+

WO2006061985A1 - Procede d’emission a luciferase et reactif d’emission pour mesure simultanee multicouleur - Google Patents

Procede d’emission a luciferase et reactif d’emission pour mesure simultanee multicouleur Download PDF

Info

Publication number
WO2006061985A1
WO2006061985A1 PCT/JP2005/021333 JP2005021333W WO2006061985A1 WO 2006061985 A1 WO2006061985 A1 WO 2006061985A1 JP 2005021333 W JP2005021333 W JP 2005021333W WO 2006061985 A1 WO2006061985 A1 WO 2006061985A1
Authority
WO
WIPO (PCT)
Prior art keywords
luciferase
luminescence
luciferin
emission
reagent
Prior art date
Application number
PCT/JP2005/021333
Other languages
English (en)
Japanese (ja)
Inventor
Masayuki Ryufuku
Chie Suzuki
Akihiro Kurita
Original Assignee
Toyo B-Net Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toyo B-Net Co., Ltd. filed Critical Toyo B-Net Co., Ltd.
Priority to JP2006547759A priority Critical patent/JPWO2006061985A1/ja
Publication of WO2006061985A1 publication Critical patent/WO2006061985A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase

Definitions

  • the present invention relates to a method for stabilizing a luminescence reaction of luciferase characterized by luminescent color, a method for using the same, and a luminescent reagent.
  • the luminescent reagent of the present invention can be applied to any luciferase atsy system using a luciferase enzyme having a different luminescent color as a reporter and a signal, and further to a luciferase atsy system using a multicolor simultaneous luminescence measuring system.
  • luciferases derived from luminescent beetles and having different luminescent colors perform a luminescent reaction with a common luminescent substrate. Therefore, instead of the Renilla luciferase, for example, a red luminescence luciferase derived from a railroad worm (Non-patent Document 3) is used to simultaneously emit yellow-green North American firefly luciferase, which has been used in the past. If the emission spectrum showing the amount of luminescence can be separated by a filter, the amount of each luminescence can be quantified in a single measurement step.
  • each luminescent reaction is different from the luciferase derived from the fireflies of North American fireflies that have been tried in the past.
  • the final objective, in vitro measurement which is a luminescent reaction system with stable luminescent kinetics that shows a constant luminescence intensity within a certain time from the start of the luminescence reaction, and ⁇ 3 Luminescent reaction system with controllable ratio of color emission intensity " Construction is required.
  • Patent Document 1 Japanese Patent No. 3171595
  • NB 2 Grentzmann, u., Ingraman, J.J.A., Kelly, P.J., uesteland, R.F., and Atkins, J.F. (1998) RNA 4, 479-486.
  • Non-Patent Document 3 Viviani, V.R., Bechara, E.J.H, and Ohmiya, Y. Biochemistry (1999) 3 8, 8271-827.
  • the present invention provides a luciferase luminescence method for simultaneous multicolor luminescence measurement by improving Kinetics of luminescence reaction of luciferin Z luciferase derived from a luminescent beetle characterized by luminescent color and controlling mutual luminescence intensity For the purpose.
  • the gist of the present invention is as follows.
  • the luminescent reagent comprising:
  • Luminescence whose fluctuation in luminescence intensity for 90 seconds within the luminescence measurement period is 10% or less means that the luminescence intensity measured at a certain point in the luminescence measurement period and the luminescence intensity measured 90 seconds after that When the absolute value of the difference is expressed as a percentage of the luminescence intensity measured at a certain time (100%), it is within 10%.
  • Green emission luciferase means that the maximum emission wavelength is 534 ⁇ ! Luciferase showing luminescence color of ⁇ 565nm and half width of 53 ⁇ 9lnm!
  • Range emission luciferase has a maximum emission wavelength of 580 ⁇ ! ⁇ 595nm, half width is 66 ⁇ 8
  • Red luminescent luciferase means a luciferase that exhibits an emission color with a maximum emission wavelength of 610 nm to 630 nm and a half-value width of 53 to 70 nm.
  • the luminescence intensity per unit time required for quantitatively and simultaneously measuring the luminescence reaction by the green luciferase, orange luciferase and red luciferase selected as the multicolor luminescence luciferase is constant.
  • Light emission Kinetics and at least 2 types, preferably 3 types of light emission intensity can be controlled and brought close to each other within the measurement range, enabling the construction of a luciferase luminescence reaction system for simultaneous multicolor luminescence measurement. It becomes ability.
  • FIG. 1 shows the time course of the luminescence intensity of a luminescence reaction with a conventional luciferase luminescence reagent and a luciferase luminescence reagent for multicolor luminescence in a luminescence reaction with irimotobotaru-derived green luminescence luciferase.
  • FIG. 2 shows the time course of the luminescence intensity of the luminescence reaction by the conventional luciferase luminescence reagent and the luciferase luminescence reagent for multicolor luminescence in the luminescence reaction by the orange luciferase derived from Irimotobotaru.
  • FIG. 3 shows the time course of the luminescence intensity of the luminescence reaction by the conventional luciferase luminescence reagent and the luciferase luminescence reagent for multicolor luminescence in the luminescence reaction by the railway worm-derived red luminescence luciferase.
  • FIG. 5 shows the correlation between the dilution rate and the amount of luminescence when serially diluted silkworm-producing enzyme preparations of green luciferase derived from Irimotobotaru.
  • FIG. 6 shows the correlation between the dilution rate and the amount of luminescence when serially diluted silkworm-producing enzyme preparation of orange luciferase derived from Irimotobotaru.
  • FIG. 7 shows the correlation between the dilution factor and the amount of luminescence when serially diluted silkworm-producing enzyme preparations of railway worm-derived luminescent luciferase.
  • FIG. 9 Quantitative determination of luminescence reaction in dilution series of red luminescence luciferase and green luminescence luciferase when orange luciferase is used as a control when simultaneously emitting three colors using a luminometer for simultaneous multicolor luminescence measurement. Indicates.
  • FIG. 10 Quantification of luminescence reaction in dilution series of orange luminescence luciferase and green luminescence luciferase when using red luminescence luciferase as a control during simultaneous emission of three colors using a multicolor luminescence luminometer .
  • FIG. 11 shows the luminescence intensity and time course of green, orange, and red luciferases when using a luminescent reagent for multicolor luminescence with a luminescence half-life of 2 hours or more in a one-liquid system.
  • the luminescence intensity for 90 seconds within the luminescence measurement period is appropriately selected according to the type of enzyme used, so long as it is within the luminescence measurement period, the luminescence intensity for 90 seconds starting at any time point after the start of the luminescence reaction is acceptable. Good.
  • orange luciferase which is a green-emitting luciferase derived from irimotobotaru and orange luciferase, a mutant of green-emitting luciferase derived from irimotobotaru, it is preferable to have a 90-second emission intensity that starts at the time when luminescence starts.
  • the two or more luciferases used in the present invention are not particularly limited, but may be those having a common substrate.
  • firefly luciferin derived from beetle that is, a multi-complex organic acid D (-)-2- (6'hydroxy 2, monobenzothiazolyl)- ⁇ 2-thiazoline-4 monostrophic rubonic acid (hereinafter not particularly described) (As long as “Luciferin”) Is an enzyme that emits photons by catalyzing this as an oxidative substrate, and includes all enzymes that originate from luminescent beetles, such as the firefly family, the mosquito family, the firefly family, and the Iriomote family, and that are responsible for the luminescent reaction. This includes enzymes in which the stability and luminescence properties of the enzyme protein itself have been artificially altered by recombinant DNA technology and mutation technology.
  • green luminescence luciferase examples include, for example, green luciferase derived from Rhagophthalmidae Ohbai (Sumiya, M., Viviani, VR, Ohba.N., and Ohmiya.Y. (1998) In Bioluminescence and Chmiluminescence.
  • orange luciferase examples include green luciferase from Rhagophthalmidae Ohbai (Sumiya, M., Viviani, VR, Ohba.N., and Ohmiya, Y. (1998) In Bioluminesce nce and Chmiluminescence. Proceeding of 10th International Symposium on Bioluminescence and Chemiluminescence (Roda, A., Pazzagli, M., Kricka, L., and Stanley, PE, Erd.), Pp.433-436, Bolona, Italy.) Luciferase (Viviani, VR, Uchida, A., Suenaga, N., Ryufoku, M., and Ohmiya, Y.
  • red luciferase examples include, for example, red luciferase derived from the railway insect Phrixothrix hirtus (Viviani, VR, Bechara, EJH, and Ohmiya, Y. Biochemistry (1999) 38, 82 71-827.), Red luciferase of a site-specific mutant of Pyrophorus. Plagiophthalamus (Wood, KV, Lam, YA, Seliger, HH, and McElroy. WD (1989) Science 244, 700-702.) JP 08-510387).
  • the concentration of luciferase in the luciferin Z luciferase luminescence reaction system is suitably 100 femto g / mL to 100 ⁇ g / mL, and preferably 1 ng / mL to 200 ng / mL.
  • the luciferin used in the present invention is not particularly limited as long as it emits light by the action of luciferase, but is preferably the above-mentioned beetle luciferin, which is directly extracted and purified from beetles, Includes chemically synthesized products. Furthermore, it is a beetle luciferin derivative and includes a luminescent substrate having a luminescent activity after digestion with an enzyme.
  • the concentration of sodium fluoride is suitably 0.1 mM to 500 mM, Preferably it is lmM-100mM.
  • the reducing agent is considered to have an action of protecting the luciferase enzyme.
  • reducing agents include dithiorubamates, xanthates, thiophosphates, thiazoles, and the like.
  • Dithiocarnomates include piperidine pentamethylenedithiocarnomate, pipecoline methylpentamethyldithiocarnomate, zinc dimethyldithiocarbamate, zinc jetyldithiocarbamate, dibutyldithiocarbamate.
  • sulfhydryl compounds such as dithiothreitol, dithioerythritol, j8 mercaptoethanol, 2 mercaptopropanol, 3 mercaptopropanol, 2, 3 dithiopropanol, glutathione, and coenzyme A are used. Moyo! /
  • the reducing agents can be used alone or in combination.
  • the concentration of the reducing agent used is suitably from 0.01 mM to 100 mM, preferably from 0.1 mM to 50 mM, but can be used at a concentration of 2 mM or less. In some cases, it is not necessary to add a reducing agent.
  • Adenosine triphosphate generates luciferyl-AMP (adenylate) from luciferin by reaction with luciferase and contributes to the luminescence reaction.
  • Adenosine triphosphate may be in the form of a salt. Examples of the salt include disodium salt, dipotassium salt, and magnesium salt.
  • concentration of adenosine triphosphate used is suitably 0.001 mM to 100 mM, preferably 0
  • Magnesium ions are considered as a cofactor in the luminescence reaction!
  • Examples of the compound containing magnesium include magnesium chloride, magnesium carbonate, magnesium sulfate, magnesium acetate, and magnesium hydrogen phosphate.
  • the concentration of magnesium ion used is suitably 0.001 mM to 200 mM, preferably 0.
  • luminescence with a constant luminescence intensity per unit time necessary for quantitatively and simultaneously measuring the luminescence reaction by the green luciferase, orange luciferase and red luciferase selected as the multicolor luciferase An excellent luminescent reaction system can be constructed for Kinetics.
  • the luminescence reaction of luciferin Z luciferase by the method of the present invention can be used for the measurement of the amount of beetle luciferase subjected to multicolor luminescence (luciferase activity).
  • Luciferase Atsey is a method for examining the transcriptional activity of a gene by measuring its luminescence activity using a luciferase gene as a reporter gene.
  • a vector in which a luciferase gene is bound downstream of the promoter to be analyzed is transferred to a culture cell and cultured. In the cell growth process, if the transcriptional activity of the promoter is strong, many luciferase enzymes are produced in the cell, and if the activity is weak, the amount of luciferase enzyme produced decreases.
  • a multicolor luciferase measurement system based on simultaneous multicolor emission measurement has been devised in order to further enhance the functionality of this luciferase assembly.
  • the conventional luciferase assay method using a single signal it has been difficult to evaluate the assay data due to variations in the luminescence reaction itself. For this reason, a different kind of luminescence reaction system that does not interact with each other is introduced, which is completely different from fireflies, and the two types of luciferase activities are individually measured, so that the transcription activity efficiency of the target reporter relative to the internal standard reporter is determined.
  • the Dual Luciferase Atsey method was introduced. However, with this method, HTS (Hig) is effective in terms of cost and operability due to the necessity of conducting an expensive luminescent substrate and a two-step reaction. Application to industrial applications such as h-Throughput Screening) has progressed!
  • luciferases derived from luminescent beetles and having different luminescent colors perform a luminescent reaction with a common luminescent substrate. Therefore, if the luciferases with different emission colors are simultaneously emitted and the emission spectra showing the difference in color tone and the amount of emission can be separated by a filter, the amount of each emission can be quantified in one measurement step.
  • the multicolor luciferase luminescence simultaneous measurement system makes it possible to measure multiple signals per reaction (one step). This system measures the two types of transcriptional activity simultaneously for each of the two luminescence levels, while the luminescence level of the remaining one color is used as an internal standard. A plurality of transcriptional activities with high accuracy can be evaluated. It is also possible to simultaneously measure three types of transcriptional activity and evaluate each.
  • the luminescence amount of the luminescence reaction of luciferin Z luciferase by the luciferase luminescence method for simultaneous multicolor luminescence measurement can be measured with a dedicated commercial luminometer.
  • Lumine sensor MCA AB-2250 type manufactured by Atoichi Co., Ltd. as a device that can measure three types of luminescence reactions by filter division.
  • This device has a built-in function to separate and quantitate the three emission colors from the transmittance and the amount of luminescence. Just like a normal measurement of luminescence, simply place a sample in the cuvette and set the sample in the cuvette. , The amount of light emitted from the three colors is automatically calculated. With the spread of luminometers with similar functions, this method can be expected to further develop.
  • the measurement of the light emission amount may be performed at a desired time interval over a desired period.
  • HTS high-through put screening
  • the amount of luminescence from each specimen is measured from 0.1 seconds up to 8 hours for the initiation of luminescence reaction to 10 seconds.
  • the present invention is a luminescent reagent for producing luminescence by the method of the present invention, and improves the luminescent reaction Kinetics by at least two, preferably three, luciferases, and at least two, preferably Also provided is the above-mentioned luminescent reagent containing a composition necessary for allowing the luminescence intensity of the three luciferases to fall within the measurement range and enabling simultaneous measurement of multicolor luciferase luminescence reaction.
  • aqueous solutions dissolved in water so that each has the same concentration as the solution used, and the lyophilized product (reagent A) is frozen. It is recommended that the aqueous solution (reagent B) be stored refrigerated or frozen. In this case, it is recommended that the reagent B is sufficiently returned to room temperature and then dissolved in the reagent A to make it into a solution state before use.
  • pyrophosphoric acid and Z or pyrophosphate or CoA luciferin, adenosine triphosphate and magnesium ion, as well as components such as cyclodextrin, sodium fluoride, saponin, reducing agent, buffer component, etc. It can also be a form.
  • the luminescent reagent of the present invention is suitable for lysis of cultured cells.
  • the required component and the component necessary for light emission may be contained in one solution (one-component system), or the solution containing the component necessary for lysis of cultured cells and the component necessary for light emission may be included. It may be in a form (two-component system) provided separately with the solution it contains.
  • Solvent Water (Milli-Q water)
  • Solvent Water (MiUi-Q water)
  • the solution having the above composition was frozen. It is recommended to supply it as a one-component frozen product. This should be stored at -70 ° C or below, and after thawing, return to room temperature.
  • lyophilized reagent A which is a relatively unstable component in solution
  • reagent B consisting of the other components are added and dissolved immediately before use. It can also be supplied as a two-component reagent kit. In this case, lyophilized reagent A is stored at -20 ° C, and reagent B is stored refrigerated (4 ° C) or frozen (-20 ° C).
  • the present invention also provides a luminescent reagent kit comprising the luminescent reagent of the present invention.
  • the kit of the present invention may contain, in addition to the luminescent reagent of the present invention, an instruction manual, a flow chart of an operation method showing extensive usage, and the like.
  • the instruction manual should contain an overview of the luminescent reaction, measurement principles, product characteristics, storage conditions, reagent preparation and operation methods, related products, troubleshooting, and so on.
  • the kit of the present invention may further contain a standard luciferase enzyme (when used for luciferase assembly), etc., depending on the application.
  • pEX-Red expressing the red luciferase gene (RED) from the firefly Phrixothrix hirtus
  • pEX-ROL expressing the green luciferase (ROL) gene derived from the Rhagophthalmidae Ohbai
  • Plasmids for each of the three luciferase expression vectors, pEX-ROLO, which expresses the site-specific mutant orange luciferase (ROLO) gene derived from Rhagophthal midae Ohbai were prepared.
  • Each expression vector is a commercially available Pitsuka Gene Control Vector 2 (Production Number: PGV-C2: manufactured by Toyo Ink Co., Ltd.), and the luc + gene at the top of the vector is replaced with the RED, ROL, and ROLO genes, respectively. Prepared.
  • DMEM Dulbecco's modified Eagle medium
  • COS7 cells cultured in 0% ushi fetal serum (FBS, Nippon Pharmaceutical Co., Ltd.) medium were used.
  • COS7 cells are cultured in a 6-well plate (NUNC No.140675) to 3 x 10 5 cells ZwellZ2mL, and 1 ⁇ g of each plasmid is added using Lipofectamine Plus reagent (Invitrogen). Introduced into COS7 (Transfusion).
  • cell lysing agent 0.005% CHAPSZ 10% glycerol Z2mM CDTA / 5mg / mL BS A / 20mM HEPES (pH 7.9) to each well to lyse the cells and dissolve luciferase enzyme. After leaving at room temperature for 5 minutes, 20 ⁇ L of lysate, which was also capable of expressing cells expressing three different plasmids, was collected at the bottom of the cuvette for luminometer (Berthold LB9506).
  • cell lysing agent 0.005% CHAPSZ 10% glycerol Z2mM CDTA / 5mg / mL BS A / 20mM HEPES (pH 7.9)
  • luciferase luminescence reagent for multicolor luminescence 530 ⁇ / 470 ⁇ luciferin Z2mM AED (jetyldithiocarbamate ammonium salt) Z5 mM MgSO /0.1%
  • the luminescence intensity tends to increase slightly for 200 seconds from the start of the luminescence reaction, and the luminescence intensity varies on average. It was 2% in 100 seconds.
  • the results of the luminescence reaction by the red luciferase derived from railway insects obtained by expressing pEX-Red are shown in Fig. 3.
  • the conventional luciferase luminescence reagent prepared with the ⁇ Pitsukagene luminescence kit '' immediately after the start of luminescence, It decayed rapidly and then gradually increased after 20 seconds.
  • the light emission starting power is also about 60% of light emission fluctuation in 20 seconds, and about 16% of light emission fluctuation in 100 seconds from 30 seconds to 130 seconds later, enabling quantitative measurement of light emission.
  • the luminescence reaction using the luciferase luminescence reagent for multicolor luminescence it decays rapidly 20 seconds after the start of the luminescence reaction, but then a constant luminescence intensity is obtained, and after 30 seconds from the start of light emission, 130 seconds later.
  • the variation in the amount of emitted light was 1.2% in 100 seconds.
  • pEX-Red expressing a red luciferase gene (RED) derived from a firefly, Phrixothrix hirtus, a green luciferase (ROL) gene derived from Rhagophthalmida e Ohbai, a red luciferase gene (RED) PEX-ROL, which expresses the luciferase (ROL) gene, which is a site-specific variant of the green luciferase (ROL) gene from Rhagophthalmidae Ohbai of the Iriomote firefly family, and pEX-ROLO, which expresses the orange luciferase (ROLO) gene.
  • a first plasmid was prepared.
  • DMEM Dulbecco's modified Eagle medium
  • COS7 cells cultured in 0% ushi fetal serum (FBS, Nippon Pharmaceutical Co., Ltd.) medium were used. 6well COS7 cells are cultured on a plate (NUNC No.140675) at 3 X 10 5 cells / well / 2 mL, and 1 ⁇ g of each plasmid is transferred to COS7 using lipofectamine plus reagent (Invitrogen). (Transfusion).
  • cell lysing agent 0.005% CHAPSZ 10% glycerol Z2 mM CDTA / 5 mg / mL BSA / 20 mM HEPES (pH 7.9) was added to each well to lyse cells and extract luciferase enzyme at room temperature for 5 minutes. After standing, 20 L of cell lysate obtained by expressing three different plasmids was collected at the bottom of a cuvette for a luminometer (Berthold LB9506).
  • luciferase luminescent reagent for multicolor luminescence (1) 530 ⁇ / 470 ⁇ luciferin / 2 mM AED / 5 mM MgSO / 0.1% a-cyclodextrin / O.lmM pyrophosphate
  • Example 3 Site-specificity of red luciferase gene (RED) from Phrixothrix hirtus, the red luciferase gene (ROL) gene from Rhagophthalmidae Ohbai, green luciferase (ROL) gene from Rhagophthalmidae Ohbai from Rhagophthalmidae Ohbai.
  • the orange luciferase (ROLO) gene was prepared using a baculovirus / silkworm protein expression system (Super worm System from Katakura Kogyo Co., Ltd.). Obtained.
  • luciferase luminescence reagent for multicolor luminescence 530 ⁇ / 470 ⁇ luciferin Z2 mM AED / 5 mM MgSO / 0.1% a-cyclodextrin ZO. LmM potassium pyrophosphate
  • [Mu] [theta] .01% saponin Z20 mM NaF / 100 mM Tris-phosphate (pH 8.0) was prepared. 100 ⁇ L each of luciferase luminescence measuring reagents for multicolor luminescence is used for 20 L of 10-fold, 100-fold, 1,000-fold, and 10,000-fold serial dilutions of three silkworm-produced luciferase crude enzyme preparations. The total amount of luminescence in 20 seconds was measured 20 seconds after the start of the luminescence reaction obtained with a luminometer (LB9506 manufactured by Berthold). Subsequently, the integrated luminescence amount obtained for 20 seconds and the dilution factor of the silkworm-producing enzyme preparation were plotted, respectively, to confirm the quantitativeness in each luminescence reaction.
  • a luminometer LB9506 manufactured by Berthold
  • Fig. 5 shows the results of plotting the amount of luminescence from the serially diluted ROL silkworm-producing enzyme preparation.
  • the dilution factor and the amount of luminescence from 10 times (0.1) to 10,000 times (0.0001) showed a high correlation.
  • Fig. 6 shows the results of plotting the amount of luminescence from the serially diluted ROLO silkworm-producing enzyme preparation.
  • the orange luciferase enzyme preparation showed a high correlation between the dilution factor and the amount of luminescence from 10 times (0.1) to 10,000 times (0.0001).
  • Fig. 7 shows the results of plotting the amount of luminescence by the serially diluted RED silkworm-producing enzyme preparation.
  • enzyme preparations of red luciferase from 10 times (0.1) to 1,000 times (0.001) The dilution factor and the amount of luminescence showed a high correlation.
  • pEX-Red expressing a red luciferase gene (RED) derived from the firefly, Phrixothrix hirtus, a green luciferase (ROL) gene derived from Rhagophthalmida e Ohbai, a red luciferase gene (RED) PEX-ROL, which expresses the luciferase (ROL) gene, which is a site-specific variant of the green luciferase (ROL) gene from Rhagophthalmidae Ohbai of the Iriomote firefly family, and pEX-ROLO, which expresses the orange luciferase (ROLO) gene.
  • a first plasmid was prepared.
  • DMEM Dulbecco's modified Eagle medium
  • NIH3T3 cells cultured in 0% urine fetal serum (FBS, Nippon Pharmaceutical Co., Ltd.) medium were used. Incubate the NIH3T3 cells at 2 ⁇ 10 4 cells / well / 500 ⁇ L in a 24-well plate (NUNC No.140675), and use 400 ng of each plate using lipofectamine plus reagent (Invitrogen). Rasmid was introduced into NIH3T3 (transfusion).
  • a cell lysing agent 0.005% CHAPSZ 10% glycerol Z2 mM CDTA / 5 mg / mL BSA / 20 mM HEPES (pH 7.9) was lysed in each well to extract the luciferase enzyme. After standing at room temperature for 5 minutes, the cell force expressing three different plasmids was mixed with the obtained lysate as follows.
  • luciferase luminescence reagent for single-component multicolor luminescence [1.06 mM ATP / 1.88 mM luciferin Z540 ⁇ M CoA / 4.9 mM DTT (dithiothreitol) Z5.34 mM MgSO /7.5 % a—Cyclodextrin Z0.01% Triton / -100 / lOOmM Tris—HCl (p
  • the luciferase luminescence method for simultaneous measurement of multicolor luminescence can simultaneously measure the amount of luminescence by a plurality of luciferases with different luminescent colors by performing a luminescence reaction once, and can output signals twice to three times that of the conventional method. It can be used for highly functional luciferase assembly. Therefore, by simultaneously using the luciferase luminescence method for simultaneous multicolor luminescence measurement of the present invention, simultaneous measurement of multigene transcriptional activity can be easily performed.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Cette invention concerne un procédé d’émission à luciférase pour une mesure simultanée multicouleur qui permet d’améliorer la cinétique et de contrôler l’intensité d’émission mutuelle en réaction à la luciférine/luciférase avec une caractéristique de couleur d’émission. Un procédé électroluminescent à luciférine/luciférase se caractérise par le fait que la réaction à l’émission par au moins deux types de luciférase, sélectionnés dans un groupe de luciférases électroluminescentes de couleur verte, orange et rouge, consiste en la réaction de la luciférase avec la luciférine afin que la variation de l’intensité d’émission en 90 sec dans la période de mesure devienne inférieure ou égale à 10 %. L’invention concerne également un procédé d’utilisation, un réactif d’émission et un kit de réactifs.
PCT/JP2005/021333 2004-12-07 2005-11-21 Procede d’emission a luciferase et reactif d’emission pour mesure simultanee multicouleur WO2006061985A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006547759A JPWO2006061985A1 (ja) 2004-12-07 2005-11-21 多色同時測定用ルシフェラーゼ発光方法および発光試薬

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004-353964 2004-12-07
JP2004353964 2004-12-07

Publications (1)

Publication Number Publication Date
WO2006061985A1 true WO2006061985A1 (fr) 2006-06-15

Family

ID=36577816

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/021333 WO2006061985A1 (fr) 2004-12-07 2005-11-21 Procede d’emission a luciferase et reactif d’emission pour mesure simultanee multicouleur

Country Status (2)

Country Link
JP (2) JPWO2006061985A1 (fr)
WO (1) WO2006061985A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009133697A (ja) * 2007-11-29 2009-06-18 Olympus Corp 発光測定方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103149199B (zh) * 2012-12-25 2015-05-27 北京城市排水集团有限责任公司 水质急性毒性高通量快速测定试剂盒制作及使用方法
EP2930497A1 (fr) * 2014-04-07 2015-10-14 Institut Pasteur Émission de photons non dépendant d'enzyme

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0847399A (ja) * 1994-08-08 1996-02-20 Eiken Chem Co Ltd 生物発光の測定方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4385135B2 (ja) * 2003-05-06 2009-12-16 独立行政法人産業技術総合研究所 マルチ遺伝子転写活性測定システム

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0847399A (ja) * 1994-08-08 1996-02-20 Eiken Chem Co Ltd 生物発光の測定方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GRENTZMANN G. ET AL: "A dual-luciferase reporter system for studying recoding signals", RNA, vol. 4, 1998, pages 479 - 486, XP002118030 *
NAKAJIMA Y. ET AL: "Bidirectional role of orphan nuclear receptor RORalpha in clock gene transcriptions demonstrated by a novel reporter assay system", FEBS LETT., vol. 565, 7 May 2004 (2004-05-07), pages 122 - 126, XP004507690 *
VADIM R. ET AL: "Cloning, Sequence Analysis, and Expression of Active Phrixothrix Railroad-Worms Luciferases: Relationship between Bioluminescence Spectra and Primary Structures", BIOCHEMISTRY, vol. 38, no. 26, 1999, pages 8271 - 8279, XP002172177 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009133697A (ja) * 2007-11-29 2009-06-18 Olympus Corp 発光測定方法

Also Published As

Publication number Publication date
JPWO2006061985A1 (ja) 2008-06-05
JP2012163577A (ja) 2012-08-30

Similar Documents

Publication Publication Date Title
Hampf et al. A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays
Branchini et al. Red-and green-emitting firefly luciferase mutants for bioluminescent reporter applications
Fan et al. Bioluminescent assays for high-throughput screening
Branchini et al. Red-emitting luciferases for bioluminescence reporter and imaging applications
da Silva et al. Kinetics of inhibition of firefly luciferase by dehydroluciferyl-coenzyme A, dehydroluciferin and L-luciferin
Leitão et al. Firefly luciferase inhibition
CA2608337C (fr) Systeme de dosage de detection de la luciferase
US10364454B2 (en) Bioluminescent assays utilising secreted luciferases
JP6120335B2 (ja) ルシフェラーゼシグナルを増強する組成物
AU2004210982B2 (en) Methods and kits for dual enzymatic assays whereby light is quenched from luminescent reactions
JP2008532517A (ja) 発光シグナルの増強
Michelini et al. Combining intracellular and secreted bioluminescent reporter proteins for multicolor cell-based assays
JP2012163577A (ja) 多色同時測定用ルシフェラーゼ発光方法および発光試薬
JP2007330185A (ja) 多検体試料中における複数のルシフェラーゼを検出する方法
JPH0847399A (ja) 生物発光の測定方法
JP2008113620A (ja) ルシフェラーゼの発光方法
JP4761150B2 (ja) ハイスループット発光活性測定のためのセレンテラジン（ウミシイタケルシフェリン）溶液の安定化組成物および安定化法
US20040219622A1 (en) Phosphine-containing formulations for chemiluminescent luciferase assays
Kim-Choi et al. Kinetic characterization and in vitro toxicity evaluation of a luciferase less susceptible to HPV chemical inhibition
Kim‐Choi et al. Creating a mutant luciferase resistant to HPV chemical inhibition by random mutagenesis and colony‐level screening
WO2006013837A1 (fr) Methode de luminescence a la luciferase et reactif luminescent
JP2008536519A (ja) 延長した直線を用いる発光ａｔｐ検出
JP2009112199A (ja) 多色ルシフェラーゼの検出方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2006547759

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05809738

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载