and the position of the protein, and locating the protein in the spore based on the signal of the label Can do. Such a label is preferably one that can specifically label the candidate protein and can visualize the position of the candidate protein in the spore. For example, a label that produces a detectable label signal (such as a light signal) can be used, such as a fluorescent protein (eg, green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow Fluorescent protein (YFP) etc.), chemiluminescent protein, etc. can be used. The label may be bound directly to the candidate protein or indirectly through a substance that specifically binds to the label. In the present invention, direct binding is preferable in that a candidate protein can be labeled and an accurate localization site can be specified while maintaining the structure without destroying the spore.

  The candidate protein and the label can be bound by a method known in the art. For example, a technique for expressing a candidate protein as a fusion protein with a label in spore-forming bacteria is used. The case where a fluorescent protein is used as a label will be described below as an example. However, in the present invention, other labels can be used as in the case of fluorescent proteins.

  The fluorescent protein used as the fusion protein is not particularly limited, and GFP, RFP, CFP, YFP, etc. for which a vector system is supplied as the fluorescent protein can be used. In order to express a fusion protein of a candidate protein and a fluorescent protein in spore-forming bacteria, it is preferable that the gene of the fluorescent protein is compatible with the codon usage of the spore-forming bacteria. In addition, it is desirable that productivity in spore-forming bacteria has been confirmed and that it has been used for spore protein research. From these viewpoints, it is preferable to use RFP or GFP, and more preferable to use GFP in spore-forming bacteria such as Bacillus bacteria, particularly Bacillus subtilis.

  The method for fusing the candidate protein and the fluorescent protein is not limited, and any method using a gene recombination technique can be used. Such a method is described in documents such as Molecular Cloning (edited by Sambrook et al. (1989) Cold Spring Harbor Lab. Press, New York). Specifically, DNA encoding a fluorescent protein is introduced into a spore-forming bacterium by homologous recombination and the candidate protein and the fluorescent protein are expressed as a fusion protein, or the DNA encoding the candidate protein and the fluorescent protein are encoded. A method in which a candidate protein and a fluorescent protein are expressed as a fusion protein after being introduced into a spore-forming bacterium by homologous recombination in a state where the DNA is fused can be used.

  Arbitrary methods well-known in this technical field can be adopted for the preparation of the DNA encoding the candidate protein and the DNA encoding the fluorescent protein. For example, when DNA encoding a candidate protein is isolated from a source (eg, a spore-forming bacterium), it can be prepared by a method of synthesizing cDNA from RNA prepared by the guanidine isothiocyanate method. It can also be prepared by amplifying genomic DNA as a template by PCR. For fluorescent proteins, commercially available or known vector systems can be used (eg, pGFG7c plasmid, etc.). The DNA encoding the candidate protein and the DNA encoding the fluorescent protein thus obtained can be used as they are depending on the purpose, or digested with a restriction enzyme or added with a linker as desired.

  Subsequently, the DNA encoding the fluorescent protein is introduced so as to be fused with the candidate protein gene of the spore-forming bacterial genome, or the DNA encoding the fluorescent protein is fused with the DNA encoding the candidate protein, and the fusion product is then sporulated. Replace with a candidate protein gene in the forming bacterial genome. Such introduction of DNA can be performed using any technique known in the art. For example, the target DNA can be introduced into the genome of a host (spore-forming bacterium) using a recombinant vector. A recombinant vector can be obtained by ligating (inserting) the target DNA into an appropriate vector. The vector for inserting the target DNA is not particularly limited as long as it can be integrated into the genome of the host, and examples thereof include plasmid DNA, bacteriophage DNA, and retrotransposon DNA.

  Examples of plasmid DNA include plasmids derived from Escherichia coli (pRFP3KP plasmid, pUC plasmid, pBluescript, ColE plasmid, p15A plasmid, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), and phage DNA Λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP), φX174, M13 phage vectors and the like. Examples of retrotransposons include Ty factor.

  In order to insert a target DNA into a vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of an appropriate vector DNA, and ligated to the vector. The target DNA needs to be incorporated into a vector so that a fusion protein of a candidate protein and a fluorescent protein is expressed by the introduced DNA. Therefore, in addition to the target DNA, elements such as homologous sequences can be linked to the recombinant vector as necessary. Moreover, you may connect the selection marker which shows that the vector is hold | maintained in the cell. Examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, a chloramphenicol acetyltransferase gene, and the like.

  As described above, a recombinant vector can be prepared so as to be compatible with the expression of the target DNA in the host. By transforming a host (spore-forming bacterium) using this recombinant vector, a fusion protein of a candidate protein and a fluorescent protein can be expressed in the host.

  The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method. Thus, the host into which the recombinant vector has been introduced selects for a strain in which the target DNA has been introduced into its genome. Specifically, a transformant is selected using the above selection marker as an index.

  As described above, candidate proteins are expressed as fusion proteins with fluorescent proteins in transformed spore-forming bacteria. Therefore, the position of the candidate protein can be specified by observing the shell formed by the candidate protein using the light signal of the fluorescent protein as an index.

  Observation of the shell formed by the candidate protein in the spore-forming bacterium can be performed by any method or means capable of detecting the label. For example, when a substance that generates an optical signal such as a fluorescent protein or a chemiluminescent protein is used as a label, the labeling can be performed with an optical microscope equipped with a fluorescence observation apparatus, an objective lens, a camera adapter lens, a CCD camera, and the like. As the objective lens, a commonly used 100 × oil immersion lens can be used, but in order to obtain accurate data, a lens having excellent optical performance is desirable. Camera adapter lenses are limited by the type of CCD camera used. The CCD camera may be used for black-and-white photography or color photography, but a camera with higher sensitivity and a larger number of pixels is desirable. Since the size of the spore is generally about 1 to several micrometers, the area of the object projected per pixel of the CCD camera can be less than 64.5nm (nanometer) by combining the objective lens and camera adapter lens. It is desirable to become.

  When a substance that generates an optical signal such as fluorescent protein or chemiluminescent protein is used as a label, the excitation wavelength and the light wavelength to be measured (for example, fluorescence wavelength) may be selected according to the label, The specifications of the cube unit used for fluorescence observation are not limited.

  The exposure time required for measuring the optical signal (for example, fluorescence) differs depending on the performance of the microscope and CCD camera used for photographing and the software for image processing, and an appropriate value can be set according to the performance of the device. In addition to manual adjustment, the value can be set automatically by software. If the exposure time at the time of fluorescence observation is too long, the influence of non-specific fluorescence becomes strong or the detection range of fluorescence becomes unclear, so that accurate position determination cannot be performed. Further, if the exposure time is too short, there is a problem that the target protein cannot be detected due to insufficient fluorescence intensity.

  When a substance that generates a light signal (preferably a fluorescent protein) is used as a label, the label is elliptical along the spore contour by irradiating excitation light of an appropriate wavelength in the resulting image (preferably a fluorescent image). In rare cases, light signals (preferably fluorescence) are emitted concentrated on both poles or monopoles (for example, C in FIG. 3). The obtained observation image is photographed, captured as image data, and the signal intensity is calculated using image analysis software. For example, when a fluorescent protein is used as a label, the fluorescence intensity (brightness) of the label is found as a maximum value at both ends with respect to the long axis direction of the spore (for example, F in FIG. 3), so the distance between the two maximum values is The protein shell diameter.

  In the present invention, the phase difference image for measuring the spore length obtained as described above and an observation image (preferably a fluorescence image) for measuring the diameter of the candidate protein shell are superimposed and evaluated. Is preferable (for example, see A in FIG. 3). Standard image analysis software may be selected for the analysis. If there is a gap between the image files as a result of the superimposition, it is corrected using an automatic correction function provided in the software or manually corrected. The format of the image file is arbitrary, but a format that can be handled by general-purpose image processing software such as Photoshop (registered trademark) (Adobe (registered trademark)), that is, TIFF, JPEG, PICT, etc. is desirable.

  In the superimposed image of the image data, the lightness (brightness in the phase contrast image, signal intensity in the observation image of the label) at an arbitrary point on the straight line overlapping the spore length is calculated. Here, if the horizontal axis is the distance from the start position with the lightness measurement start position as a reference, the lightness can be uniquely displayed as a graph (for example, D to F in FIG. 3). In this graph, since the signal intensity (brightness) of the label is found as a maximum value at both ends with respect to the long axis direction of the spore, the distance between the two maximum values is defined as the diameter of the shell formed by the candidate protein. In addition, at the periphery of the spore, both poles are found as minimum values with respect to the long axis direction of the spore. The distance between the two minimum points is the spore length. 1/2 of the value obtained by subtracting the spore length from the diameter of the shell formed by the candidate protein is defined as the position of the candidate protein (distance from the spore periphery of the protein). In addition, the absolute ratio of the candidate protein (position from the periphery of the spore) is obtained by obtaining the ratio of the diameter of the shell formed by the protein when the spore length is 1, subtracting the diameter ratio from 1, and dividing by 2 Can be obtained (FIG. 2).

  In addition, instead of the spore length and the protein shell diameter, the position of the protein is determined by measuring i) spore periphery and protein periphery, ii) spore, protein surface area, iii) spore, protein volume. It can also be estimated by evaluation.

  In addition, by visually observing the above superimposed image, or by visually measuring the diameter and spore length of the protein shell from the graph displaying the coordinates and lightness described above, the candidate protein is either inside or outside the spore periphery. Can be estimated.

  Function to continuously measure light signal intensity and brightness of spore phase contrast image from a certain direction, function to measure protein shell diameter and spore length, phase contrast image and multiple label images (fluorescence image) Examples of software having a function for creating an image in which images are superimposed include ImageJ, Image-Pro (registered trademark) (Media Cybernetics), MetaMorph (registered trademark) (Molecular Devices), RsImage, etc. The type is not limited.

  If the candidate protein position (pixels) determined as described above is between −1 and +1, preferably the protein position is greater than 0 or the numerical range of protein positions as measured for multiple spores Is distributed positively and negatively across 0, it can be presumed that the protein is present on the surface of the spore. In addition, the absolute position obtained from the ratio of the diameter of the shell formed by the protein when the spore length is 1 is preferably the absolute position of the protein when the determined absolute position of the candidate protein is 0.06 or less. Is less than 0, or when the numerical range of the absolute position of the protein as measured for multiple spores is distributed across 0 (preferably −0.03 to +0.03), the protein is a spore It can be estimated that it exists in the surface layer. Furthermore, even when the protein shell diameter is larger than the spore length, it is presumed that the protein exists in the spore surface. For example, when the diameter ratio of the shell formed by the candidate protein when the spore length is 1 is 0.88 or more, preferably 0.95 to 1.05, it can be estimated that the candidate protein is present on the surface of the spore.

  Here, the larger the candidate protein shell diameter, the more the protein will be located away from the center of the spore. In the first place, whether the spore surface is uniformly covered with a single protein or multiple proteins There is a problem that it is not clear whether it is covered in a mosaic shape. In addition, since the surface structure of the spore shown by electron microscope observation is uneven, several different proteins may be exposed on the surface of the spore. Therefore, for the detection of spore-forming bacteria as described later, it is only necessary that the reagent be exposed on the surface of the spore so that the reagent can come into contact with the protein, and it is essential that the protein is positioned so as to cover the outermost layer of the spore. Absent. Moreover, you may couple | bond with the film | membrane which wraps a spore in a bag shape like exosporium.

  When evaluating the positional relationship between multiple proteins that are estimated to be located on the surface of the spore, compare the absolute position of the proteins between the proteins, and if the difference is not significant, determine that all of these proteins are present on the surface. Can do. When the difference is significant, it is presumed that the protein having a larger value is located on the surface layer. At this time, it is preferable to measure a plurality of spores and compare the measurement value groups between the proteins (for example, see FIG. 4).

  When evaluating the relative positional relationship of multiple (N types) proteins, each of the N types of candidate proteins is labeled with a different label (for example, N types of fluorescent proteins having different fluorescence wavelengths), and the above method is used. Then, a superimposed image is created, and the protein shell diameter of each candidate protein is measured by analyzing the image. It can be inferred that the larger the diameter is, the more outside it is.

  Furthermore, it was verified that the protein presumed to be present on the spore surface as described above was present on the spore surface of spore-forming bacteria by immunostaining using an antibody that specifically binds to the protein or its labeled protein. May be. As an antibody that can be used in such an immunostaining method and a reaction procedure thereof, those known in the art can be used, and will be described in detail in the section “2. Utilization of Spore Surface Protein” described later. is doing.

  The method according to the present invention makes it possible to easily, efficiently and reliably determine the proteins present on the spore surface in spore-forming bacteria. Further, according to the method of the present invention, it is possible not only to estimate whether the candidate protein is present on the surface layer of the spore but also to determine in detail in which position of the spore-forming bacterium.

2. Use of Spore Surface Protein As described above, it is possible to detect, identify, remove or purify spore-forming bacteria using a protein presumed to be present on the spore surface according to the present invention (also referred to as “spore surface protein”). It is. Specifically, the spore-forming bacterium is detected, identified, removed or purified based on the reaction between the substance that specifically binds to the spore-surface protein and the spore surface protein on the spore-forming bacterium.

  For example, as shown in Examples 3 and 4 of the present specification, it was confirmed that CotZ protein and CgeA protein are present in the spore surface of Bacillus subtilis. Therefore, Bacillus subtilis can be detected, identified, removed or purified by using a substance that specifically binds to the CotZ protein and / or a substance that specifically binds to the CgeA protein. An example of the base sequence of the gene encoding the CgeA protein of Bacillus subtilis and an example of its amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively. An example of the base sequence of the gene encoding the CotZ protein of Bacillus subtilis and an example of its amino acid sequence are shown. The numbers 3 and 4 are shown. In addition, CgeA protein has Bacillus amyloliquefaciens (accession number YP_001421555), Bacillus pumilis (accession number YP_001487126), B. subtilis 168 (accession number AAB81150). CotZ protein is known to exist in Bacillus amyloliquefaciens (accession number YP_001420770.1), Bacillus licheniformis (YP_090868.1), Bacillus cereus (B. cereus) (accession number YP_002529012.1), B. pumilis (accession number YP_001486347.1), Lysinibacillus sphaericus (accession number YP_001696970.1), B. core philensis (B. coahuilensis) (Accession number ZP_03226709.1) , B. thuringiensis (accession number YP_035462.1), B. subtilis 168 (accession number NP_389056.1), B. subtilis subsp. Natto (accession number) BAI84730.1) etc. are known.

  The substance that specifically binds to the spore surface protein is not particularly limited as long as it is known to bind specifically to the protein. For example, antibodies, nucleic acids or peptide aptamers, enzymes and the like can be mentioned. Preferably, the substance that specifically binds to the spore surface protein is an antibody or an antibody fragment.

  An antibody or antibody fragment that specifically binds to the spore surface protein can be prepared according to a conventional method. As the protein or peptide serving as an antigen, a protein or peptide extracted from a spore, a chemically synthesized peptide, or a protein produced by a method using a gene recombination technique can be used.

  In the case of chemical synthesis, an antigenic peptide can be synthesized according to a known peptide synthesis method, for example, using a commercially available peptide synthesizer or a commercially available peptide synthesis kit.

  In the case of using a genetic recombination technique, the nucleic acid encoding the antigenic peptide is a spore surface protein using mRNA purified from RNA extracted from spore-forming bacteria (which may be either spore bacteria or germs). From a cDNA library using reverse transcription polymerase chain reaction (RT-PCR) with primers designed based on the sequence of the encoding gene or using probes designed based on the sequence of the gene encoding the spore surface protein It can be obtained by screening. Alternatively, a nucleic acid encoding an antigenic peptide can be obtained by performing a nucleic acid amplification reaction (for example, PCR) using a primer designed based on the sequence of a gene encoding a spore surface protein using DNA extracted from spore-forming bacteria as a template. Can be obtained. An expression vector for recombinantly expressing the antigenic peptide can be obtained by ligating the nucleic acid to an appropriate vector. Moreover, a transformant can be produced by introducing the nucleic acid or expression vector into a host cell so that the target antigen peptide can be expressed. Production of such transformants is well known in the art, and those skilled in the art can carry out by appropriately selecting a vector, a host cell and the like to be used. The antigenic peptide of the spore surface protein can be obtained by culturing a transformant introduced with a nucleic acid encoding the antigenic peptide and collecting it from the culture. After the culture, when the antigenic peptide is produced intracellularly or in cells, the peptide is extracted by disrupting the cells or cells. In addition, when the antigen peptide is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like.

  Antigen peptides produced by chemical synthesis or recombinant techniques can be obtained by general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. Alternatively, it can be isolated and purified by appropriately combining them. Whether or not the target antigen peptide has been obtained can be confirmed by polyacrylamide gel electrophoresis or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

  An antibody against the spore surface protein can be produced using the antigenic peptide prepared as described above. In that case, in order to enhance antigenicity, it may be combined with a carrier protein (for example, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), etc.). These carrier proteins are known in the art and commercially available kits are also sold. The antigen peptide thus prepared can also be used to purify antibodies against spore surface proteins.

  The immunogen is prepared by dissolving the antigen peptide of the spore surface protein obtained as described above or the antigen peptide bound to the carrier protein as an antigen in a buffer. If necessary, an adjuvant (Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), etc.) may be added for effective immunization.

  When producing polyclonal antibodies, the immunogen is administered to animals such as mammals and birds, such as mice, rabbits, rats, goats, chickens and ducks. Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, or into the footpad. The interval between immunizations is not particularly limited, and immunization is performed 1 to 5 times at intervals of several days to several weeks. Thereafter, serum or egg yolk (in the case of birds) is collected 14-90 days after the last immunization, and immunoassays such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) ), Etc., and collect on the day when the maximum antibody titer was shown. Thereafter, the reactivity of the polyclonal antibody specific to the spore surface protein present in the serum or egg yolk is measured by the above immunoassay or the like.

  For example, a method for producing a rabbit polyclonal antibody (antiserum) against the CgeA protein is described in Kuwana, R. et al., Microbiol, 150 (2): 163-170, 2004. Antibodies against spore surface proteins can be made.

  Antiserum can also be used directly in immunological measurement methods, but the antibody in the antiserum can be purified by affinity chromatography using spore surface protein or antigen peptide, protein A or protein G affinity chromatography, etc. It is preferable to use it.

  When producing monoclonal antibodies, the immunogen is administered to mammals such as mice, rabbits, rats and the like. Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, or into the footpad. The interval between immunizations is not particularly limited, and immunization is performed 1 to 5 times at intervals of several days to several weeks. Then, antibody-producing cells are collected 14 to 90 days after the final immunization day. Examples of antibody-producing cells include lymph node cells, spleen cells, peripheral blood cells and the like.

  In order to obtain a hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. Generally available cell lines can be used as myeloma cells to be fused with antibody-producing cells. The cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. Examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) and NS-I. Cell fusion between myeloma cells and antibody-producing cells is performed by mixing antibody-producing cells and myeloma cells in an animal cell culture medium such as serum-free DMEM, RPMI-1640 medium, etc. The fusion reaction is carried out in the presence of glycol or the like. Alternatively, cell fusion can be performed using a commercially available cell fusion device utilizing electroporation. The target hybridoma is selected from the cells after cell fusion treatment. For example, the cell suspension is appropriately diluted with a fetal bovine serum-containing RPMI-1640 medium or the like and then spread on a microtiter plate. Selective medium (for example, HAT medium) is added to each well, and thereafter cell culture is performed by appropriately replacing the selective medium. As a result, cells that grow in about 10 to 30 days after the start of culture in a selective medium can be obtained as hybridomas.

  Next, the culture supernatant of the hybridoma that has proliferated is screened for the presence of antibodies that react with spore surface proteins or spores of spore-forming bacteria. The screening of hybridomas may be carried out in accordance with a usual method, and for example, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), or radioimmunoassay (RIA) can be employed. Cloning of the fused cells is performed by a limiting dilution method or the like to establish a hybridoma that produces the target monoclonal antibody.

  As a method for collecting the monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed. When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.

  In the present invention, when it is desirable to detect with high sensitivity including spores of spore-forming bacteria of the same or related species, it is preferable to use a polyclonal antibody, limited to a specific strain or strain. When it is desired to obtain a reactive antibody, it is preferable to use a monoclonal antibody.

Furthermore, from the antibody molecules that specifically bind to the spore surface protein produced as described above, single-chain antibodies (US Pat. No. 4,946,778), F (ab ′) ₂ fragments, Fab fragments, etc. It can be produced using techniques known in the field. Such antibody derivatives and antibody fragments can also be used in the present invention as long as the desired activity, that is, reactivity with the spore surface protein is retained.

  Using a substance (preferably an antibody) that specifically binds to a spore surface protein produced as described above, spore-forming bacteria can be detected, identified, excluded, or purified. This detection, identification, exclusion and / or purification is known in the art, which can detect the binding of substances that specifically bind to spore surface proteins to spore surface proteins present in the spores of spore-forming bacteria. It can be carried out by any method.

  In the method according to the present invention, the sample is brought into contact with a substance that specifically binds to the spore surface protein, and the reaction between the spore-forming protein present in the spore-forming bacteria in the sample and the substance is detected. Detecting the presence of spore-forming bacteria in the sample or identifying the test bacterium as a spore-forming bacterium. In the present invention, “detection” includes not only detecting the presence or absence of spore-forming bacteria, but also quantitatively detecting spore-forming bacteria and immunostaining spore-forming bacteria.

  In the present invention, the term “contact” means that the spore surface protein of the spore-forming bacterium present in the sample and a substance that specifically binds to the spore surface protein can be brought into close proximity so that they can bind to each other. Meaning, for example, mixing a liquid sample and a solution containing the substance, applying the liquid sample to a solid support on which the substance is immobilized, and a solution containing the substance for a solid sample. Operations such as application and immersion of a solid sample in a solution containing the substance are included.

  In addition, as the substance that specifically binds to the spore surface protein, one kind may be used, or a plurality of kinds may be used in combination. For example, one substance or a plurality of substances that specifically bind to one spore surface protein can be used, or a plurality of substances that specifically bind to a plurality of spore surface proteins can be used. be able to.

  The assay for detecting the reaction between the spore surface protein present in the spore-forming bacterium and a substance that specifically binds to the protein may be performed in either a liquid phase system or a solid phase system, and the assay conditions Are also known to those skilled in the art.

  In the method according to the present invention, the sample is contacted with a substance that specifically binds to the spore surface protein, and the spore-forming bacteria having the spore surface protein bound to the substance are separated, thereby forming a spore from the sample. Bacteria can be removed or purified.

  EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to these Examples.

Example 1: Preparation of a fluorescent fusion protein expression strain For genes encoding CotB, CotC, CotD, CotF, CotZ, GerQ, YaaH, YmaG, YsnD, YtxO known as spore shell proteins, using a primer sandwiching them, hay The genomic DNA of fungus 168 was amplified by PCR. The amplified product was treated with BamHI and XhoI, ligated with a pGFP7C plasmid having a gfp gene using a DNA ligation enzyme, and transformed into Escherichia coli JM109 according to a conventional method. The target plasmid was extracted from the recombinant E. coli and introduced into the chromosomal DNA of Bacillus subtilis 168 by homologous recombination using competent cells. Transformants were selected by resistance to chloramphenicol.

  When the spores of the obtained recombinant Bacillus subtilis were observed with a fluorescence microscope, fluorescence derived from GFP could be confirmed in 91% or more of the spores.

Example 2: Observation with a fluorescence microscope The fluorescence microscope used was Olympus, the television camera used was CoolSNAP ES / OL manufactured by Roper Scientific, and the objective lens used was AUPlanApo100x. The superimposed image of the phase contrast image and the fluorescence image was analyzed using RSImagePro ver.4.5. The contrast and balance were adjusted using CANVAS8 software from DENEBA.

  The exposure time for fluorescence imaging of GFP was 0.5-2 seconds. One pixel in the image corresponded to 64.5nm.

Example 3: Position determination of spore shell protein Spores were prepared for 168 strains of Bacillus subtilis expressing spore shell protein as a fusion protein with GFP produced in Example 1, and microscopic observation was performed. Microscopic observation was performed using 40 or more spores for each protein to be examined.

  The results are shown in FIG. Using the image analysis software, the brightness from X1 to X2 of the superposition image (A in Fig. 3) of the phase contrast image (B in Fig. 3) and the fluorescence observation image (C in Fig. 3) of the GFP fusion protein-producing spore Continuously measured. The horizontal axis of the graph shown to DF of FIG. 3 shows length, and a vertical axis | shaft shows the brightness.

  In the phase contrast microscope, the periphery of the spore was observed as a dark outline (B in FIG. 3). In the graph (E in Fig. 3) where the horizontal axis shows the position on a straight line passing through the spore center and connects the two poles, and the vertical axis shows the brightness (brightness) of the phase contrast image, a and b are the darkest parts of the phase contrast image. (Negative peak) was shown, and the spore periphery was recognized as a negative peak (minimum value). The distance between the abs indicates the spore length. In addition, non-spore cells and mother cells were darker than the visual field background, whereas new spore was observed as bright.

  In the fluorescence microscopic image, strong fluorescence was observed at the edge of the shell (protein shell) formed by the protein, which was particularly remarkable at both poles (C in FIG. 3). In the graph (F in FIG. 3) showing the fluorescence intensity (brightness and expression) of the fluorescence image on the vertical axis, c and d indicate the peak of the fluorescence intensity emitted by the fluorescently labeled protein, and the protein bipolar is the peak (maximum). Value). The distance between cd indicates the protein shell diameter.

  A graph is created from the superimposed image of the phase contrast image and the fluorescence image (A in FIG. 3), the spore length and the protein shell diameter are obtained from this graph (D in FIG. 3), and the protein position (unit: pixel) is obtained from these values. )

  The positions of various spore shell proteins are shown in Table 1 and FIG. Table 1 shows the actual protein positions measured in pixels and nm (nanometers). In addition, the diameter ratio of the protein shell when the spore length is 1 and the absolute position of the protein calculated based on the ratio are also shown. In FIG. 4, the average (pixel unit) of the position of the spore shell protein calculated from the above formula is shown with the spore periphery as the reference (0). That is, the position of the spore shell protein is represented by [(d-c)-(b-a)] × 1/2. One pixel corresponds to 64.5 nanometers.

  As shown in Table 1 and FIG. 4, only CotZ-GFP had an average value of the positions of fluorescently labeled proteins of “positive” or a diameter ratio greater than 1. On the other hand, CotZ-GFP and CgeA-GFP were observed on the spore periphery or outside by visual inspection. The protein shell diameter was large in the order of CotZ-GFP and CgeA-GFP.

  From the examination of the absolute position described above, CotZ-GFP and CgeA-GFP are derived from GFP fusion proteins of CotA, CotB, and CotC, which have been reported to exist on the spore surface in previous studies (Non-Patent Documents 5 and 6) Was also suggested to be outside.

When the positions of the respective proteins were compared, it was found that CotZ-GFP and CgeA-GFP were located in the outermost layer among the proteins measured this time, as shown in FIG. However, since these distance measurements included standard deviations, statistical processing was performed on the positions (measurements) of these two types of proteins, and it was considered that there was a significant difference when P value <0.01. It was. For example, the P value was 0.055 for CotZ-GFP and CgeA-GFP, but 7.8 × 10 ⁻¹² for CgeA-GFP and CotC-GFP. From the above, there was no significant difference in the positions of CotZ and CgeA, suggesting that both are located in the outermost layer of the spore shell. Therefore, when the absolute position of the protein is 0.06 or less, preferably 0.03 or less, it can be estimated that the protein is present on the spore surface.

  Similar analysis revealed that YxeE was located between the outer and inner layers of the spore shell, and that CotT, YmaG, YsnD, CotF, YaaH, CotD, GerQ, and YeeK existed in the inner layer of the spore shell.

Example 4: Verification using anti-GFP antibody The analysis of Example 3 showed that CotZ-GFP and CgeA-GFP were located in the outermost layer of the spores (spoor coat). Therefore, in order to verify that these outermost layer proteins are exposed on the surface of the spore, fluorescence microscopic observation using an anti-GFP antibody was performed (FIG. 5). That is, for the spores prepared by the methods of Examples 1 to 3, a rabbit-derived anti-GFP antibody as a primary antibody and a goat-derived fluorescent-labeled anti-rabbit IgG antibody (Invitrogen Alexa Fluor 594 goat anti-anti- Rabbit IgG (H + L)) was used for immunostaining. Stained spores were subjected to microscopic observation. A phase contrast image (blue) and a fluorescence labeled anti-GFP antibody fluorescence (red) were photographed, and a superimposed image was created using software. The results are shown in FIG.

  As shown in FIG. 5, since the Bacillus subtilis wild strain does not have GFP, binding of anti-GFP antibody was not observed (no spore was observed in the fluorescence image), but the CotZ-GFP strain and CgeA-GFP strain. Anti-GFP antibody binding was observed in the spore (the spore was confirmed by a fluorescence image). On the other hand, even when the same operation was performed on other GFP fusion strains such as CotA-GFP, the binding of the anti-GFP antibody could not be observed (spores could not be observed in the fluorescence image).

  These results indicate that CgeA-GFP and CotZ-GFP are exposed on the surface of the spore, which is consistent with the analysis result of Example 3 that these proteins are located in the outermost layer of the spore coat. To do.

Example 5: Verification using anti-CgeA antibody Using an anti-CgeA antibody, it was verified that CgeA was exposed on the surface of spores of Bacillus subtilis wild strains. That is, for the spores prepared by the methods of Examples 1 to 3 (spores of Bacillus subtilis 168 strain and cgeA gene-disrupted strain), the rabbit-derived anti-CgeA antibody as the primary antibody and the goat-derived fluorescent label as the secondary antibody Immunostaining was performed using an anti-rabbit IgG antibody (Alexa Fluor 594 goat anti-rabbit IgG (H + L) manufactured by Invitrogen). Stained spores were subjected to microscopic observation. The anti-CgeA antibody used in this example was prepared by the inventors, and the method of preparation was disclosed in Kuwana, R. et al., Microbiol, 150 (2): 163-170, 2004. Yes. The phase contrast image (blue) after immunostaining and the fluorescence (red) of the fluorescence-labeled anti-CgeA antibody were photographed, and a superimposed image was created using software.

  The result is shown in FIG. Anti-CgeA antibody binding was observed in wild-type spores (spores were observed in the fluorescence image). This binding was lost in the CgeA-disrupted strain, indicating that it is a specific binding between the anti-CgeA antibody and CgeA. The above analysis revealed that CgeA was exposed on the surface of Bacillus subtilis spores.

  The present invention provides a technique for efficiently and reliably searching for proteins present in the spore surface of spore-forming bacteria. The present invention also contributes to the development and improvement of a spore detection method based on this technology, the removal method of spores, and the development of applied technologies such as pharmaceutical production using spores. Therefore, the method for detecting and removing spore-forming bacteria provided by the present invention is useful for hygiene management in the field of production of food and beverages and pharmaceuticals, and basic research techniques for bacteria. In addition, the identification of spore surface proteins can contribute to the development of genetically modified vaccines and the development and improvement of genetically modified bioreactors in the field of bacterial applied technology.

Claims (8)

A method for estimating whether a candidate protein is present on a spore surface in a spore-forming bacterium, comprising:
(A) measuring the spore length of spore-forming bacteria;
(B) measuring a diameter of a shell formed by the candidate protein;
(C) The ratio of the diameter of the shell formed by the candidate protein when the diameter of the spore is 1 is obtained, and the value obtained by subtracting the diameter ratio from 1 and dividing by 2 is defined as the absolute position of the candidate protein. Estimating the candidate protein in the spore surface when the target position is 0.06 or less.   The spore length is obtained as a distance between minimum values or maximum values of the brightness of an observed image when the lightness on a straight line connecting both poles through the center of the spore is evaluated using an optical microscope. the method of.   The distance between the labeled signal maximum values of the obtained image when the diameter of the shell constituted by the candidate protein is evaluated using a microscope for the candidate protein labeled while maintaining the shape and location of the spore. The method according to claim 1 or 2 obtained as follows.   4. A method according to claim 3, wherein the label is a fluorescent protein.   The candidate protein estimated to be present on the spore surface in step (c) further comprises the step of verifying that the candidate protein is present on the spore surface by immunostaining. The method described.   Bacteria belonging to the genus Bacillus, bacteria belonging to the genus Clostridium, bacteria belonging to the genus Sporolactobacillus, bacteria belonging to the genus Geobacillus, bacteria belonging to the genus Thermonerobacter, bacteria belonging to the genus Thermonerobacterium, des 6. The method according to any one of claims 1 to 5, wherein the method is selected from bacteria belonging to the genus Ruphotomacrum, bacteria belonging to the genus Morella, bacteria belonging to the genus Alicyclobacillus and bacteria belonging to the genus Ricinibacillus.   The method according to claim 6, wherein the spore-forming bacterium is Bacillus subtilis. A method for detecting spore-forming bacteria in a sample, comprising:
(A) contacting the sample with a substance that specifically binds to a protein presumed to be present on the spore surface by the method according to any one of claims 1 to 7, and (b) in the sample A method comprising a step of detecting a reaction between the substance present in a spore-forming bacterium and the substance.

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