JP2006343207A - Probe for microscope analysis, and microscopic analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a probe for microscopic analysis capable of labeling specifically a specific portion of a target molecule in a sample, labeling and analyzing an analysis object without damaging a space resolution of a used microscope, and analyzing localization, the direction (aspect), the shape (stereoscopic structure) and kinetics of the analysis object in the functional state, and a microscopic analysis method using the probe for microscopic analysis. <P>SOLUTION: This probe for microscopic analysis is characterized by having a bar-like structure having a contact part capable of contacting with the specific portion of the microscopic analysis object. This microscopic analysis method is characterized by including at least a process for preparing a sample for microscopic analysis including the probe for microscopic analysis, and a process for detecting the probe for microscopic analysis in the sample for microscopic analysis. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microscope analysis probe capable of maintaining the highest spatial resolution of a microscope used for analysis, and a microscope analysis method using the microscope analysis probe.

As a method for analyzing the structure of a biopolymer such as protein, for example, an X-ray crystal diffraction method or a multidimensional NMR method is generally used.
However, since the X-ray crystal diffraction method requires that the molecule to be subjected to structural analysis is crystallized, it is necessary to search for crystallization conditions and obtain a high-quality protein crystal. In addition, the multidimensional NMR method is limited in the molecular weight of a molecule to be subjected to structural analysis, and it is necessary to obtain a large amount of a soluble sample having high purity and high solubility for analysis.
In addition, the structure determined by these methods is an average of the structure of a large number of uniform molecules in terms of time and space, so that the instantaneous changes of individual molecules that vary with function. It is not suitable for analyzing the structure of the structure or the structure when incorporated in the composite.

  On the other hand, a direct observation method using an electron microscope is known as a method for photographing real images of individual molecules. For example, when a protein complex is an object of structural analysis, a sample in which cells and tissues containing the protein complex are rapidly frozen and ice-embedded is observed with an electron microscope, and the obtained image is appropriately grouped and After averaging, a single particle analysis method is used in which an inclination angle is estimated by an appropriate method and an image is reconstructed.

  However, a biological sample consisting mostly of water and light elements has a low contrast with the background, and the focus must be removed to obtain an image of the target molecule. On the other hand, since it is extremely weak, there is a problem that it is difficult to image a target molecule multiple times from many directions. In addition, since the S / N ratio is low, it is essential to average a large number of images. Therefore, even when a purified uniform sample is used, the structure of individual molecules depends on the activation state. If the difference is significant, there is a problem that precise structural analysis becomes impossible.

  In order to solve these problems, rapid-frozen deep-etch replica electron microscopy has been proposed as a method for analyzing the structure of proteins in cells in situ. The rapid-frozen deep-etch replica electron microscopy is a method of lightly scraping the surface of a rapidly-frozen biological sample, sublimating (etching) water molecules around the target structure, vapor deposition with heavy metal, and coating with carbon. In this method, the replica obtained by dissolving the biological sample portion is observed with an electron microscope.

  In the rapid-frozen deep-etch replica electron microscopy, it is possible to physically fix a functioning protein within 1 millisecond by changing the structure at the site of a life phenomenon by rapidly freezing a biological sample. In addition, since the contrast of the sample is high, the shape of each protein molecule can be clearly observed, the structure can be directly analyzed, and the spatial resolution of the obtained image is high. Furthermore, since the sample is composed only of heavy metal and carbon, the sample is resistant to electron beam irradiation, and a large number of tilted images are taken, and image processing is applied using an appropriate image processing apparatus (for example, Patent Documents 1 to 3). 3), the three-dimensional structure of the protein to be analyzed can be accurately reconstructed.

By the way, in the analysis of the protein in the cell in situ, unlike the case where the purified protein in the solution is used as the target molecule to be observed, it is difficult to estimate the localization position and shape. In order to identify the localization position of a protein as a target molecule in a cell, a means for directly labeling the molecule with a label that can specifically recognize and interact with the molecule is necessary.
As a conventional labeling method, for example, a method using spherical heavy metal colloidal particles having an antibody or the like adsorbed on the surface is known. When such a label is used, since the heavy metal particle size is usually larger than the target molecule, the target molecule and heavy metal particle images overlap, and the structure and detailed localization of the target molecule cannot be analyzed. There is a problem. In order to analyze without impairing the spatial resolution of a microscope or the like to be used, it is necessary to reduce the particle diameter, but there is a problem that detection becomes extremely difficult by reducing the particle diameter.
Further, since the replica sample is given contrast using a heavy metal such as platinum, there is a problem that it is extremely difficult to recognize minute heavy metal particles used as a label.

  In this way, it is possible to label and analyze an analysis target without impairing the spatial resolution of the microscope to be used, and to analyze the localization, direction (orientation), shape, and dynamics of the analysis target in a functional state. At present, a microscope analysis probe and a microscope analysis method using the microscope analysis probe are not yet provided.

International Publication No. WO02 / 48961 Pamphlet International Publication No. WO02 / 056259 Pamphlet International Publication No. WO02 / 075658 Pamphlet

An object of the present invention is to solve the conventional problems and achieve the following objects.
That is, the present invention can label and analyze an analysis object without impairing the spatial resolution of the microscope to be used, and the localization, direction (orientation), shape (three-dimensional structure), and dynamics of the analysis object can be functioned. An object of the present invention is to provide a microscope analysis probe that can be analyzed in a state, and a microscope analysis method using the microscope analysis probe.

Means for solving the above problems are as follows. That is,
<1> A probe for microscopic analysis, which has a rod-like structure, and the rod-like structure has a contact portion that can contact a specific part to be analyzed. The probe for microscopic analysis described in <1> has a rod-like structure, and the rod-like structure has a contact portion that can come into contact with a specific part to be analyzed, so that the rod-like structure is visually recognized. By tracking the rod-like structure so as to follow, it is possible to identify the contact portion and analyze the specific part of the microscope analysis target. Further, by making the diameter of the rod-like structure extremely thin, unlike conventional markers such as colloidal gold particles, analysis can be performed without blocking the microscope analysis target, that is, without impairing the spatial resolution of the microscope. it can.
<2> The microscope analysis probe according to <1>, wherein the contact portion is disposed at one end of the rod-like structure, and a marker portion is disposed at the other end of the rod-like structure. In the probe for microscopic analysis described in <2>, the contact portion is disposed at one end of the rod-shaped structure, and the labeling portion is disposed at the other end of the rod-shaped structure. Therefore, the microscope analysis probe can be easily detected.
<3> The microscope analysis probe according to any one of <1> to <2>, wherein an end portion of the rod-like structure is branched and has at least one of a contact portion and a label portion.
<4> The microscopic analysis according to any one of <1> to <3>, wherein the rod-like structure is at least one selected from proteins, filamentous phages, metals, semiconductors, carbon nanohorns, carbon nanotubes, and amylose. Probe.
<5> The microscopic analysis probe according to any one of <1> to <4>, wherein the labeling portion is at least one selected from a fluorescent substance, a dye, a heavy metal compound, and a heavy metal colloid.

<6> The probe for microscopic analysis according to any one of <1> to <5>, wherein at least the rod-like structure is a protein.
<7> The probe for microscopic analysis according to <6>, wherein the rod-like structure is a protein having an antiparallel coiled coil structure.
<8> The probe for microscopic analysis according to any one of <6> to <7>, comprising a single molecule recombinant fusion protein.

<9> The probe for microscopic analysis according to any one of <1> to <8>, wherein the contact portion is made of a substance capable of interacting with a specific region to be analyzed.
<10> The probe for microscopic analysis according to <9>, wherein the interaction is an antigen-antibody reaction.
<11> The probe for microscopic analysis according to any one of <1> to <10>, wherein the contact portion is configured in a state of being coupled to a specific site to be analyzed.
<12> The probe for microscopic analysis according to <11>, which is expressed integrally with a target for microscopic analysis.

<13> The above-mentioned used for the preparation of a sample for microscopic analysis by at least one selected from a quick freeze deep etch replica method, a negative staining method, a cryo-negative staining method, a resin-embedded section method, and an ice-embedded method The probe for microscopic analysis according to any one of <1> to <12>.
<14> Any one of <1> to <13>, which is used for analysis by at least one selected from a fluorescence microscope, a transmission electron microscope, a scanning electron microscope, a scanning probe microscope, and an atomic force microscope The probe for microscopic analysis described.

<15> A microscope expression probe expression vector comprising at least a gene encoding the microscope analysis probe according to any one of <6> to <14>.
<16> The probe expression vector for microscopic analysis according to <15>, which is used by being introduced into a cell expressing a target for microscopic analysis.
<17> A vector for expressing the microscope analysis probe according to any one of <12> to <14>, comprising at least a specific site to be analyzed and a gene encoding the microscope analysis probe This is a probe expression vector for microscopic analysis.
<18> The probe expression vector for microscopic analysis according to <18>, which is used by being introduced into a knockout cell from which a gene encoding a target for microscopic analysis has been deleted.

<19> A step of preparing a microscope analysis sample including the microscope analysis probe according to any one of <1> to <14>, and a step of detecting the microscope analysis probe in the microscope analysis sample; Is a microscope analysis method characterized by including at least.
<20> a step of expressing the microscope analysis vector according to any one of <15> to <18>, a step of preparing a sample for microscope analysis including the probe for microscope analysis, and the sample for microscope analysis And a step of detecting the probe for microscope analysis.
<21> The sample according to <19> to <20>, wherein the sample for microscopic analysis is prepared by any one selected from a rapid freezing deep etch replica method, a negative staining method, a cryo-negative staining method, and an ice embedding method The microscope analysis method according to any one of the above. In the microscopic analysis method according to <21>, the microscopic analysis sample is obtained from a quick freeze deep etch replica method, a negative staining method, a cryo negative staining method, a resin-embedded section method, and an ice embedding method. It is produced by at least one selected. As a result, for example, the three-dimensional structure of the protein is accurately reconstructed by taking a large number of tilted images using the sample for microscopic analysis and applying image processing using an appropriate image processing apparatus or the like.
<22> The above <19> to <21>, wherein the microscopic analysis is performed using at least one selected from a fluorescence microscope, a transmission electron microscope, a scanning electron microscope, a scanning probe microscope, and an atomic force microscope. The microscope analysis method according to any one of the above.

  According to the present invention, conventional problems can be solved, and an analysis object can be labeled and analyzed without impairing the spatial resolution of the microscope to be used. The localization, direction (orientation), shape ( It is possible to provide a microscope analysis probe capable of analyzing the three-dimensional structure) and dynamics in a functional state, and a microscope analysis method using the microscope analysis probe.

(Probe for microscope analysis)
The probe for microscopic analysis of the present invention has a rod-like structure, and the rod-like structure has a contact portion that can contact a specific part to be analyzed, and further improves the visibility if necessary. For example.
Since the rod-like structure has a strange shape with respect to the shape of general protein molecules and intracellular organelles, the object of microscopic analysis is either purified molecules in solution or biological samples such as cells / tissues. Are easily recognized and detected in the object.

  The structure of the probe for microscopic analysis is not particularly limited as long as it is a structure that can identify the contact portion by following at least the rod-like structure, and can be appropriately selected according to the purpose. It is preferable that the visibility under the microscope of the joining area | region of the said contact part and the said rod-shaped structure is improved because the said contact part exists in the edge part of the said rod-shaped structure. The visibility may be enhanced by a method such as changing the length or thickness of the molecules constituting the structure or the fine shape as long as it has a thin tip. Furthermore, it is more preferable that the diameter of the contact portion is the thinnest in the rod-like structure. Note that the rod-like structure may be linear or curved.

Moreover, it is preferable to make it the structure which has the said contact part in the end of the said rod-shaped structure, and has a marker part in the other end. With such a structure, the rod-like structure can be detected more easily and visually recognized.
Furthermore, it is preferable that the microscope analysis probe has a structure in which an end portion of the rod-like structure is branched and at least one of the contact portion and the marker portion is two or more. By adopting such a structure, it becomes easier to label and detect the analysis target.

The microscopic analysis target is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include proteins, peptides, nucleic acids, sugars, lipids, and semiconductors, metals, and other inorganic materials. Of these, proteins are preferred.
The protein to be analyzed is not particularly limited and can be appropriately selected depending on the purpose. For example, the protein in a living tissue such as a cell / tissue, the purified intracellular organelle dispersion Examples include proteins and proteins in solution.

  As an aspect of the microscope analysis target, (1) a first aspect which is an external detection target detected by interaction with a contact portion of the microscope analysis probe, and (2) integrated with the microscope analysis probe The 2nd aspect which is is mentioned.

  The microscope is not particularly limited as long as the target molecule can be analyzed at a specific site of the microscope analysis target, and can be appropriately selected according to the purpose. For example, a stereo microscope, a fluorescence microscope, a laser scanning Examples include an optical microscope such as a microscope, an electron microscope such as a transmission electron microscope and a scanning electron microscope, a scanning probe microscope such as an atomic force microscope, and a scanning tunnel microscope. Among these, a fluorescence microscope and a transmission electron Preferred examples include a microscope, a scanning electron microscope, and an atomic force microscope.

-Rod structure-
The rod-shaped structure is not particularly limited as long as it has a size that does not impair the spatial resolution of the microscope used for analysis and can indicate the localized position and direction (orientation) of the microscope analysis target. However, the diameter is preferably smaller than the length, although it can be appropriately selected according to the purpose.
In general, the length of the rod-like structure is, for example, preferably 15 nm or more, and more preferably 20 nm or more. If the length is less than 15 nm, the rod-like structure itself may not be visually recognized. However, if the object of microscopic analysis is even smaller, a smaller structure is suitable.
Moreover, as a diameter of the said contact part in the said rod-shaped structure, it is preferable that it is 5 nm or less, for example, and it is preferable that it is 3 nm or less. When the diameter exceeds 5 nm, the specific part of the microscope analysis target including the joint portion is covered widely, and the localization and direction (orientation) may not be specified.

  The material of the rod-shaped structure may be either an organic molecule or an inorganic molecule, and examples thereof include proteins, filamentous phages, metals, semiconductors, carbon nanohorns, carbon nanotubes, and amylose. Among these, proteins, And filamentous phages are preferred, and proteins are more preferred.

The protein as the material of the rod-like structure is preferably a so-called rod-like protein (fibrous protein), and more specifically, an antiparallel coiled-coil protein is more preferred.
As such a protein, for example, an antiparallel coiled coil region of a stalk (Stalk or B-link) site constituting a dynein of a motor protein is particularly preferably mentioned.

  Examples of the filamentous phage as the material of the rod-shaped structure include f1 phage, fd phage, M13 phage and the like, which have been genetically manipulated so as to be shortened. Among these, a shape excellent in visibility and discrimination From the viewpoint of being, fd phage subjected to genetic manipulation is preferable.

-Contact part-
The contact portion is appropriately selected according to the aspect of the microscope analysis target.
When the microscope analysis target is an external detection target that is detected by interaction with the contact portion of the microscope analysis probe, the contact portion is made of a substance that can interact with the specific part of the microscope analysis target. It is preferable that it is a 1st aspect which is an interaction area | region (contact area | region) with the said specific site | part of the said microscope analysis object.
When the microscope analysis target is integrated with the microscope analysis probe, the contact portion is configured to be coupled with a specific part of the microscope analysis target, and is coupled with the specific part of the microscope analysis target It is preferable that it is a 2nd aspect which is a coupling | bonding end (contact end).

(1) 1st aspect The 1st aspect of the said contact part is an aspect of the interaction area | region (contact area | region) with the said specific site | part of the said microscopic analysis object.
In the present invention, the “interaction” between the specific site to be analyzed by the microscope and the contact portion represents an affinity and site-specific interaction, for example, adhesion, binding, adsorption, fusion, etc. Specifically, an antigen-antibody reaction is preferable.
In this case, the contact portion is preferably made of a protein capable of antigen-antibody reaction with the external detection target. Note that a protein synthesized by genetic engineering may be used as the microscope analysis target, a protein capable of antigen-antibody reaction with the microscope analysis target may be searched, and this may be used as the contact portion.

  Examples of the substance constituting the contact portion include antigens against a specific site to be analyzed by the microscope, antibody binding sites, natural ligands such as lectin, biotin, and nucleotides, tags such as GST tag, His tag, and FLAG tag, enzymes, Examples include pharmaceutical compositions and reagents, among which ligands and tags are preferred.

  The antigen-antibody-reactive protein combination is not particularly limited and can be appropriately selected according to the purpose. Allergen, enzyme, enzyme substrate, coenzyme, enzyme inhibitor, hormone receptor, protein, blood protein, Tissue cells, cell debris, nuclear materials, viruses, virus particles, various metabolites, neurotransmitters, haptens, drugs, nucleic acids, microorganisms, parasites, bacteria, biotin, avidin, lectins, sugars, bioactive substances, bioactivity Examples include combinations of substance receptors, environmental substances, chemical species or derivatives thereof, and molecules that recognize ligands of these molecules. Specifically, plasma proteins, tumor markers, apoproteins, viral antigens, self Combinations of antibodies, coagulation / fibrinolytic factors, hormones, blood drugs, HLA antigens, etc. with molecules that recognize ligands of these molecules, actin As myosin, can be selected combination of proteins specific interactions are known.

Examples of the plasma protein include immunoglobulins (IgG, IgA, IgM, IgD, IgE), complement components (C3, C4, C5, C1q), CRP, α ₁ -antitrypsin, α ₁ -microglobulin, β Examples include _2- microglobulin, haptoglobin, transferrin, ceruloplasmin, and ferritin.

  Examples of the tumor marker include α-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9, CA125, CA15-3, SCC antigen, prostatic acid phosphatase (PAP), PIVKA-II, γ-semi Noprotein, TPA, elastase I, nerve specific enolase (NSE), and immunosuppressive acidic protein (IAP).

  Examples of the apoprotein include apo AI, apo A-II, apo B, apo C-II, apo C-III, and apo E.

Examples of the viral antigen include hepatitis B virus (HBV) -related antigen, hepatitis C virus (HVC) -related antigen, HTLV-I, HIV, rabies virus, influenza virus, and rubella virus.
Examples of the HCV-related antigen include HCVc100-3 recombinant antigen, pHCV-31 recombinant antigen, and pHCV-34 recombinant antigen, and mixtures thereof can be preferably used. Examples of the HIV-related antigen include virus surface antigens, and the like, for example, HIV-I env. gp41 recombinant antigen, HIV-I
env. gp120 recombinant antigen, HIV-I gag. p24 recombinant antigen, and HIV-II env. Examples include p36 recombinant antigen.
Examples of antigens other than viruses include MRSA, ASO, toxoplasma, mycoplasma, and STD.

  Examples of the autoantibodies include anti-microsomal antibodies, anti-thyroglobulin antibodies, antinuclear antibodies, rheumatoid factors, anti-mitochondrial antibodies, and myelin antibodies.

Examples of the coagulation / fibrinolytic factor include fibrinogen, fibrin degradation product (FDP), plasminogen, α ₂ -plasmin inhibitor, antithrombin III, β-thromboglobulin, factor VIII, protein C, and protein S. It is done.

Examples of the hormone include pituitary hormone (LH, FSH, GH, ACTH, TSH, prolactin), thyroid hormone (T ₃ , T ₄ , thyroglobulin), calcitonin, parathyroid hormone (PTH), adrenal cortex hormone (aldosterone) , Cortisol), gonadal hormones (hCG, estrogen, testosterone, hPL), pancreatic / gastrointestinal hormones (insulin, C-peptide, glucagon, gastrin), and others (renin, angiotensin I, II, enkephalin, erythropoietin) It is done.

  Examples of the blood drug include antiepileptic drugs such as carbamazepine, primidone and valproic acid, cardiovascular disease drugs such as digoxin, quinidine, digitoxin and theophylline, and antibiotics such as gentamicin, kanamycin and streptomycin.

As the protein capable of reacting with an antigen-antibody, for example, a specific single-chain antibody for a specific site to be analyzed with a microscope and a single-chain variable fragment (scFv: single chain fragment-V) of the antibody are preferable. Can be mentioned.
The microscopic analysis target is a recombinant protein in which a foreign peptide or the like (FLAG tag or the like) is inserted in advance at an arbitrary site, so that the contact portion includes an anti-tag single chain antibody against the microscopic analysis target foreign peptide. , And a single chain variable region fragment (scFv) of the antibody can also be used.
The scFv can be prepared by a method such as a biopanning method using phage display.

As the method for preparing the contact part, for example, after preparing only the substance constituting the contact part, it may be added to the end part of the rod-shaped structure prepared separately by a chemical reaction or the like. When both rod-like structures are proteins, amino acids may be synthesized as a single molecule protein, and as a single molecule recombinant fusion protein using a vector comprising the contact portion and a gene sequence encoding the rod-like structure. It may be synthesized.
The method of synthesizing the single molecule recombinant fusion protein will be described in detail later.

  Examples of the method of preparing only the substance constituting the contact portion and then adding it to the end of the rod-shaped structure prepared separately by chemical reaction or the like include, for example, the peptide method, diazo method, alkylation method, cyanogen bromide activity Method, immobilization method using Ugi reaction, immobilization method using thiol-disulfide exchange reaction, Schiff base formation method, chelate bond method, tosyl chloride method, biochemical specificity Bonding methods and the like can be mentioned, but for more stable bonds such as covalent bonds, there are methods using reaction of thiol group and maleimide group, reaction of pyridyl disulfide group and thiol group, reaction of amino group and aldehyde group, etc. Preferably, a method using a chemical binder or a crosslinking agent is more preferable.

Examples of such chemical binder / crosslinking agent include carbodiimide, isocyanate, diazo compound, benzoquinone, aldehyde, periodic acid, maleimide compound, pyridyl disulfide compound, and the like. Among these, glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, N′-polymethylene bisiodoacetamide, N, N′-ethylene bismaleimide, ethylene glycol bissuccinimidyl succinate, bisdiazobenzidine 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) N-sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl) aminobenzoate, N-succinimidyl 4- ( -Maleimidophenyl) butyrate, iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4'-dithiopyridyl) propionimidate, methyl-4-mercaptobutyrylimidate, methyl-3-mercaptopropionimidate N-succinimidyl-S-acetylmercaptoacetate and the like.

(2) 2nd aspect The 2nd aspect of the said contact part is an aspect of the coupling end (contact end) of the state couple | bonded with the said specific site | part of the said microscope analysis object.
In this case, the contact portion is not particularly limited and can be appropriately selected according to the purpose. For example, the contact portion is preferably composed of a protein, and is a single molecule of the microscope analysis target and the microscope analysis probe. More preferably, it is a part of the recombinant fusion protein.
The method of synthesizing the single molecule recombinant fusion protein will be described in detail later.

-Marking part-
It is preferable that the said marker part exists in the other end with respect to one end part which has the said contact part of the said rod-shaped structure.

  The substance constituting the labeling part is not particularly limited as long as it consists of a substance that can be detected with a microscope, and can be appropriately selected according to the purpose. For example, fluorescent substance, dye, heavy metal compound, heavy metal colloid, oxidation Examples include reductase.

  When a stereomicroscope and an electron microscope are used as the microscope, the substance constituting the labeling part is preferably a fluorescent substance, and more preferably a fluorescent protein. Further, biotin is used for the labeling part, and the visibility of the probe for microscopic analysis is improved by using a complex of fluorescently labeled biotin and avidin, fluorescently labeled streptavidin, development sensitizer, etc. May be.

  The fluorescent substance is not particularly limited as long as it has fluorescence, and can be appropriately selected from known substances according to the purpose. Examples thereof include fluorescein series, rhodamine series, eosin series, NBD series, and the like. Specifically, fluorescein-5-isothiocyanate, diacyl (such as isobutyryl, acetyl or pivaloyl) fluorescein-5 and / or 6-carboxylic acid pentafluorophenyl ester, 6- (diacyl-5 and / or 6-carboxamide-fluorescein ) Aminohexanoic acid pentafluorophenyl ester, Texas Red (trademark of Texas Probes, Inc.), tetramethylrhodamine-5 (and 6) isothiocyanate, eosin-isothiocyanate, erythrosine- -Isothiocyanate, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole, 3- (7-nitrobenz-2-oxa -1,3-diazol-4-yl) methylaminopropionitrile, 6- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminohexanoic acid, succinimidyl 12- (N-methyl- N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminododecanoate, 7-diethylamino-3- (4′-isothiocyanatophenyl) -4-methylcoumarin (CP), 7-hydroxycoumarin-4-acetic acid, 7-dimethylaminocoumarin-4-acetic acid, succinimidyl 7-dimethylaminocoumarin-4-acetate, 7-methoxy bear Phosphorous-4-acetic acid, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 9-chloroacridine, succinimidyl 3- (9-carbazole) propionate, succinimidyl 1-pyrenebutyrate Succinimidyl 1-pyrene nonanoate, p-nitrophenyl 1-pyrene butyrate, 9-anthracene propionic acid, succinimidyl anthracene-9-propionate, 2-anthracene sulfonyl chloride, and the like.

  Examples of the fluorescent protein include green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and enhanced yellow fluorescent protein (EYFP). , Cyan fluorescent protein (CFP) -enhanced cyan fluorescent protein (ECFP), blue fluorescent protein (BFP), and enhanced blue fluorescent protein (EBFP). Among these, enhanced green fluorescent protein (EGFP) is preferred.

  Here, when the labeling portion is made of a fluorescent material, the detection target is labeled by using the microscopic analysis probe to which another fluorescent material that overlaps the excitation wavelength or emission wavelength of the fluorescent material is added. The structural change can be analyzed by measuring the change in fluorescence energy transfer and distance between molecules, and the orientation of the label can be directly analyzed by detecting the polarization of fluorescence using a fluorescence microscope. can do.

  On the other hand, when an atomic force microscope is used as the microscope, the substance constituting the labeling part is not particularly limited as long as it functions as a marker indicating the position of the target molecule, and can be appropriately selected according to the purpose. it can.

Examples of a method for binding the labeling portion to the end of the rod-shaped structure include, for example, a method in which only the substance constituting the labeling portion is prepared and then added to the end of the rod-shaped structure prepared separately by a chemical reaction or the like A combination of single molecules using a method of synthesizing amino acids as a single molecule protein when the labeling portion and the rod-like structure are both proteins, and a vector comprising a gene sequence encoding the labeling portion and the rod-like structure. Examples include a method of synthesizing as a replacement fusion protein.
After preparing only the substance constituting the labeling part and adding it to the end of the rod-shaped structure prepared separately by chemical reaction or the like, for example, reactive substitution capable of forming a bond such as a covalent bond or an ionic bond And a method in which a group is introduced into the skeleton of a fluorescent substance and introduced using these bonds.
The method of synthesizing the single molecule recombinant fusion protein will be described in detail later.

<How to use the probe for microscope analysis>
When the microscope analysis target is the first aspect that is an external detection target that is detected by interaction with the contact portion, a method for using the microscope analysis probe is as follows. As long as the probe for microscopic analysis is administered and interacts with the specific site, there is no particular limitation, and it can be appropriately selected according to the purpose. For example, the microscopic analysis probe prepared in advance can be selected for the microscopic analysis target. A method of administering and using a microscopic analysis probe prepared in advance in a form that can be analyzed, a method of administering and using the previously prepared microscope analysis probe on an unprocessed microscopic analysis target, and a code for the microscope analysis probe An expression vector containing a sequence to be analyzed is introduced into a cell expressing the target for microscopic analysis, and the probe for microscopic analysis expressed in the cell is used. Method, and the like.

  When the microscopic analysis target is the second aspect integrated with the microscopic analysis probe, the method for using the microscopic analysis probe is not particularly limited and can be appropriately selected depending on the purpose. For example, a method for preparing a sample solution in which a molecule in which the probe for microscopic analysis and the target for microscopic analysis are integrated is suspended, a vector for expressing the probe for microscopic analysis and the target for microscopic analysis, Examples thereof include a method of introducing a gene into a knockout cell from which a gene encoding the subject has been deleted and expressing it.

  In addition, for the purpose of analyzing two or more types of analysis objects at the same time, for example, two or more types of the microscope analysis probes having different contact portions and the different contact portions may be used for the microscope analysis probes. Accordingly, two or more kinds of the microscope analysis probes having different lengths or shapes of the rod-like structures can be used at the same time. Thereby, a plurality of analyzes can be performed at the same time in the same sample, and in this case, it is not necessary to perform another control experiment, which is advantageous.

<Preparation method of probe for microscope analysis>
As the probe for microscopic analysis, it is preferable that at least the rod-like structure is composed of a protein, more preferably a single recombinant fusion protein molecule, which is expressed using the probe expression vector for microscopic analysis of the present invention. It is particularly preferred to consist of a single molecule recombinant fusion protein.

  The probe expression vector for microscopic analysis of the present invention is clarified through the method for preparing the probe for microscopic analysis comprising the single-molecule recombinant fusion protein.

  When the microscope analysis target is an external detection target detected by interaction with the contact portion, and the contact portion is the interaction region (contact region) of the first aspect, the microscope analysis probe For example, the coiled coil region of the dynein stalk site has the rod-like structure, and as the contact portion, at least one of the N-terminus and C-terminus of the rod-like structure is fused with any protein as the contact portion, and Suitable examples include those in which a fluorescent protein is inserted into a region where a coiled coil is not formed at the substantially central portion of the rod-like structure.

  The probe expression vector for microscopic analysis in the first aspect preferably includes a sequence formed by linking genes encoding these proteins, specifically, a gene encoding a dynein stalk region, A gene obtained by linking a gene encoding the contact portion and a gene encoding a fluorescent protein is preferable.

  The method for constructing the probe expression vector for microscopic analysis can be appropriately selected from known methods. For example, (1) cDNA encoding the stalk site of dynein is prepared, and this is subcloned into a plasmid. 2) A substantially central region (region not forming a coiled coil) of the cDNA encoding the stalk site is removed, and a sequence encoding the fluorescent protein is inserted therein, and (3) an end of the stalk site (N-terminal and Examples include a method of inserting a sequence encoding a protein as the contact portion into at least one of the C terminals.

  When the protein at the contact portion is a protein capable of antigen-antibody reaction, an antibody variable region is preferably exemplified as the protein. Examples of the antibody variable region include scFv (single chain Fv). The scFv is a single chain variable region fragment in which a variable region composed of VH and VL, which are the minimum units necessary for an antibody to recognize an antigen, is bound by a flexible peptide linker. It is known that the order of linking VH and VL, the length and sequence of the peptide linker, the amino acid substitution in the V region, etc. may cause differences in ease of expression, stability in solution, morphology, etc. .

  Examples of the scFv cDNA preparation method include a biopanning method using phage display. Specifically, scFv is displayed on the outer shell protein of a filamentous phage, a library is prepared, and it is made to bind | bond with the solid-phased desired antigen, and performs selection operation.

The probe for microscopic analysis can be prepared by a known method for producing a recombinant protein using the probe expression vector for microscopic analysis. For example, the probe for microscopic analysis is used for cells that express the probe for microscopic analysis. It can be prepared by introducing a probe expression vector for transformation, culturing the obtained transformant, collecting the probe for microscopic analysis from the culture, and purifying it.
Examples of the purification method include subcloning cDNA encoding the probe for microscopic analysis into a GST fusion protein expression vector, and using the GST column for the probe for microscopic analysis synthesized as the obtained GST fusion protein. And a method of removing GST after purification.

The cell into which the probe expression vector for microscopic analysis is introduced is not particularly limited and can be appropriately selected according to the purpose. For example, when the microscopic analysis target is a biological sample, the identification of the analysis target A cell constituting the site is preferred.
By expressing the probe for microscopic analysis in cells that express the specific site of the microscopic analysis target, the step of adding and interacting with the probe for microscopic analysis is unnecessary. In this state, and under normal physiological conditions, the localization, direction (orientation), structure, shape (three-dimensional structure), dynamics, and the like of the target molecule can be analyzed.

  When the microscopic analysis target is the second aspect integrated with the microscopic analysis probe, the microscopic analysis probe composed of the single molecule recombinant fusion protein may be, for example, a dynein stalk site. A region in which the coiled coil region is the rod-shaped structure, the protein to be analyzed is fused to at least one of the N-terminal and C-terminal of the rod-shaped structure serving as the contact end, and a coiled coil is not formed in the substantially central portion of the rod-shaped structure. Suitable examples include those obtained by inserting a fluorescent protein into.

  When the microscope analysis target is integral with the microscope analysis probe, and the contact portion is a coupling end (contact end) in a state of being coupled to the specific site of the microscope analysis target, For example, the coiled-coil region of the dynein stalk site is the rod-shaped structure, and an arbitrary protein to be analyzed with the microscope is fused to at least one of the N-terminal and C-terminal of the rod-shaped structure serving as the contact portion, and further required Accordingly, a material obtained by inserting a fluorescent protein into a region where the coiled coil is not formed at the substantially central portion of the rod-like structure is preferably mentioned.

  The method for constructing the probe expression vector for microscopic analysis can be appropriately selected from known methods. For example, (1) cDNA encoding the stalk site of dynein is prepared, and this is subcloned into a plasmid. 2) A substantially central region (region not forming a coiled coil) of the cDNA encoding the stalk site is removed, and a sequence encoding the fluorescent protein is inserted therein, and (3) an end of the stalk site (N-terminal and Examples include a method of inserting a sequence encoding the protein to be analyzed by microscopic analysis by linking to the contact portion which is at least one of C-terminal.

  The cell into which the probe expression vector for microscopic analysis is introduced is not particularly limited and can be appropriately selected according to the purpose. For example, when the microscopic analysis target is a biological sample, the microscopic analysis target is encoded. It is preferably a knockout cell from which the gene to be deleted is deleted.

The probe for microscopic analysis of the present invention is preferably used for the preparation of various microscopic analysis samples, for example, quick freeze deep etch replica method, negative staining method, cryo-negative staining method, resin-embedded section method, and It is more preferably used in the preparation of a sample by an ice embedding method or the like, and among these, it is particularly preferably used for the preparation of a sample by a quick freezing deep etch replica method.
In the sample preparation by the quick-frozen deep-etch replica method, using the microscope analysis probe expression vector, combining the method for expressing and using the microscope analysis probe in cells that express the specific site to be analyzed This makes it possible to analyze proteins that change their structure during the performance of functions in the field of biological phenomena.

(Microscope analysis method)
The microscope analysis method of the present invention is a method including at least a step of preparing a microscope analysis sample including a microscope analysis probe and a step of detecting the microscope analysis probe in the microscope analysis sample.
The microscope analysis method using the microscope analysis probe expression vector of the present invention includes a step of expressing the microscope analysis vector, a step of preparing a microscope analysis sample including the microscope analysis probe, and the microscope. And a step of detecting the probe for microscopic analysis in the analysis sample.

  The microscope used for the analysis is not particularly limited as long as analysis of a specific part of the microscope analysis target is possible, and can be appropriately selected according to the purpose. For example, a stereo microscope, a fluorescence microscope, a laser scanning microscope Examples include an optical microscope such as a transmission electron microscope, an electron microscope such as a scanning electron microscope, a scanning probe microscope such as an atomic force microscope, and a scanning tunneling microscope. Among these, a fluorescence microscope and a transmission electron microscope , Scanning electron microscope, and atomic force microscope are preferable.

When the microscope analysis target is an external detection target that is detected by interaction with the contact portion of the microscope analysis probe, a method for preparing the microscope analysis sample is to use the microscope analysis probe as the microscope A step of interacting with a specific part to be analyzed.
The interaction method is not particularly limited and may be appropriately selected depending on the purpose. For example, the previously prepared microscope analysis probe is applied to a sample processed into a form in which the microscope analysis target can be analyzed. A method for administering the microscopic analysis probe prepared in advance to an untreated target for microscopic analysis, and an expression vector containing a sequence encoding the probe for microscopic analysis as a specific site of the target for microscopic analysis. Examples thereof include a method of introducing into a cell or the like to be expressed and allowing the cell to express.

Examples of the method for administering the probe for microscopic analysis include a method of mixing the probe for microscopic analysis and the object of microscopic analysis in a solution.
Examples of the method for introducing the probe expression vector for microscopic analysis into cells include a method using a carrier peptide, calcium phosphate, polyethylene glycol, and the like, and an electroporation method.

  When the target for microscopic analysis is integral with the probe for microscopic analysis, the method for preparing the sample for microscopic analysis is to prepare the probe for microscopic analysis as a single fusion protein with the target for microscopic analysis, Can be prepared as the sample for microscopic analysis.

The sample for microscopic analysis can be appropriately selected from known methods according to the microscope used for analysis and the type of the target for microscopic analysis.
When the microscope is an electron microscope, for example, rapid freezing deep etch replica method, negative staining method, cryo negative staining method, ice embedding method and the like, among these, rapid freezing deep etch replica method is preferable .

  As the rapid freezing deep etch replica method, for example, after the probe for microscopic analysis and the target molecule interact with each other, a heavy metal (for example, platinum) is deposited on at least a specific part of the microscopic analysis target. In addition, there is a method in which carbon is further coated to dissolve and remove the biological sample.

-Analysis method-
As a method for analyzing the specific portion of the microscope analysis target, there is no particular limitation as long as it is a method capable of detecting at least one of the label portion and the rod-like structure of the probe for microscopic analysis and detecting the contact portion. The analysis conditions can be appropriately selected according to the type of microscope, the type of sample for microscope analysis, the substance constituting the labeling portion, and the like.
For example, an electron microscope sample prepared by a method of analyzing the localization and direction (orientation) of the detection target, a quick freezing method, etc., by detecting the fluorescence of the labeling portion made of a fluorescent substance using a fluorescence microscope In particular, a method for analyzing the localization, direction (orientation), and shape of a specific part (detection target) of the microscope analysis target, and a method for analyzing the dynamics of the sample in the solution containing the detection target with an atomic force microscope Etc.

  When an electron microscope is used as the microscope, the analysis method includes, for example, the methods described in International Publication No. WO02 / 48961, Pamphlet of International Publication No. WO02 / 056259, Pamphlet of International Publication No. WO02 / 075658, and the like. Preferably mentioned.

  Since the microscope analysis probe of the present invention has high visibility and does not impair the spatial resolution of the microscope, the microscope analysis method of the present invention using the microscope analysis probe can be used, for example, for a specific site of the microscope analysis target. The presence, direction (orientation), shape (three-dimensional structure), dynamics, and the like can be analyzed in detail without averaging. Further, according to the microscopic analysis method of the present invention, it is possible to analyze the change over time of the specific site under physiological conditions, the structure in the active state, and the like.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1
Using the synthetic FLAG peptide as the microscope analysis target, the microscope analysis probe for detecting the FLAG peptide and the microscope analysis probe expression vector were prepared by the following method.
The probe for microscopic analysis includes, as the rod-shaped structure, a coiled coil region of a stalk site of dynein (Human Cytoplasmic Dynein), an scFv having an antigen-specific binding ability to the FLAG peptide as the contact portion (contact region), and The labeling portion was prepared as a single molecule recombinant fusion protein consisting of the fluorescent protein EGFP (A206K).

<Preparation of probe expression vector for microscopic analysis>
(1) Subcloning of stalk site cDNA into plasmid -Preparation of stalk site cDNA-
CDNA corresponding to the stalk site of Human Cytoplasmic Dynein (the 9350th to 10369th base sequence portion of the DNA sequence reported as Genebank Accession No. AB002323: 1019 bases) was isolated from SK-N-MC cells (human neuroblastoma). From the total RNA of (derived cell line), it was prepared by RT-PCR method.
The total RNA of the SK-N-MC cells was extracted from 1 × 10 7 cells using TRIzol reagent (manufactured by Invitrogen).
The RT-PCR method used SuperScript II Reverse Transcriptase (manufactured by Invitrogen) as a reverse transcriptase and Oligo (dT) _12-18 as a primer to synthesize First Strand cDNA.

Using the First Strand cDNA as a template, as thermostable DNA Polymerase, KOD plus DNA Polymerase (manufactured by Toyobo Co., Ltd.), Forward primer (SEQ ID NO: 5′-GCGAATTCATGCGCCATACCCCTCTC-3 ′), Reverse primer (SEQ ID NO: 5′-) PCR was performed using GCATCTCGAGGAGAGAGCTCCCAGC-3 ′).
In order to confirm that the obtained PCR product was a cDNA corresponding to a stalk site, the sequence was analyzed using a sequencer (ABI PRISM 3100-Avant Genetic Analyzer, Applied Biosystems). The above-mentioned Genebank Accession No. It was confirmed that the DNA sequence of AB002323 was completely identical.

-Subcloning into plasmid-
The probe for microscopic analysis is expressed downstream of GST cDNA of GST fusion protein expression vector pGEX-6P-1 (Amersham) for fusion expression to the C-terminus of Glutathione-S-Transferase (GST). In order to construct the GST cDNA and the microscopic analysis probe cDNA so that their reading frames match within the existing multicloning site, between the EcoRI site and the XhoI site in the multicloning site of pGEX-6P-1. Then, cDNA corresponding to the stalk site was inserted.

(2) Insertion of fluorescent protein EGFP cDNA From the cDNA corresponding to the stalk site subcloned into pGEX-6P-1, a microtubule binding site sandwiched between antiparallel coiled coil regions was removed, and the fluorescent protein EGFP Was inserted by the following method.

The cDNA corresponding to the stalk site of the Genebank Accession No. In the sequence of AB002323, site-specific gene mutation was performed using QuickChange Site-Directed Mutagenesis Kit (manufactured by Stratagene) so that the sequence corresponding to positions 9707 to 9712 in the sequence of AB002323 and the sequence corresponding to positions 100070 to 10075 become Aor13HI sites. It was. The sequence of the primer used for mutagenesis is shown below.
(SEQ ID NO: 3)
5'-CAAAGAAGATCTTGATAAGGGTGACACCTGGGTCCGGACCCTACCGCAATGAGCTGCAGAAGCTGG-3 '
(SEQ ID NO: 4)
5'-CCAGCTTCTGCAGCCTCATTGCGTAGGGGTCCGGACCCAGGTCCACCTTTACAAGATCTTTCTTTG-3 '

  The EGFP cDNA having Aor13HI sites at both ends uses pEGFP-C1 (manufactured by Clontech) as a template, KOD plus DNA Polymerase (manufactured by Toyobo) as a thermostable DNA Polymerase, and Forward primer (SEQ ID NO: 5 ' -GTCAACTAGTGGAGCAAGGGCGAGG-3 ') and a reverse primer (SEQ ID NO: 6: 5'-CAGTACTAGTCCCCTTGTACAGCTCGTCC-3') were used for synthesis. The obtained EGFP cDNA having Aor13HI sites at both ends was inserted into the region where the stalk site was removed so that the reading frame would match.

  Further, in order to prevent the dimerization of EGFP (Zacharias DA., Science 2002, 296: 913-916), QuickChange Site-Directed Mutagenesis Kit (manufactured by Stratagene) was used and a primer (SEQ ID NO: 7: 5′- A206K mutation was introduced using CTGAGCACCCATCTCAGCGGAGCAAAGACCCC-3 ′ and SEQ ID NO: 8′—GGGGTCTTTGCTCAGCCTGGACTGGGGTCTCAG-3 ′).

(3) Insertion of ScFv cDNA-Cloning of ScFv-expressing phage by panning method-
(I) Antigen preparation Synthetic FLAG peptide (SEQ ID NO: 9: DYKDDDDKGC, MW: 1174.18, manufactured by Sigma) and BSA (manufactured by Sigma) are combined with a crosslinker MBS (manufactured by Sigma). Created.

(Ii) Mouse immunization A mixture of the antigen (FLAG-BSA) solution 2 mg / mL prepared in (i) above and Freund Complete Adjuvant was administered intraperitoneally to the mouse, and two weeks later, the antigen A mixture of 2 mg / mL of the solution and Incomplete Adjuvant 1: 1 was administered in the same manner. One week later, blood was collected from the mice, and the antibody titer was measured by ELISA using FLAG-BSA as an antigen.

(Iii) Extraction of mRNA from immunized mouse As a result of (ii), the spleen was extracted from the mouse in which the antibody titer was increased, and the total RNA was extracted from the extracted spleen using TRIzol reagent (Invitrogen). Then, mRNA was extracted using Oligotex-dT30-super (Takara Shuzo).

(Iv) Synthesis of antibody cDNA cDNA was synthesized from the mRNA obtained in (iii) above, and this was used as a template to obtain antibody heavy chain cDNA and antibody light chain cDNA by PCR.
In addition, the synthesis of ScFv was performed using a Recombinant Page Antibody System (manufactured by Amersham, hereinafter referred to as “RPAS”). As primers, Heavy Primer 1 & 2 and Light Primer Mix were used, and AmpliTaq (manufactured by Applied Biosystems) was used as thermostable DNA Polymerase.

(V) Insertion of linker (ScFv cDNA synthesis)
The antibody heavy chain cDNA and antibody light chain cDNA obtained in (iv) above were mixed using the RPAS Linker-Primer Mix, and PCR was performed for only 7 cycles using AmpliTaq. The obtained PCR product was purified using the RPAS Sephacryl S-400 HR Spun-Column to obtain ScFv cDNA.

(Vi) Addition of SfiI site and NotI site to cDNA of ScFv The ScFv obtained in (v) above was subjected to PCR using AmpliTaq using RS Primer Mix of RPAS as a primer. The obtained PCR product was cleaved with SfiI and NotI, and then purified using the RPAS Sephacryl S-400 HR Spin-Column.

(Vii) Subcloning into pCANTAB 5E (creation of ScFv expression library)
The ScFv cDNA having the SfiI site and NotI site obtained in (vi) was subcloned into the SfiI site and NotI site of pCANTAB 5E.
Escherichia coli (JM109) was transformed with the obtained pCANTAB 5E-ScFv vector and cultured on an LB plate, and then the plasmid was recovered and used as an ScFv expression library.

(Viii) Preparation of ScFv expression phage library Escherichia coli (TG-1) was transformed using the ScFv expression library obtained in (vii). The Escherichia coli was infected with helper phage M13KO7 to obtain a ScFv-expressing phage library having 1 × 10 ¹¹ phage particles.

(Ix) Cloning of antigen-specific ScFv-expressing phage by panning method After adsorbing and fixing 20 g of antigen (FLAG-BSA) to a 3 cm diameter plastic dish and blocking with BSA (or casein), (viii The ScFv expression phage library obtained in (1) was added and reacted. Thereafter, the plate was washed with a PBS solution (pH 7.4) containing Tween 20, and 0.1 M glycine hydrochloride buffer (pH 2.2) was added to elute the phage bound to the dish. Immediately after the phage elution, neutralization was performed using 1M Tris-HCl (pH 9.1).
The recovered phages were infected with E. coli (TG-1), and the E. coli was propagated, then infected with helper phage M13KO7, and ScFv-expressing phages were again recovered and used as a secondary library. Using the obtained secondary library, the above operation (panning operation) was further repeated twice to concentrate antigen-specific ScFv expression phage.
E. coli (TG-1) was infected with the concentrated antigen-specific ScFv expression phage to obtain a cloned phage. The obtained phage clones were confirmed for antibody titers by ELISA.

Separately, an ScFv-expressing phage clone having antigen specificity was cloned from the ScFv-expressing phage library using the panning method, and a ScFv expression vector (pCANTAB 5E) was rescued from the antigen-specific ScFv-expressing phage clone.
Using the obtained pCANTAB 5E as a template, KOD plus DNA Polymerase (manufactured by Toyobo Co., Ltd.) as heat-resistant DNA Polymerase, Forward primer (SEQ ID NO: 10: 5′-GACTGCCGCGCGCGTGTGTCTGGATCGAGATCTGC-3se) : 5'-CGATGCCGCCGCTGGAGAGACGGTGACCGTGGTCCCTG-3 ') to synthesize ScFv cDNA having NotI sites at both ends.
The obtained ScFv cDNA was inserted into the NotI site in pGEX-6P-1MCS in such a way that the reading frame matched forward with the C-terminus, and the above-mentioned probe expression vector for microscopic analysis (pGEX-SMP) was obtained. . A schematic diagram is shown in FIG.

<Preparation of probe for microscopic analysis>
(1) Introduction of pGEX-SMP Escherichia coli BL21 CodonPlus (DE3) RIL was used as the cell for expression of the probe for microscopic analysis, and transformed with the probe expression vector for microscopic analysis (pGEX-SMP). Several clones were picked up from the LBG-amp (containing 2% glucose) plate, cultured in about 15 ml of medium, protein was extracted from the obtained E. coli, SDS-PAGE was performed, and expression induction by IPTG was normal. A clone that can be confirmed was selected and used as a transformant.

(2) Mass culture of transformant After inoculating 10 mL of E. coli culture solution of the transformant obtained in (1) above into 1000 mL of 2 × YTG (containing 2% glucose) and culturing at 25 ° C. for 2 hours In order to induce expression, a final concentration of 0.5 mM IPTG (manufactured by Sigma) was added, and further cultured for 1 hour, followed by centrifugation at 4700C and 7700 × G to collect E. coli.

(3) Preparation of cell extract 50 mL of ice-cold 1 × PBS (including Roche Complete-EDTA Free table) as a protease inhibitor was added to the E. coli pellet recovered in (2) above, and pipetting was performed. Then, the pellet was completely suspended, and the cells were disrupted by an ultrasonic treatment device to obtain a cell extract. The ultrasonic treatment was performed several times on ice in order to suppress the generation of heat. Centrifugation was performed at 4 ° C. and 12,000 × G, and the supernatant (cell extract) was transferred to a new container.

(4) Purification of probe for microscopic analysis After connecting a GSTrapFF 1 mL column to a high-performance liquid chromatography system (AKTA explorer 10S, Amersham Biosciences) and equilibrating the column with 5 mL of a binding buffer (1 × PBS) Then, 50 mL of the cell extract obtained in (3) above was added to the column, and the column was washed with 10 mL of the binding buffer.
Subsequently, the column was washed with 10 mL of PreScission cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0).
A mixture of 80 μL of PreScission Protease and 920 μL of PreScission cleavage buffer was prepared and added to the column, and then the column was sealed and allowed to stand for 12 hours.

  Further, another equilibrated GSTrapFF 1 mL column was connected to the end of the column to make a filter for adsorbing and removing the cleaved GST tag and undigested GST fusion protein (probe for microscopic analysis). After digestion treatment with PreScission Protease, the protein (probe for microscopic analysis) whose GST tag was cleaved was eluted with 15 mL of PreScission cleavage buffer. The eluate was collected as 0.5 mL fractions, and the three fractions with the highest OD280 values were subjected to SDS-PAGE to confirm that a single protein molecule was detected as a single band. Thus, the probe for microscopic analysis of the present invention was obtained. A schematic diagram of the obtained probe for microscopic analysis is shown in FIG.

From Example 1, it was found that the probe for microscopic analysis composed of a single molecule can be efficiently prepared.
Even when another antigen is selected as the target molecule, the desired probe for microscopic analysis can be prepared by the above method.

(Example 2)
Myosin was selected as the microscope analysis target, and the microscope analysis probe integrated with the myosin capable of analyzing a specific region in myosin, and the microscope analysis probe expression vector for obtaining the probe were prepared.
The microscopic analysis probe of Example 2 was prepared by preparing the microscopic analysis probe expression vector obtained by inserting the myosin cDNA into the contact end region and using the myosin heavy chain as the vector. It was prepared in the same manner as in Example 1 except that it was introduced into a gene knockout strain (cellular slime mold Dictyosterium discoideum ) and expressed.

(1) Insertion of myosin cDNA Primer (SEQ ID NO: 12: 5′-TCATGATGCAAAACCGTTTCCACGAGAAGCG-3 ′ and SEQ ID NO: 13: 5 ′) using as a template a plasmid containing the rod-like structure of the microscope analysis probe and the labeled portion cDNA. -CCATGGAGACAGTCCCCCAGCAATGG-3 '), PCR was performed with LA-Taq (manufactured by Takara Bio Inc.), and BspHI site and NcoI site were added to the ends, respectively, and this was added to pGEM-T easy vector (Promega). Inserted. After confirming the sequence with a sequencer (ABI3200 Genetic Analyzer, Applied Biosystems), the sequence containing the rod-shaped structure and the cDNA of the labeling portion was excised with BspHI and NcoI.

On the other hand, pTIKLMyDΔB was treated with BspHI and alkaline phosphatase, and the sequence containing the rod-shaped structure and the cDNA of the labeling portion was inserted. The pTIKLMyDΔB is a myosin heavy chain obtained by removing two of the three BspHI sites in pTIKLMyD (Liu et al., 1998) in the vector backbone derived from pBluescript by site-directed mutagenesis. The BspHI site near 5 ′ of the gene is unique.
The inserted cDNA can be inserted in both directions, but the one with the BspHI site inserted on the 5 ′ side and the NcoI site inserted on the 3 ′ side was selected based on the restriction enzyme cleavage pattern. As a result, a plasmid (pTIKLprobeMyo) was obtained in which the cDNA was inserted into the N-terminus of the myosin heavy chain (the site of “HD” in the amino acid sequence “MNPIHDRT...”).

(2) Purification of probe for microscopic analysis The myosin heavy chain gene knockout strain HS1 (Ruppel et al., 1994) of the cellular slime mold Dictyostelium discoideum was introduced by the electroporation method (Egelhoff et al., 1991) and the above pTIKLprobeMyo. Then, the cells were cultured in 12 μg / mL G418 (manufactured by BIBCO BRL) and HL5 medium (Sussman, 1987) containing penicillin and streptomycin, and transformants were selected. The obtained transformant was cultured at 21 ° C. using 12 pieces of 30 cm square plastic petri dishes.

The purification of myosin from the transformed cells was performed by a modification of the method of Ruppel et al. (Ruppel et al., 1994), and all operations were performed at a temperature of 0 to 5 ° C.
The transformant was recovered in 10 mM Tris buffer (pH 7.4) and collected by low-speed centrifugation (2000 rpm, 2 minutes). This was resuspended in the same Tris buffer and centrifuged twice to remove the medium.
To 1 g of the obtained cells, 4 mL of lysis buffer (25 mM Hepes pH 8.0, 50 mM NaCl, 2 mM EDTA, 1 mM DTT) was added and suspended. Furthermore, 5 mL of a lysis buffer containing 1% Triton X-100 and a proteolytic enzyme inhibitor (PMSF, TPCK, TLCK, benzamidine, and leupeptin) was added per 1 g of the microbial cells and allowed to stand on ice for 5 minutes.
This was centrifuged at 18,000 rpm for 30 minutes, the supernatant was discarded, and the obtained cells were suspended in 2 mL of buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM DTT) per 1 g.
This was centrifuged at 18,000 rpm for 20 minutes, the supernatant was discarded, and the resulting cells were suspended in 2 mL of buffer (10 mM Hepes, 300 mM NaCl, 5 mM MgCl ₂ , 4 mM ATP, 1 mM DTT) per gram, Immediately, the mixture was centrifuged at 55,000 rpm for 15 minutes.
To the resulting supernatant, 5 μg / mL RNase A was added and dialyzed overnight against buffer (10 mM Pipes pH 6.8, 40 mM NaCl, 10 mM MgCl ₂ , 0.1 mM DTT). The resulting precipitate was collected by centrifugation at 18,000 rpm for 30 minutes, and this was homogenized in a buffer (10 mM Hepes pH 7.4, 200 mM NaCl, 3 mM MgCl2, 2 mM ATP, 1 mM DTT). Immediately after centrifugation at 55,000 rpm for 10 minutes, the insoluble fraction was removed to obtain a probe fraction for microscopic analysis.
The purity of the obtained fraction was assayed by SDS-PAGE and found to be about 90%.
The concentration of the probe for microscopic analysis was 10 mg / mL as measured by Advanced Protein Assay Reagent (manufactured by Cytoskeleton).

(3) Exercise assay The probe for microscopic analysis integrated with myosin obtained in (2) above was used as 1 mg / mL F-actin, 4 mM ATP, 10 mM imidazole (pH 7.4), 250 mM KCl, 4 mM MgCl ₂ , 1 mM DTT. Then, the mixture was immediately centrifuged at 68,000 rpm for 6 minutes to remove molecules that did not dissociate from F-actin even in the presence of ATP, and then subjected to an actin sliding movement type movement assay.
The motility assay was performed according to the method of Uyeda et al. (Uyeda et al., 1996). The motility assay comprises a buffer consisting of 0.1 mg / mL glucose oxidase, 0.02 mg / mL catalase, 3 mg / mL glucose containing 10 mM imidazole (pH 7.4), 25 mM KCl, 4 mM MgCl2, 1 mM ATP, 1 mM DTT. Used and performed under temperature conditions of 25 degrees.

  In the kinematic assay, F-actin on the cover glass covered with myosin expressed integrally with the probe for microscopic analysis exhibits normal sliding movement at a speed not greatly different from that when wild type myosin is used. I woke up. From this, it has been clarified that the addition of the rod-shaped structure has a small influence on the function of myosin, which is the object of analysis, and the probe for microscopic analysis of the present invention is suitable for the analysis of functional proteins. It was.

(Example 3)
The myosin with a probe for microscopic analysis prepared in Example 2 was observed with a transmission electron microscope by the following method.
10 mg / ml of myosin with a probe for microscopic analysis was added to 0.4 M ammonium acetate / 40% glycerol solution and mixed well to prepare a diluted solution of 15 μg / mL. The obtained diluted solution (5 μL) was sucked into a plastic pipette tip, and directly inserted into a small hole opened at the tip of a spray paint brush (spray gun).
Place a target slide glass 30 cm in front of the paint brush, and prepare a piece of mica with a double-sided tape with a fresh cleaved surface facing forward in the center of the paint brush. The tube was connected to a nitrogen cylinder, and the myosin dilution with the microscope analysis probe was sprayed at once.

  After confirming the distribution of fine water droplets on the mica surface with a stereomicroscope, the slide glass was inserted into a freeze etching apparatus (Balzers 300 type) and waited for the vacuum level to rise. After 1-2 hours, platinum / carbon rotary shadowing was applied from a height of 10 ° C., and coating with pure carbon was performed thereon. The carbon film on the mica surface was peeled off using the surface tension of the water surface, and the film was floated on the water surface.

  The carbon film was scooped up with a copper grid for electron microscope (# 150 mesh), inserted into a transmission electron microscope (JEM-2000ES), and observed and photographed at an acceleration voltage of 80 kV. The results are shown in FIG.

As shown in FIG. 3, the probe for microscopic analysis of Example 2 in which the spherical portions of the myosin head are integrated is expressed two by two along with the expression form of the myosin molecule having two spherical portions. . As is apparent from FIG. 3, the microscope analysis probe of the present invention is sufficiently thin, and all of them grow from the middle position of the myosin head. It was revealed that the high resolution of the microscope can be maintained.
In the construction of the expression vector for the microscopic analysis probe, the rod-shaped cDNA was linked to the N-terminus of the inserted myosin molecule, so that the end of the rod-shaped structure in the expressed microscopic analysis probe was the myosin. It was clarified that the contact portion with the N-terminus of the molecule was constituted and was visible under a microscope, and the position and orientation of the N-terminus of the myosin molecule in the three-dimensional structure could be clearly analyzed. It was also confirmed that the N-terminal position of the myosin molecule was consistent with the localized position of the site in the atomic model.

Example 4
An atomic force microscope (model number: model number) was prepared by diluting the probe for microscope analysis prepared in Example 2 with an assay solution (250 mM KCl, 25 mM HEPES, 4 mM MgCl ₂ , 1 mM DTT) at 4 μg / mL. NVB500 (manufactured by Olympus Corporation) was used to analyze the behavior of the myosin molecule. The results are shown in FIG.

  The probe for microscopic analysis of the present invention can label and analyze an analysis object without impairing the spatial resolution of the microscope to be used. The localization, direction (orientation), shape (three-dimensional structure), and dynamics of the analysis object Therefore, the microscope analysis method of the present invention using the microscope analysis probe is unlikely to be a target of a conventional structural analysis method. It is suitable for analysis of biological samples, analysis of samples with structural changes over time, and the like.

FIG. 1 is a schematic view showing the probe expression vector for microscope analysis (pGEX-SMP) of the present invention constructed in Example 1. FIG. FIG. 2A shows a microscope analysis probe suitable for the case where the microscope analysis target is the first aspect that is an external detection target detected by interaction with the contact portion of the microscope analysis probe. FIG. 2B is a schematic diagram (probe for microscopic analysis of Example 1), and FIG. 2B is a microscopic analysis suitable for the case where the microscopic analysis target is the second aspect integrated with the microscopic analysis probe. It is a schematic diagram of a probe (probe for microscope analysis of Example 2). FIG. 3 shows an electron microscope image obtained by observing the probe for microscopic analysis of the present invention prepared in Example 2 in Example 3. A transmission electron micrograph is shown on the left, a schematic diagram explaining the position of the probe for microscopic analysis in the left photo on the right, and the position of the N-terminal site in the atomic model of the myosin head is shown below it. . The sign indicates the exact position. FIG. 4 shows the behavior of the probe for microscopic analysis of the present invention prepared in Example 2, analyzed by an atomic force microscope, and still images extracted from moving images taken every about 80 milliseconds are arranged in time series. FIG.

Explanation of symbols

10 Probe for microscope analysis 20 Bar-shaped structure 21 Contact part (contact area)
22 Contact part (contact end)
30 Microscope analysis target 40 Labeling part

Claims (19)

  A probe for microscopic analysis, which has a rod-like structure, and the rod-like structure has a contact portion that can come into contact with a specific portion to be analyzed.   The probe for microscopic analysis according to claim 1, wherein the contact portion is disposed at one end of the rod-like structure, and a labeling portion is disposed at the other end of the rod-like structure.   The probe for microscopic analysis according to claim 1, wherein at least the rod-like structure is made of protein.   The probe for microscopic analysis according to claim 3, wherein the rod-like structure is a protein having an antiparallel coiled coil structure.   The probe for microscopic analysis according to any one of claims 3 to 4, comprising a single molecule recombinant fusion protein.   The probe for microscopic analysis according to any one of claims 1 to 5, wherein the contact portion is made of a substance capable of interacting with a specific part to be microscopically analyzed.   The probe for microscopic analysis according to claim 6, wherein the interaction is an antigen-antibody reaction.   The probe for microscopic analysis according to any one of claims 1 to 5, wherein the contact portion is configured in a state of being coupled to a specific part to be microscopically analyzed.   The probe for microscopic analysis according to claim 8, wherein the probe for microscopic analysis is expressed integrally with a target for microscopic analysis.   The method is used for preparing a sample for microscopic analysis by at least one selected from a quick-frozen deep-etch replica method, a negative staining method, a cryo-negative staining method, a resin-embedded section method, and an ice-embedded method. The probe for microscopic analysis according to any one of 9 above.   The probe for microscopic analysis according to any one of claims 1 to 10, which is used for analysis by at least one selected from a fluorescence microscope, a transmission electron microscope, a scanning electron microscope, a scanning probe microscope, and an atomic force microscope. .   A probe expression vector for microscopic analysis, comprising at least a gene encoding the probe for microscopic analysis according to any one of claims 3 to 11.   The probe expression vector for microscopic analysis according to claim 12, which is used by being introduced into a cell expressing a target for microscopic analysis.   A vector for expressing the probe for microscopic analysis according to any one of claims 9 to 11, comprising at least a specific region to be analyzed and a gene encoding the probe for microscopic analysis, Probe expression vector.   The probe expression vector for microscopic analysis according to claim 14, which is used after being introduced into a knockout cell from which a gene encoding a target for microscopic analysis has been deleted.   A method for preparing a microscope analysis sample including the probe for microscope analysis according to claim 1, and a step for detecting the probe for microscope analysis in the sample for microscope analysis. Microscopic analysis method. (Vector use)
A step of expressing the microscope analysis vector according to any one of claims 12 to 15, a step of preparing a microscope analysis sample including the microscope analysis probe, and the microscope analysis probe in the microscope analysis sample A microscopic analysis method, comprising:   The sample for microscopic analysis is prepared by at least one selected from a rapid freezing deep etch replica method, a negative staining method, a cryonegative staining method, a resin embedding section method, and an ice embedding method. The microscope analysis method according to any one of the above. The microscope analysis is performed using at least one selected from a fluorescence microscope, a transmission electron microscope, a scanning electron microscope, a scanning probe microscope, and an atomic force microscope. Microscope analysis method.