WO2010004736A1 - Biocapteur utilisant l'adn en tant qu’élément - Google Patents

Biocapteur utilisant l'adn en tant qu’élément Download PDF

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WO2010004736A1
WO2010004736A1 PCT/JP2009/003168 JP2009003168W WO2010004736A1 WO 2010004736 A1 WO2010004736 A1 WO 2010004736A1 JP 2009003168 W JP2009003168 W JP 2009003168W WO 2010004736 A1 WO2010004736 A1 WO 2010004736A1
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protein
dna
arsr
gfp
sensor
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PCT/JP2009/003168
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English (en)
Japanese (ja)
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前田勇
井上浩一
川上泰生
宮坂均
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国立大学法人宇都宮大学
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Priority to CN2009801259561A priority Critical patent/CN102083978A/zh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a method for detecting and / or quantifying an analyte in a test sample by using a sensor protein that specifically binds to the analyte and a nucleic acid specifically recognized by the sensor protein. .
  • a biosensor that uses a test molecule such as an enzyme, antibody, or receptor derived from a living organism that identifies a detection target substance as a sensor element is also attracting attention as one of such simple detection methods.
  • the detection reaction is based on biochemical reaction and binding specificity, so high-sensitivity measurement is possible, measurement and detection is quick and simple, the running cost is low, and the sensor itself is small and excellent in portability.
  • biochemical reactions such as enzyme reactions, antigen-antibody reactions, and ligand binding to receptors enables analysis with high specificity to specific substances. It is possible to reduce the size.
  • a recombinant microbial biosensor consists of three elements: a host cell, an inducible promoter that promotes transcription to a downstream gene in response to a specific substance, and a reporter gene downstream of the promoter, and optionally detects the signal emitted by the reporter
  • a system combining devices is used.
  • luciferase derived from bacteria and fireflies see Non-Patent Documents 1 to 3
  • GFP derived from Echinella jellyfish see Non-Patent Documents 2 and 4
  • LacZ derived from E. coli non-patent documents
  • Rhodobram sulphidophilum has a spheroidene pathway, a kind of carotenoid synthesis system, and originally accumulates the final product spheroidenone and is a red bacterium.
  • this sensor uses a crtA-deficient strain obtained by destroying crtA encoding spheroidene monooxygenase (CrtA) that catalyzes the final step of the spheroidene pathway, it accumulates the spheroidene precursor of spheroidene. It is yellow. Therefore, CrtA activity is restored in the presence of arsenic by introducing a plasmid containing crtA as a reporter gene and incorporating an arsenic-inducible promoter upstream thereof into Rhodobram sulphidophilum, so that the color changes from yellow to red. Color change occurs. The presence of arsenic can be determined using such a change in color tone as an index.
  • Rhodopseudomonas pultris which is a freshwater red bacterium that can be cultured under anaerobic conditions
  • Rhodopseudomonas pultris phytoene-to-lycopene phytoene desaturase (CrtI) in the spiroloxanthin pathway possessed by Rhodopseudomonas pultris.
  • Rhodopseudomonas pultris which is a freshwater red bacterium that can be cultured under anaerobic conditions
  • Rhodopseudomonas pultris phytoene-to-lycopene phytoene desaturase
  • a plasmid incorporating a reporter gene crtI downstream of an arsenic-inducible operator / promoter region derived from E. coli was introduced into Rhodopseudomonas pulsetris, and CrtI activity was carried out in the presence of arsenic.
  • Biosensors using microorganisms such as those described above have problems such as the need for culturing equipment and time, and large variations among cells. To solve these problems, cell-free biotechnology is required. Sensor development is required.
  • An object of the present invention is to detect and / or quantify an analyte in a test sample using a sensor protein that specifically binds to the analyte and a nucleic acid that is specifically recognized by the sensor protein. It is to provide an excellent method in terms of cost, operability, and speed.
  • FRET Fluorescence, Resonance, Energy, Transfer
  • the excitation energy excites the acceptor before light emission from the donor occurs, and the light emission can be detected. Since FRET is more likely to occur as the distance is shorter, it is used as a means for detecting interaction between molecules and structural change of molecules.
  • E. coli has a gene zntA that encodes an enzyme that functions to discharge heavy metals out of the cell.
  • the base sequence of promoter region DNA (hereinafter referred to as promoter DNA) exists in the region of ⁇ 10 bases and ⁇ 35 bases upstream from the transcription start point of zntA, and lead and cadmium sensor protein ZntR binds to this promoter DNA.
  • promoter DNA exists in the region of ⁇ 10 bases and ⁇ 35 bases upstream from the transcription start point of zntA, and lead and cadmium sensor protein ZntR binds to this promoter DNA.
  • a ZntR protein-promoter DNA complex is formed. In the absence of heavy metals such as lead and cadmium compounds, the promoter DNA is bent as a higher-order structure.
  • FIG. 25 shows the principle devised for externally presenting this higher-order structural change due to the presence or absence of heavy metals using FRET.
  • FITC fluorescein isothiocyanate
  • TAMRA tetramethylrhodamine
  • Escherichia coli has an arsenic resistance operon that gives cells arsenic resistance.
  • the expression of the arsenic resistance operon is controlled by the arsenic sensor protein ArsR encoded by the gene arsR.
  • ArsR protein binds to promoter DNA in the absence of an arsenic compound to form an ArsR protein-promoter DNA complex. Therefore, RNA polymerase cannot bind to promoter DNA, and transcription of the arsenic resistance operon is suppressed.
  • TAMRA is bound to one end of the promoter DNA.
  • an ArsR-GFP fusion protein in which a green fluorescent protein (Green) Fluorescent Protein (GFP) was fused to the ArsR protein was used to fluorescently modify the ArsR protein. At this time, it was thought that TAMRA absorbed and excited the fluorescence emitted by GFP by FRET.
  • Green green fluorescent protein
  • the promoter DNA and the ArsR-GFP fusion protein are in a combined state, and TAMRA and GFP are close to each other, so TAMRA is excited by the fluorescence of GFP emitted when irradiated with GFP excitation light. Then, mainly TAMRA fluorescence is detected.
  • the promoter DNA chain and the ArsR-GFP fusion protein are in a dissociated state, so that the average distance between TAMRA and GFP becomes long, and TAMRA hardly absorbs GFP fluorescence. Therefore, TAMRA is difficult to excite and GFP fluorescence is mainly detected. It was considered that the arsenic compound can be detected or quantified by measuring the change in the fluorescence wavelength due to the presence or absence of the arsenic compound with a fluorometer.
  • the FRET method cannot solve the problem of the present invention. Accordingly, the present inventors are forced to develop different approaches, and pay attention to ArsR protein, which is a sensor protein that binds to arsenic, and CadC protein, which is a sensor protein that binds to cadmium and lead. ArsR-GFP and CadC-GFP fused with fluorescent protein (GFP) were prepared. It was confirmed that these fusion proteins bind to each specific recognition sequence DNA and that their binding is inhibited by metal ions.
  • ArsR protein which is a sensor protein that binds to arsenic
  • CadC protein which is a sensor protein that binds to cadmium and lead.
  • ArsR-GFP and CadC-GFP fused with fluorescent protein (GFP) were prepared. It was confirmed that these fusion proteins bind to each specific recognition sequence DNA and that their binding is inhibited by metal ions.
  • the present invention (1) A method for detecting or quantifying an analyte in a test sample in an aqueous system, comprising the following steps (A) and (B): (A) Specific binding to the analyte Reacting a sensor protein with a nucleic acid immobilized on a support containing a sequence specifically recognized by the sensor protein in the presence of a test sample; (B) detecting or measuring a sensor protein bound to the immobilized nucleic acid; (2) The method according to (1) above, wherein the sensor protein is labeled with a detectable marker, (3) The method according to (1) above, wherein the sensor protein is a fusion protein with a detectable marker protein, (4) A method for detecting or quantifying an analyte in a test sample in an aqueous system, comprising the following steps (A) and (B): (A) The above-mentioned solid-phased on a support Reacting a sensor protein that specifically binds to an analyte and a
  • the present invention also provides (6) The method according to any one of (1) to (5) above, wherein the binding between the sensor protein and the nucleic acid is inhibited by the analyte, (7) The method according to any one of (1) to (6) above, wherein the sensor protein is a protein encoded by a metal responsive operon, (8) The method according to (7) above, wherein the protein encoded by the metal responsive operon is ArsR protein or CadC protein, (9) The method according to any one of (1) to (8) above, wherein the analyte is a heavy metal compound, (10) The method according to (9) above, wherein the heavy metal compound is selected from arsenic (As) compounds, cadmium (Cd) compounds, and lead (Pb) compounds, (11) The method according to (10) above, wherein the pentavalent arsenic (As (V)) compound is reduced to a trivalent arsenic (As (III)) compound in advance, (12) The above (9) to (9), wherein the reaction between
  • the present invention provides (14) A test protein comprising: a sensor protein that specifically binds to an analyte; and a nucleic acid immobilized on a support that includes a sequence that is specifically recognized by the sensor protein.
  • a kit for detecting or quantifying an analyte in a sample (15) A test sample comprising a sensor protein that specifically binds to an analyte immobilized on a support, and a nucleic acid containing a sequence that is specifically recognized by the sensor protein.
  • the present invention relates to a kit for detection or quantification of an analyte therein.
  • the embodiment of the present invention is characterized in that the sensor protein is labeled with a detectable marker, or the sensor protein is a fusion protein with a detectable marker protein.
  • the kit characterized in that the nucleic acid is labeled with a detectable marker.
  • any of the kits described above wherein the binding between the sensor protein and the nucleic acid is inhibited by the analyte, or the sensor protein is encoded by a metal responsive operon.
  • the kit is characterized by being a protein, or the kit encoded by a metal-responsive operon is an ArsR protein or a CadC protein, or the analyte is arsenic (As ), Cadmium (Cd), lead (Pb), and other heavy metal compounds.
  • FIG. 4 is a diagram showing the results of analyzing the protein after addition to Pars -DNA-immobilized wells by electrophoresis. Lane 1, fraction obtained by adding the crude protein extract to the well and removing the extract after the reaction and washing the well, then recovering the bound protein; Lane 2, adding the protein crude extract to the well and extracting after the reaction The fraction from which the solution was removed and the well was washed, and then the protein was collected; Lane 3, protein extract; Lane 4, molecular weight marker.
  • FIG. 6 is a diagram showing the results of detection of arsenous acid by a biosensor using Pars -DNA as an element. * Indicates P ⁇ 0.05 and ** indicates a significant difference of P ⁇ 0.005. It is a figure which shows the change of the Pars-ArsR-GFP complex amount by washing
  • R 2 0.95.
  • the method for detecting or quantifying the analyte in the test sample of the present invention includes [1] a sensor protein that specifically binds to the analyte in the test sample, and a sequence that is specifically recognized by the sensor protein.
  • a nucleic acid immobilized on a support containing a nucleic acid is reacted in the presence of a test sample, and a sensor protein bound to the immobilized nucleic acid is detected and / or measured in an aqueous system (in the presence of water).
  • a sensor protein that is immobilized on a support and specifically binds to the analyte, and a nucleic acid containing a sequence that is specifically recognized by the sensor protein
  • the method is not particularly limited as long as it is a method for detecting and / or measuring a nucleic acid bound to a solid-phased sensor protein in the presence of an aqueous solution. In the method of [1] above, detection is possible.
  • the sensor protein that did not bind to the immobilized nucleic acid is discharged out of the system, so that the immobilized nucleic acid
  • the bound sensor protein can be detected and / or measured in an aqueous system.
  • a nucleic acid labeled with a detectable marker is used to bind to the immobilized sensor protein.
  • the test sample is not particularly limited, but preferable examples include river water containing heavy metals, sewage, well water, industrial effluent, and contaminated soil. In the case of solid matter such as contaminated soil, Liquid or water extract can be used.
  • the kit for detecting or quantifying an analyte in a test sample of the present invention includes [3] a sensor protein that specifically binds to the analyte, and a sequence that is specifically recognized by the sensor protein.
  • a kit for detection or quantification of an analyte in a test sample characterized by comprising a solid phase immobilized nucleic acid on a support, and [4] binding specifically to the analyte and fixing to the support.
  • kits for detecting or quantifying an analyte in a test sample characterized by comprising a phased sensor protein and a nucleic acid containing a sequence specifically recognized by the sensor protein
  • a sensor protein labeled with a detectable marker or a sensor protein fused with a detectable marker protein is used.
  • the sensor protein bound to the phased nucleic acid can be detected / measured, and in the kit of [4] above, the sensor protein immobilized by using a nucleic acid labeled with a detectable marker The nucleic acid bound to can be detected / measured.
  • the sensor protein used in the method for detecting and / or quantifying the analyte in the test sample of the present invention is a protein that can specifically bind to a nucleic acid containing a predetermined sequence, and depends on the predetermined analyte.
  • a sensor protein encoded by a metal-responsive operon can be cited as a good example, as long as it is a protein that inhibits binding to the nucleic acid.
  • the metal response operon is not particularly limited, and specifically, ars encoding ArsR protein, czc encoding CzcR, cad encoding CadC, chr encoding ChrB, Examples include mer that encodes MerR1 and Merer2, pbr that encodes pbrR, and the like, and among them, ars and cad can be preferably indicated.
  • Each sensor protein encoded by the metal responsive operon recognizes a specific metal responsive operon region DNA sequence, but in the presence of a specific metal ion, these sensor proteins bind to the metal ion and have a higher order structure. By changing, it becomes impossible to bind to DNA.
  • the ArsR protein binds to ars region DNA, but the binding is inhibited by arsenic.
  • the CadC protein binds to cad region DNA, but the binding is inhibited by cadmium and lead.
  • the sensor protein is preferably labeled with a detectable marker, and as the detectable marker,
  • the marker is not particularly limited as long as it is a conventionally known marker for peptide labeling.
  • radioactive isotopes such as 32 P, 3 H, 14 C, 125 I, FITC, TAMRA, Cyber Green, dansyl chloride
  • fluorescent substances such as tetramethylrhodamine isothiocyanate, biologically relevant binding structures such as biotin and digoxigenin, bioluminescent compounds, and chemiluminescent compounds.
  • the sensor protein may be a fusion protein fused with a detectable marker protein.
  • the marker protein include enzymes such as alkaline phosphatase and HRP, antibody Fc regions, and fluorescent substances such as GFP.
  • Specific examples include marker proteins such as MBP, binding proteins having binding ability such as MBP, lectin, and avidin, and epitope tag proteins such as HA, FLAG, and Myc.
  • the fusion protein ARSR-GFP the ARSR protein and GFP, as well as capable of binding P ars-DNA are the recognition sequences ARSR protein, that the binding is inhibited in a concentration-dependent manner by arsenite Therefore, it can be used as an excellent arsenite biosensor protein in the method for detecting and / or quantifying arsenite of the present invention.
  • the nucleic acid containing a sequence specifically recognized by the sensor protein is labeled with a detectable marker.
  • the detectable marker is not particularly limited as long as it is a conventionally known marker for nucleic acid labeling, for example, radioactive such as 32 P, 3 H, 14 C, 125 I, etc. Isotopes, fluorescent materials such as FITC, TAMRA, Cyber Green, dansyl chloride, tetramethylrhodamine isothiocyanate, biologically relevant binding structures such as biotin and digoxigenin, bioluminescent compounds, chemiluminescent compounds, etc. Can be specifically mentioned.
  • the support for immobilizing the sensor protein or nucleic acid is not particularly limited as long as it is a known support. Instead, specifically, plastic (polystyrene, polyamide, polyethylene, polypropylene, etc.), agarose, cellulose, hydrophilic polyvinyl alcohol, acrylate polymer, polyacrylamide glass, metal, and the like can be exemplified.
  • the method for immobilizing the sensor protein or nucleic acid on these supports is not particularly limited as long as it is a known method.
  • a covalent bonding method such as a carrier bonding method using a cross-linking reagent can be employed, and a physical adsorption method is particularly preferable.
  • the sensor protein or nucleic acid is immobilized on the support, it may be indirectly immobilized via another substance.
  • nucleic acid can be immobilized on a support by labeling the nucleic acid with biotin and binding biotin and the support (plastic plate) by physical adsorption. it can.
  • a biosensor for detecting arsenic, lead / cadmium using bacterial promoter region DNA and sensor protein as elements.
  • This sensor is a cell-free sensor that directly captures changes that occur in bacterial transcriptional switches. Green fluorescence changes between the two states, binding and dissociation, that occur between the sensor protein that constitutes the transcriptional switch and the promoter region DNA. It is characterized by capturing using fluorescence of protein (GFP) or the like. More specific description will be given below.
  • a sensor element is immobilized in a well of a 96-well microplate in order to make the sensor part a replaceable / detachable module.
  • the DNA can be immobilized by reacting a promoter region DNA labeled with biotin at the 3 'end in a 96-well microplate on which streptavidin is immobilized.
  • a washing operation is performed after reacting the sensor protein-GFP solution, and the fluorescence intensity of the sensor protein-GFP that maintains a binding state with the promoter region DNA in the well.
  • E. coli strains that produce sensor proteins ArsR-GFP and CadC-GFP, respectively are bred to confirm that these recombinant proteins bind to promoter region DNA and heavy metals
  • ArsR-GFP In biosensor development using ArsR-GFP, it has been clarified that the fluorescence intensity decreases when ArsR-GFP is mixed with a sample containing As (III).
  • conditions for stabilizing the binding between the promoter region DNA and the sensor protein-GFP, or conditions for conspicuous changes in the state of binding to the complex and dissociation of the complex were examined.
  • CadC-GFP it was clarified that the fluorescence intensity decreased by mixing CdC-GFP with a sample containing Pb (II) or Cd (II).
  • the detection test procedure it is important to mix the crude protein extract containing the sensor protein-GFP with the sample, salmon sperm DNA, and buffer and incubate at room temperature or on ice for 30 minutes. It was shown that there is. Although a method of adding the sensor protein-GFP to the well and first forming the DNA-sensor protein-GFP complex and then adding the sample to measure the degree of protein dissociation was considered, this procedure is effective. Didn't work. In addition, it was shown that the presence of 40 mM or more NaCl during the 30-minute incubation is important in making the fluorescence intensity variable noticeable during subsequent measurements.
  • phosphate buffer is suitable for ArsR-GFP and trishydroxymethylaminomethane buffer is suitable for CadC-GFP. confirmed.
  • the lower limit of detection was 5 ⁇ g / L for arsenic, 10 ⁇ g / L for lead, and 1 ⁇ g / L for cadmium, indicating that detection is possible below the drinking water reference value set by the World Health Organization. Depending on future research, higher sensitivity may be achieved.
  • the time required for detection is expected to be completed within 40 minutes, including the initial 15 minute incubation of the sample and protein extract. Both ArsR-GFP and CadC-GFP did not show cross-responsiveness to other metals of 100 ⁇ M, and were found to selectively respond to the target metal.
  • a 96-well microplate modified with streptavidin (Reacti-Bind Streptavidin High Binding Capacity Coated 96-Well Plates: PIERCE) was adopted as the substrate.
  • the fluorescence intensity was measured using a corona fluorescence microplate reader MTP-601Lab (Hitachi High-Technologies Corporation). The measurement conditions were 490 nm for the excitation filter and 530 nm for the fluorescence measurement filter, and the detection sensitivity was “AUTO”.
  • ParsR-50-S-3-B (SEQ ID NO: 1 shows the ParsR-50-S sequence) shown in Table 1 and ParsR-50-A (SEQ ID NO: 2) are mixed.
  • a double-stranded DNA labeled with biotin at the 3 ′ end downstream of the promoter sequence (indicated as Pars-biotin) was formed.
  • Pars-biotin was prepared to a concentration of 25 pmol / 100 ⁇ L and used for immobilization.
  • the cadA gene encoding the extracellular efflux pump of lead and cadmium and the cadC gene encoding the CadC sensor protein form an operon in the order of cadC-cadA, and its transcription is controlled by the CadC protein.
  • a CadC-GFP fusion protein and a promoter region DNA (P cad -DNA) consisting of 34 base pairs centered on the CadC protein binding site (operator sequence) upstream of the CadC gene were selected. .
  • PcadC34-S-3-B shown in Table 2 SEQ ID NO: 3 shows the PcadC34-S sequence
  • PcadC34-A SEQ ID NO: 4
  • a double-stranded oligonucleotide labeled with biotin at the end was formed.
  • Pcad34-biotin was prepared and immobilized so as to have a concentration of 20 pmol / 100 ⁇ L.
  • LB medium was used for culturing the transformant, and if necessary, ampicillin (Amp) and chloramphenicol (Cm) were added to final concentrations of 50 ⁇ g / mL and 34 ⁇ g / mL, respectively.
  • Plasmid DNA For cloning DNA fragments amplified by PCR, pGEM-T vector plasmid DNA (manufactured by Promega) is used, and for preparing an expression unit for recombinant protein production, pET-3a plasmid DNA (manufactured by Novagen) is used. Each was used.
  • Recombinant protein production conditions and preparation method BL21 (DE3) pLysS transformant for expression of recombinant protein was cultured at 37 ° C overnight in 5 mL of medium supplemented with Amp and Cm (Amp + Cm + LB). After culturing, the obtained overnight culture solution was inoculated into 0.5 mL of Amp + Cm + LB. This was cultured at 37 ° C. for about 12 hours without adding isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the cells were collected and washed twice with 50 mM Tris-HCl buffer (Tris-HCl, pH 7.4), and then 4 mL of cell disruption buffer (Tris-HCl containing 15% glycerol). And freeze-thawed once at -80 ° C. After sonication, the mixture was centrifuged at 15000 rpm for 15 minutes, and the resulting supernatant was used as a recombinant protein crude extract. This crude protein extract was stored frozen at ⁇ 80 ° C., and thawed before use.
  • Tris-HCl buffer Tris-HCl, pH 7.4
  • cell disruption buffer Tris-HCl containing 15% glycerol
  • the crude protein extract and arsenous acid solution were mixed in the composition shown in Table 5 and incubated on ice for 2 hours. Thereafter, the mixed solution was added to the plate and incubated at room temperature for 1 hour. The supernatant was removed, washed three times with 200 ⁇ L of washing buffer (10 mM PBS containing 0.05% Tween 20, pH 6.0), and finally 150 ⁇ L of 50 mM Tris-HCL (pH 7.9) was added, After 10 minutes, the fluorescence intensity was measured with a Fluorescent Microplate Reader MTP-601Lab (Corona-Hitachi High-Technologies Corporation). In this case, a bandpass filter having a wavelength of 492 nm on the excitation light side and a wavelength of 530 nm on the detector side was used.
  • pGEMarsRgfp was obtained by inserting and cloning arsRgfp into pGEM-T vector plasmid DNA (FIG. 4).
  • the arsRgfp excised from the plasmid DNA by digesting pGEMarsRgfp with NdeI was separated and recovered by agarose gel electrophoresis.
  • pET-3a was digested with Nde1 and joined to the collected arsRgfp to obtain pETarsRgfp.
  • An ArsR-GFP fusion protein-producing strain was bred by transforming E. coli BL21 (DE3) pLysS with pETarsRgfp.
  • a crude extract was prepared by concentrating an amount corresponding to 40 mL of the above-described crude protein extract to 5 mL.
  • the crude protein extract and arsenous acid were mixed at a ratio of 5:95, and the fluorescence intensity was measured after 3 hours according to the procedure described in [Experimental materials and methods] of Example 2.
  • a significant decrease in fluorescence intensity was observed with the addition of arsenous acid, and the decrease width was observed to be larger at 100 ⁇ g / L than at 50 ⁇ g / L. It was shown that a biosensor for detection of arsenite using DNA using a microplate as an element can detect 50 ⁇ g / L arsenite in principle (FIG. 8).
  • Fluorescent proteins such as GFP are known to have a property that the fluorescence intensity changes depending on the pH.
  • GFP it is known that the fluorescence intensity increases as the pH increases near neutrality.
  • the fluorescence intensity of the 1st to 3rd washings with PB-T was measured under a weakly acidic pH of 6.0, and thus obtained by washing with TBS-T under a weakly basic pH of 7.4. It is conceivable that it is lower than the fluorescence intensity. A decrease in fluorescence intensity was observed with each washing of both buffers, but after the fourth washing operation, TBS-T with pH 7.4 was added to adjust the fluorescence intensity to 7.4.
  • the sensor protein ArsR has different affinity for trivalent arsenic As (III) and pentavalent arsenic As (V), which are inorganic arsenic, respectively, and As (III ) Is known to have a high affinity.
  • the arsenic indicated by the drinking water reference value does not distinguish between arsenous acid and arsenic acid, and the arsenic of As (III) and As (V) is equivalent in sensitivity with a sensor using ArsR-GFP as an element. It is preferable to detect. Therefore, it was investigated whether As (V) can be detected as equal to As (III) by reducing As (V) to As (III). In the test, sodium arsenate was used as As (V), sodium arsenite as As (III), and sodium thiosulfate as the reducing agent.
  • the sample reduction treatment was performed according to the procedure shown in FIG. 20.0 ⁇ L of 2N hydrochloric acid solution was added to 450 ⁇ L of the sample to bring the pH to around 1. Next, 1.0 ⁇ L of a 250 mM sodium thiosulfate aqueous solution prepared for use was added, followed by incubation at room temperature for 10 minutes. Ten minutes later, 16.0 ⁇ L of 2.5N sodium hydroxide solution was added for neutralization. The pH was confirmed using a pH test paper.
  • a fluorescence measurement buffer solution 50 mM Tris-HCl pH 7.9, 1 M NaCl, 0.1% (w / v) Tween-20 was added to the well, and fluorescence measurement was performed.
  • the developed arsenic sensor ArsR-GFP showed no change in fluorescence intensity even at 100 ⁇ g / L As (V), confirming that ArsR-GFP is extremely insensitive to As (V). It was. However, the reduction treatment with sodium thiosulfate showed a change in fluorescence intensity equivalent to that of As (III). Therefore, the sensor was connected to As (V) through the reduction from As (V) to As (III). Was also found to be detected in the same manner as As (III). Although not shown in the data, it has become clear that As (V) cannot be detected unless the sodium thiosulfate treatment is performed after setting the pH to a strongly acidic range in the reduction treatment. Therefore, it is considered essential to add an acid such as hydrochloric acid in the reduction treatment.
  • Reduction treatment was performed by sequentially adding 1.0 ⁇ L of sodium thiosulfate and 16.0 ⁇ L of 2.5N sodium hydroxide. Thereafter, 25.0 ⁇ L of the buffer solution shown in Table 7 was added, and the influence of the pH condition of the sample was examined. In order to increase the buffer capacity of the buffer, the concentration was 1 M (final concentration 50 mM).
  • the phosphate buffer solution having a high buffer capacity of about 1M is used to bring the pH to around 7.4 and the final phosphate buffer concentration to 50 mM or more. It is deemed appropriate to perform an arsenic detection test after adjustment.
  • CadC-GFP fusion protein producing Escherichia coli strain
  • a CadC-GFP fusion protein was produced in the same manner as the ArsR-GFP fusion protein described in Example 3.
  • pGEMrsRgfp was digested with SphI and BamHI, and pGEMgfp was obtained by removing the excised fragment (FIG. 16).
  • PCR was performed using pI258 plasmid DNA as a template, and a DNA fragment containing cadC was amplified.
  • pGEMcadCgfp was obtained by digesting cadC with SphI and BamHI and joining with pGEMgfp.
  • the cadCgfp excised from the plasmid DNA by digesting pGEMcadCgfp with NdeI was separated and recovered by agarose gel electrophoresis.
  • pETcadCgfp was obtained by digesting pET-3a with NdeI and ligating it with the recovered cadCgfp.
  • a CadC-GFP fusion protein-producing strain was bred by transforming E. coli BL21 (DE3) pLysS with pETcadCgfp.
  • Pcad34-biotin having a CadC binding sequence was immobilized on the E1 and E2 wells shown in FIG. No DNA strand was immobilized on the E3 well.
  • a crude protein extract containing 100 ⁇ L of CadC-GFP was added to the wells from E1 to E3, and shaken at room temperature for 2 hours. After 2 hours, excess protein crude extract was removed.
  • 100 ⁇ L of protein solubilization buffer (Table 8) was added to the well of E1, and after 30 minutes of shaking at room temperature, the buffer was recovered and loaded into lane 3 of electrophoresis (SDS-PAGE).
  • 200 ⁇ L of PB-T (Table 6) was added to and removed from wells E2 and E3, and washing was repeated twice.
  • CadC-GFP was concentrated in the remaining protein by contacting the protein crude extract with the well where Pcad34 was immobilized. It can be seen that it has been purified (lane 3). This is judged from the fact that the protein was recognized as the main band at the position of the molecular weight determined from the calculation of CadC-GFP. It can also be seen that washing of the wells further reduced the contaminating protein band and concentrated and purified CadC-GFP (lane 4). On the other hand, when Pcad34 is not immobilized on the well, it is understood that CadC-GFP was washed away by washing (lane 1).
  • this mixed solution was diluted with TG (50 mM trishydroxymethylaminomethane-HCl pH 7.4, 15% (v / v) glycerol) so that the volume ratio with respect to the total amount of the mixed solution became 0-100 ⁇ L / 100 ⁇ L. Then, this diluted sample was added to the Pcad34-immobilized well, and fluorescence measurement was performed (upper diagram in FIG. 21). Thereafter, the mixture was shaken at room temperature for 1 hour, and then the excess diluted sample was removed, followed by washing with 200 ⁇ L of PB-T (Table 6) twice for 5 seconds. After washing, 150 ⁇ L of 50 mM Tris-HCl, pH 7.9 was added to the well, and fluorescence measurement was performed (the lower diagram in FIG. 21).
  • TG 50 mM trishydroxymethylaminomethane-HCl pH 7.4, 15% (v / v) glycerol
  • the volume ratio of the solution added to the well was about 80 to 90 ⁇ L / 100 ⁇ L
  • the amount of CadC-GFP bound to Pcad34 reached saturation.
  • CadC-GFP is excessive with respect to 20 pmol of immobilized Pcad34 at a volume ratio of 80-90 ⁇ L / 100 ⁇ L or more.
  • the lead / cadmium sensor was constructed based on the CadC-GFP concentration in the crude protein extract used in this test.
  • the crude protein extract containing CadC-GFP also contained E. coli containing contaminating proteins. Since it is a E. coli cell lysate, it is difficult to accurately calculate the CadC-GFP concentration. Therefore, it was decided to simply determine the concentration of CadC-GFP based on the fluorescence intensity of the crude protein extract at pH 7.4.
  • CadC-GFP The major difference in comparing CadC-GFP and ArsR-GFP is that the binding to a certain amount of promoter region DNA strand is saturated if the fluorescence intensity of each protein solution is considered as the concentration of sensor protein-GFP. The amount required for this is different.
  • CadC-GFP it is necessary to add a crude protein extract so that the product of the fluorescence intensity and the liquid volume becomes larger. This may be due to the fact that the binding constants of each sensor protein-GFP and the promoter region DNA chain are different, or the number of bases of Pcad34 used is insufficient. Therefore, the influence of the number of bases of Pcad in forming the complex and the influence of the salt concentration were examined.
  • Pcad50-S-3-B shows the sequence of Pcad50-S
  • Pcad50-A shows in Table 9
  • the mixture was mixed to form a double-stranded oligonucleotide Pcad50-biotin labeled with biotin at the 3 ′ end of the promoter sequence.
  • Pcad34-biotin and Pcad50-biotin were prepared to 20 pmol / 100 ⁇ L and used for immobilization.
  • the crude protein extract containing CadC-GFP was prepared by suspending cells in 300 mL of the culture in 4 mL of TG2 (200 mM trishydroxymethylaminomethane-HCl, pH 7.4, 15% (v / v) glycerol) on ice.
  • TG2 200 mM trishydroxymethylaminomethane-HCl, pH 7.4, 15% (v / v) glycerol
  • UD-201 Tomy Seiko Co., Ltd.
  • This mixed solution was added to Pcad34 and Pcad50 fixed wells, and shaken at room temperature for 2 hours. After 2 hours, the mixed solution in the well was removed and washed once with 200 ⁇ L of PB-T (Table 6) for 5 seconds. After washing, 150 ⁇ L of a fluorescence measurement buffer was added to the well, and fluorescence measurement was performed. As a result, the fluorescence intensity was 2.10 in the Pcad34-immobilized well and 2.06 in the Pcad50-immobilized well. Even when Pcad50 with an increased CadC binding sequence at another site was immobilized, no change was observed in the binding amount of CadC-GFP.
  • the influence of the salt concentration on the formation of the complex was examined.
  • sodium chloride was added to adjust the salt concentration.
  • Pcad50-biotin was prepared and immobilized at 20 pmol / 100 ⁇ L in the well of the microplate.
  • the lead detection test in order to examine the influence of the salt concentration on the formation of the Pcad-CadC-GFP complex, first, a mixture of the sample and the crude protein extract was prepared with the composition shown in Table 10. In this case, under the sodium chloride addition condition, the lead (II) acetate solution: 4M sodium chloride becomes 99: 1 (v / v). Thereafter, a series of operations were performed in the same manner as in the previous detection tests, and the fluorescence intensity was measured.
  • the standard values of drinking water recommended by the World Health Organization are 10 ⁇ g / L for Pb (II) and 3 ⁇ g / L for Cd (II). Therefore, it was shown that the developed lead / cadmium sensor can detect lead at the reference value and cadmium below the reference value. Although not shown in the data, the sensors are cross-responsive to 1, 10, and 100 ⁇ M Ca (II), Mg (II), Fe (II), Mn (II), Fe (III), As (III). It became clear that almost no.
  • FRET fluorescence resonance energy transfer
  • the culture is centrifuged at 8000 rpm for 5 minutes to collect the cells, the supernatant is discarded, and freeze-thaw (freeze at ⁇ 80 ° C. for 30 minutes and thaw for 30 minutes at room temperature) three times to obtain an appropriate amount of Tris buffer A. Resuspended in (50 mM Tris-Cl, pH 8.0, 2 mM EDTA, 5 mM dithiothreitol) and incubated at room temperature for 10 minutes.
  • the crude extract of ZntR protein was obtained by centrifugation at 4 ° C., 12,000 rpm for 10 minutes, and then developed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the expression of the ZntR protein.
  • An Escherichia coli transformant for expression of ArsR protein was cultured overnight at 37 ° C. in 5 mL of a medium supplemented with Amp and Cm, and then inoculated into 250 mL of LB medium supplemented with Amp. This was cultured with shaking at 37 ° C. until the OD 600 was 0.8. Thereafter, IPTG was aseptically added to a final concentration of 1 mM, and cultured at 37 ° C. for 3 hours. The culture was centrifuged at 8000 rpm for 15 minutes to collect and discard the supernatant, and then resuspended in 30 mL of Tris buffer A and subjected to ultrasonic disruption to obtain an ArsR protein crude extract.
  • This ArsR protein crude extract was developed by SDS-PAGE to confirm the expression of ArsR protein.
  • DNase I treatment was performed. DNase I treatment was performed using RNase-free DNase I (TaKaRa), and the method was performed according to the product protocol.
  • the expression of the ArsR-GFP fusion protein was performed in the same manner as the expression of the ArsR protein. However, only Amp was added to the medium.
  • compositions of SDS-PAGE gel, electrophoresis buffer, and sample buffer are shown in Table 11, Table 12, and Table 13, respectively.
  • the protein solution and sample buffer were mixed 20 ⁇ L each to make 40 ⁇ L, and boiled in boiling water for 5 minutes was added to the gel and subjected to electrophoresis.
  • molecular weight markers Prestained Protein Marker and Broad Range (Bio Labs) were used.
  • the gel after electrophoresis is immersed in a staining solution in which 0.2% (w / v) Coomassie Brilliant Blue R-250 is dissolved in 40% (v / v) methanol-10% (v / v) acetic acid aqueous solution. Then, the sample was dyed by gently shaking for 30 minutes.
  • Decolorization was performed by gently immersing in 20% (v / v) methanol-5% (v / v) acetic acid aqueous solution (decolorizing solution). Decolorization was repeated by replacing with a new decolorizing solution until the gel was decolorized and the protein band was clear.
  • Tris buffer A was used as the buffer and elution buffer used for column equilibration.
  • affinity chromatography was performed for the purification of the ZntR protein, and affinity chromatography and ion exchange chromatography were performed for the purification of the ArsR-GFP fusion protein.
  • affinity chromatography Hi Prep 16/10 Heparin FF (GE Healthcare) was used, and for ion exchange chromatography, Hi Trap SP HP (GE Healthcare) was used. The method was performed according to each protocol. Each purification condition is shown in Tables 14 and 15.
  • the ZntR protein after purification was quantified by the Bradford method (measurement of absorbance at 595 nm (A 595 ) after Coomassie brilliant blue staining).
  • Smart Spec TM Plus Spectrophotometer (manufactured by Bio Rad) was used for absorbance measurement.
  • Determination of ARSR-GFP fusion protein after purification was carried out by measuring the A 280. At the same time, GFP fluorescence (518 nm) was also measured.
  • An F-2500 type spectrofluorometer (manufactured by Hitachi) was used for fluorescence measurement.
  • Detection of fluorescently labeled DNA in EMSA was performed by photographing a gel irradiated with LED light source visualizes (ATTO, AE-6935B (blue light source), AE-6935GL (green light source)) with a digital camera under dark conditions.
  • the promoter DNA used was double-stranded DNA labeled with FITC and TAMRA at both ends, as used in EMSA.
  • the promoter DNA was mixed with a single-stranded DNA probe previously labeled with FITC and TAMRA and treated by the method used in EMSA to form a double-stranded DNA.
  • lead acetate (II); Pb (CH 3 COOH) 2 hereinafter referred to as Pb
  • ZnSO 4 zinc sulfate
  • Zn zinc sulfate
  • sodium chloride; NaCl (hereinafter referred to as Na), calcium ion; CaCl 2 ⁇ 2H 2 O (hereinafter referred to as Ca) were each prepared at a concentration of 10 nM.
  • Samples were examined under 5 conditions including 1 nM Pb, 1 nM Zn, 1 nM Na, 1 nM Ca, and Tris buffer A (50 mM Tris-Cl pH 8.0, 5 mM dithiothreitol) without EDTA added.
  • Reaction solutions were prepared with the compositions shown in Table 21 and incubated at 37 ° C. for 30 minutes under dark conditions. Thereafter, 1 mL of Tris buffer A to which EDTA was not added was added to the reaction solution and mixed, and then fluorescence measurement was performed under the conditions shown in Table 22.
  • Arsenic Compound Detection Test GFP derived from pAcGFP1 plasmid fused with ArsR protein is a fluorescent protein emitting green fluorescence with an excitation wavelength of 475 nm and a fluorescence wavelength of 505 nm.
  • a reaction solution was prepared by mixing three different fluorescently labeled DNA probes with ArsR-GFP fusion protein, sodium arsenite; NaAsO 2 (hereinafter referred to as As). The three fluorescently labeled DNA probes used are shown in Table 23.
  • the wavelengths of 350 nm and 360 nm at which the peaks of the fluorescence intensity of TAMRA and GFP changed when As was not added and when As was added were determined.
  • the fluorescence measurement conditions excluding the excitation wavelength, the fluorescence start wavelength, and the fluorescence end wavelength were the conditions shown in Table 22. All incubations were performed under light shielding conditions.
  • ParsR-350 probe DNA SEQ ID NO: 9 is the sequence of ParsR-S3, SEQ ID NO: 10 is the sequence of EcorsR prm ext prm, SEQ ID NO: 11 is the sequence of R773-50-S, SEQ ID NO: 12 shows the sequence of R773-50-A).
  • the double-stranded DNA formed by ParsR-50-S-5-TAMRA and ParsR-50-A shown in Table 23 is composed of a 50-bp base sequence of promoter DNA on genomic DNA to which ArsR protein binds (hereinafter referred to as “DNA”). , Written as ParsR-50 probe DNA).
  • the double-stranded DNA formed by R773-50-S-5-TAMRA and R773-50-A shown in Table 23 is 50 bp base of promoter DNA on R-773 plasmid DNA to which ArsR protein binds. It consists of a sequence (hereinafter referred to as R773-50 probe DNA). These two fluorescently labeled DNA probes were incubated at 80 ° C. and then incubated at room temperature to form double-stranded DNA.
  • Table 25 shows the reaction composition and the detection procedure in the detection test using ParsR-50 probe DNA or R773-50 probe DNA. The detection of fluorescence was performed three times for 480 to 650 nm.
  • the resulting plasmid solution was reacted overnight using restriction enzymes NdeI and BamHI.
  • the restriction enzyme reaction solution was developed by agarose gel electrophoresis, and the band of the target zntR gene fragment was cut out and purified. Ligation was performed by using this as insert DNA and incubating with the pET3a vector at 16 ° C. overnight.
  • Escherichia coli JM109 strain was transformed using the ligation reaction solution. Five colonies selected after the transformation were designated as a, b, c, d, and e. Each of them was inoculated into 5 mL of LB medium supplemented with Amp, and cultured overnight at 37 ° C. with shaking.
  • fractions were subjected to protein staining by the Bradford method, and the absorbance values at 595 nm were measured (FIG. 29). Since the peaks observed up to fraction 5 were considered to be non-adsorbed fractions, fractions 9 to 15 forming peaks observed after fraction 5 were developed by SDS-PAGE.
  • fractions 10-13 a band thought to be a ZntR protein could be confirmed at the position indicated by the arrow (FIG. 30, position indicated by the arrow). Therefore, fractions 10-13 were combined and concentrated by ultrafiltration, protein quantification was performed by the Bradford method, and then gel filtration was performed for desalting. Furthermore, the ZntR protein was confirmed by SDS-PAGE (FIG. 31). As a result, since a band seen as a ZntR protein could be confirmed at the position indicated by the arrow, the subsequent experiment was performed using this ZntR protein partial purified solution.
  • the band recognized in lane 1 is considered to be a single-stranded DNA, and the bands recognized in lanes 2 and 6 are considered to be double-stranded DNA bands.
  • a band shift considered to be a band of the complex of the ZntR protein and the probe DNA was further observed, and the amount of the shifted band increased with the increase in the amount of the added protein.
  • the probe DNA labeled with FITC or TAMRA was detectable as green or red fluorescence. From these results, it was revealed that the prepared recombinant ZntR protein and the probe DNA used specifically bound to form a complex.
  • Table 27 shows a list of samples loaded on the gel for electrophoresis in EMSA using probe DNA labeled with FITC and TAMRA, and FIG. 33 shows the experimental results.
  • the gel was photographed through an orange film irradiated with blue excitation light to excite FITC.
  • a sample shifted with a ZntR protein partially purified solution showed an upwardly shifted band that was considered to be a complex of ZntR protein and probe DNA. .
  • the expected change in the fluorescence spectrum was that when Pb or Zn was present, the FITC peak at 518 nm increased and the TAMRA peak at 580 nm decreased.
  • the FITC peak at 518 nm increased and the TAMRA peak at 580 nm decreased.
  • Na, Ca, and control it was expected that there was no change in the ratio between the 518 nm peak of FITC and the 580 nm peak of TAMRA.
  • ArsR protein which is a sensor protein for arsenic
  • ArsR protein was prepared as a recombinant protein. If a recombinant ArsR protein can be prepared, it is considered that the ArsR can be directly labeled with a fluorescent substance even if ArsR is not in the form of a fusion protein with GFP.
  • Primers for arsR gene fragment amplification shown in Table 28 coli ArsR-S and E. coli. coilArsR-A was designed based on the DNA sequence data of E. coli K12 strain (SEQ ID NO: 13 shows the sequence of E.
  • E. coli ArsR-S and SEQ ID NO: 14 shows the sequence of E. coli ArsR-A).
  • the arsR gene was amplified by polymerase chain reaction (PCR) with Pfx50 TM DNA Polymerase using E. coli K12 strain genomic DNA as a template and primers E. coli ArsR-S and E. coli ArsR-A. After confirming amplification by electrophoresis, TaKaRa LA Taq TM with GC Buffer was added to the PCR reaction solution, and dATP was added to the 3 ′ end. This reaction solution was purified and ligated with a TA cloning vector and a pGEM-T vector to transform Escherichia coli JM109 strain (FIG.
  • PGEMarsR was extracted from the transformant, digested with XhoI and NdeI, and the inserted fragment was confirmed by electrophoresis.
  • pGEmarsR and pET16b were treated with XhoI and NdeI, respectively, and for pGEMarsR, the band of the target arsR gene fragment (about 360 bp) was excised after electrophoresis and purified.
  • pET16b was directly purified from the reaction solution and ligated with the arsR gene fragment. Escherichia coli JM109 strain was transformed with the ligation reaction solution.
  • the pET16arsR and pET3a vectors were treated with BamHI and NdeI, respectively.
  • the band of the desired arsR gene fragment was excised after electrophoresis and purified.
  • the arsR gene fragment was ligated with the pET3a vector to prepare pET3arsR. Sequencing was performed in the same manner as described above, and it was confirmed that there was no mutation in the arsR gene on pET3arsR by comparing with Gen Bank's Escherichia coli K12 strain DNA sequence data.
  • the Escherichia coli BL21 (DE3) pLysS for protein expression was transformed with the plasmid solution of pET3arsR, and the Escherichia coli strain for expressing ArsR protein was bred (FIG. 35).
  • the protein crude extract was treated with DNaseI for the purpose of removing DNA bound to the protein, and then the ArsR protein band was confirmed again by SDS-PAGE. However, no difference was observed in the band at the position predicted by the presence or absence of IPTG addition. Although the cause has not been specified, a recombinant ArsR protein has not been prepared. Therefore, it was decided to fluorescently label ArsR by preparing it in the form of a fusion protein with GFP.
  • the arsR gene fragment was amplified by PCR using primers Ec-arsR-pAcGFP-S and Ec-arsR-pAcGFP-A using the genomic DNA of Escherichia coli K12 strain as a template (FIG. 36). After confirming amplification by electrophoresis, the arsR gene fragment and the pAcGFP1 vector were treated with HindIII and PstI, respectively, and for pAcGFP1, the band at the target vector site (about 3.3 kb) was excised and purified after electrophoresis. Moreover, the reaction liquid was purified as it was for the arsR gene fragment.
  • FIG. 42 shows the results of mixing a partially purified solution of ArsR-GFP fusion protein and ParsR-350 labeled with TAMRA and measuring the fluorescence spectrum at excitation light of 350 nm and 360 nm in the presence and absence of As. In any excitation light, the fluorescence intensity of TAMRA near 580 nm was slightly decreased when added than when As was not added.
  • the probe DNA chain length was set to 50 bp, and the base sequence of the arsenic-responsive promoter on the genome was selected. It has become clear that this causes a change. Although the chain length and the base sequence of the probe DNA were changed, the expected fluorescence spectrum change via FRET could not be brought about.

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Abstract

L'invention porte sur un procédé qui permet de détecter et/ou de quantifier un analyte dans un échantillon à l'aide d'une protéine capteur qui peut se lier à l'analyte de façon spécifique et d'un acide nucléique qui peut être reconnu par la protéine capteur de façon spécifique, et qui est excellent en termes de coût, d'opérabilité et de rapidité. La protéine ArsR (qui est une protéine capteur capable de se lier à l'arsénique) et la protéine fluorescente verte (GFP) sont fusionnées ensemble pour donner ArsR-GFP. Il est confirmé que cette protéine de fusion peut se lier à une séquence de reconnaissance spécifique (Pars-ADN), et il est également confirmé que la liaison entre la protéine de fusion et Pars-ADN peut être inhibée par l'acide arsénieux. Ensuite, une plaque sur laquelle a été immobilisée Pars-ADN est préparée. Il a été découvert que la quantité d'ArsR-GFP liée à la plaque sur laquelle est immobilisé Pars-ADN est diminuée d'une manière dépendante de la concentration en acide arsénieux.
PCT/JP2009/003168 2008-07-10 2009-07-07 Biocapteur utilisant l'adn en tant qu’élément WO2010004736A1 (fr)

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