JP2020150830A - Method for measuring saliva protease activity and method for evaluating oral bacterial activity - Google Patents

Abstract

[Problem] To provide a method for measuring protease activity in saliva. [Solution] A method for measuring protease activity in saliva, which involves contacting saliva with a protease activity measuring reagent that produces luminescence or color when digested by protease, and then measuring the protease activity of the saliva based on the intensity of the luminescence or color produced; and a method for evaluating the activity of bacteria in the oral cavity, which involves measuring the protease activity of saliva collected from a subject animal using the method for measuring protease activity in saliva, and evaluating the activity of bacteria in the oral cavity of the subject animal based on the measurement results. [Selected Figures] None

Description

The present invention relates to a method for measuring saliva protease activity by utilizing a color-developing reaction or a luminescence reaction.

As one of the methods for detecting a microorganism, there is a method for measuring the activity of an enzyme produced by the microorganism to be detected. By utilizing the enzymatic activity, the microorganism can be detected in a shorter time than the method of culturing and identifying the microorganism itself. For example, Patent Document 1 discloses a method for diagnosing periodontal disease and a kit thereof by detecting an enzyme produced by a bacterium of a sample from the oral cavity of a patient.

As a method for measuring enzyme activity, a color development test method is widely used. In this method, a chromogenic substrate that produces a chromogenic substance by an enzyme produced by a microorganism is mixed with a sample suspected of having a microbial presence and reacted for a predetermined time, and the amount of the chromogenic substance produced is quantified by colorimetric analysis. The method. In addition, there is also a method in which a protease substrate such as gelatin is used as a thin film formed on a support, and digestion marks are formed on the thin film to measure protease activity. For example, in Patent Document 2, a biological sample such as a tissue or a cell is placed on a flat plate such as glass, and a protease substrate thin film is adhered thereto, so that the biological sample is decomposed by the action of a protease. Discloses a method capable of reliably decomposing a substrate and observing cell nuclei in a biological sample at the same time without dropping. However, cell incubation and staining and measurement of stained cells require specialized equipment and some time.

On the other hand, beetle luciferase is an enzyme that catalyzes the oxidation of firefly luciferin in the presence of adenosine triphosphate (ATP), magnesium ions and oxygen and causes it to emit light. Therefore, the beetle luciferase and the luciferin-luciferase luminescence reaction using the same are widely used in tests for the purpose of detecting microorganisms in a sample using ATP as an index, for example, microbial contamination ATP test and endotoxin luminescence test.

Various mutant beetle luciferases have been produced so far in order to increase the utilization of beetle luciferase in the detection of ATP. Examples of such mutant beetle luciferases include those having improved thermal stability (see, for example, Patent Document 3), those having improved substrate affinity (see, for example, Patent Document 4), and those having changed emission wavelengths (for example, see Patent Document 4). , Patent Document 5), improved emission sustainability (for example, see Patent Document 6), surfactant resistance (for example, see Patent Document 7), improved emission intensity (for example, Patent Document 7). (Refer to Document 8), those that are not easily affected by the inhibition of luminescence by sodium chloride (see, for example, Patent Document 9) and the like are known.

Special Table 2007-591923 Japanese Unexamined Patent Publication No. 2003-79395 Japanese Patent No. 30484666 Special Table 2001-518799 Japanese Patent No. 2666561 Japanese Unexamined Patent Publication No. 2000-197484 Japanese Unexamined Patent Publication No. 11-2394993 JP-A-2007-97577 Japanese Unexamined Patent Publication No. 2017-225372

Originally, saliva of animals such as humans does not contain protease, but oral bacteria produce protease. An object of the present invention is to provide a method for measuring protease activity in saliva.

As a result of diligent research to achieve the above object, the present inventor added luciferin to a reaction solution containing an oligopeptide-derived luciferin derivative containing a protease recognition site, ATP and luciferase, and found that luciferin was produced by the protease in the saliva. , And luciferin-luciferase luminescence reaction produces luminescence, and the intensity of this luminescence depends on the amount of protease in saliva, and has completed the present invention.

That is, the method for measuring the protease activity of saliva and the method for evaluating the amount of activity of oral bacteria according to the present invention are as follows [1] to [5].
[1] After contacting saliva with a protease activity measuring reagent that causes luminescence or color development by being digested by a protease, the protease activity of the saliva is measured based on the luminescence or color development intensity generated. A method for measuring saliva protease activity.
[2] The method for measuring saliva protease activity according to the above [1], wherein the protease activity measuring reagent contains a luciferin derivative in which luciferin is produced by being digested by a protease, ATP, and luciferase.
[3] The method for measuring saliva protease activity according to the above [2], wherein the luciferase is a mutant beetle luciferase in which a mutation is introduced into the wild-type beetle luciferase.
[4] The method for measuring saliva protease activity according to the above [3], wherein the luciferase is a mutant luciferase having a luminescence intensity higher than that of the wild-type luciferase.
[5] The saliva protease activity of saliva collected from a test animal is measured by the method for measuring saliva protease activity according to any one of [1] to [4], and the oral cavity of the test animal is based on the measurement result. A method for evaluating the activity of oral bacteria, which evaluates the activity of bacteria in the oral cavity.

According to the method for measuring the protease activity of saliva according to the present invention, the protease activity in saliva can be measured relatively easily and quantitatively. In addition, protease in saliva is produced by oral bacteria, and the amount of activity of oral bacteria can be evaluated by the measurement method.

It is a figure which showed the calibration curve which showed the relationship between the protease activity (BAEE U / mL) of trypsin and the luminescence intensity value, which was prepared in Reference Example 1. In Reference Example 2, it is a figure which showed the time-dependent change of the luminescence amount of each reaction solution which mixed trypsin and the reagent for measuring a protease activity.

[Measurement method of saliva protease activity]
The method for measuring protease activity of saliva according to the present invention (hereinafter, may be referred to as "method for measuring protease activity according to the present invention") is a protease activity that causes luminescence or color development by digesting saliva with a protease. After contact with the measurement reagent, the protease activity of saliva is measured based on the intensity of luminescence or color development generated. The stronger the protease activity in saliva, the stronger the intensity of luminescence or color development. That is, the intensity of protease activity in saliva is proportional to the intensity of luminescence or color development. Therefore, the intensity of protease activity in saliva, and thus the content of protease in saliva, can be quantitatively measured from the intensity of luminescence or color development generated by the reaction. Moreover, since the protease reaction is very rapid, the method for measuring protease activity according to the present invention can be carried out very quickly and in an extremely short time.

The reagent for measuring protease activity used in the present invention is a reagent that causes luminescence or color development when digested by a protease. Examples of the reagent include substances that produce a luminescent substance or a coloring substance when digested by a protease. Further, the reagent may include a substance that produces a substrate for a luminescence reaction or a color-developing reaction by being digested by a protease, and other substances necessary for a luminescence reaction or a color-developing reaction using the substrate. Good.

The substance that produces a luminescent substance by digestion with a protease is a peptide derivative of a luminescent substance in which a peptide containing a protease recognition site is linked, and the peptide does not emit light before being digested by the protease and is not emitted by the protease. Examples thereof include substances in which a luminescent substance released only after digestion emits light. Here, the peptide may be an oligopeptide or a protein (polypeptide).

The peptide derivative contained in the reagent for measuring protease activity used in the present invention may be a derivative of a peptide containing a protease recognition site recognized by any protease. For example, the protease activity of a specific microorganism or a group of microorganisms can be measured by using a derivative of a peptide containing a protease recognition site digested by a protease produced relatively specifically by the specific microorganism or the group of microorganisms. .. On the other hand, the total activity of the protease contained in the sample can be measured by using a peptide derivative containing a protease recognition site that is digested by a protease in a wide variety of microorganisms. As the protease recognition site digested by the protease, a known protease recognition site or a modified sequence thereof can be appropriately selected and used according to the target protease for measuring the activity. As the peptide derivative contained in the reagent for measuring protease activity used in the present invention, it is preferable to use a peptide derivative containing a protease recognition site recognized by a protease produced by an oral bacterium.

As the peptide derivative of the luminescent substance to which the peptide containing the protease recognition site is linked, the peptide derivative of the fluorescent substance is preferable because of its excellent sensitivity. As the fluorescent substance, known fluorescent substances such as fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), and variants thereof can be used. Examples of the peptide derivative of the fluorescent substance to which the peptide containing the protease recognition site is linked include a derivative in which a large number of fluorescent substances are bound to a protein serving as a protease substrate such as casein. The derivative does not fluoresce due to concentration quenching, or the fluorescence emitted is clearly smaller than that of a free fluorescent substance. In addition, a peptide derivative in which a peptide containing a protease recognition site is linked to methylcoumarin amide (MCA) or aminotrifluoromethylcoumarin (AFC), and a peptide that produces aminomethylcoumarin (AMC), which is a fluorescent substance, by protease digestion. Derivatives can also be used as reagents for measuring protease activity used in the present invention.

The peptide derivative of the color-developing substance in which the peptide containing the protease recognition site is linked may be a peptide derivative that does not develop color with the peptide derivative but develops color for the first time by protease digestion, and the peptide derivative and the peptide derivative in a free state after protease digestion are colored. May be peptide derivatives of different color-developing substances. Examples of the peptide derivative of such a coloring substance include a peptide derivative such as pNA (paranitroanilide).

The reagent for measuring protease activity used in the present invention may contain a peptide derivative of a bioluminescent luminescent substrate and other components necessary for the luminescence reaction. Among them, those containing a luciferin derivative in which a peptide containing a protease recognition site is linked to luciferin, which is a luminescent substrate for a luciferin-luciferase luminescence reaction, ATP, and luciferase are preferable because of their good sensitivity. Examples of the luciferin derivative that produces luciferin by protease digestion include a derivative in which a peptide containing a protease recognition site is bound to a phenolic hydroxyl group or a carboxyl group of luciferin.

The luciferase contained in the reagent for measuring protease activity may be any protein having luciferase activity, may be wild-type luciferase, and is a mutant in which a mutation is introduced into a region of wild-type luciferase having luciferase activity. It may be beetle luciferase. Examples of the mutant beetle luciferase include the mutant beetle luciferase described in Patent Documents 3 to 9. Further, the enzyme may have various tags added to the N-terminal and C-terminal of the region having luciferase activity of wild-type beetle luciferase and mutant beetle luciferase. As the tag, for example, a tag widely used in the expression / purification of a recombinant protein such as a histidine tag, a HA (hemagglutinin) tag, a Myc tag, and a Flag tag can be used.

In the present invention and the present specification, "wild-type beetle luciferase" refers to North American firefly (Photinus pyralis) luciferase (SEQ ID NO: 1), Heike firefly (Luciola lateralis) luciferase (SEQ ID NO: 2), and Genji firefly (Luciola cruciata) luciferase (sequence). No. 3), East European firefly (Luciola mingrelica) luciferase, Tuchi firefly (Lampyris noctiluca) luciferase, Hikarikometsukimushi (Pyrophorus plagiophthalamus) luciferase (SEQ ID NO: 4) and the like. The amino acid sequences of various wild-type beetle luciferases can be searched in a database (for example, EMBL-EBI Database (http://www.ebi.ac.uk/queries/)).

The luciferase used in the present invention is preferably a mutant beetle luciferase that is less susceptible to the inhibition of luminescence by sodium chloride than the wild-type beetle luciferase. The mutant beetle luciferase, which is not easily affected by the inhibition of luminescence by sodium chloride, is specifically in a 0.9% by mass sodium chloride solution when the luminescence intensity in a sodium chloride-free solution is 100%. Relative value of luminescence intensity ([Emission intensity in 0.9% by mass sodium chloride solution] / [Emission intensity in sodium chloride-free solution] × 100) (%) (hereinafter referred to as “residual activity (%)” .) Means high variant beetle luciferase. The luciferase activity of the mutant beetle luciferase used in the present invention preferably has a residual activity of 50% or more, more preferably 60% or more, further preferably 70% or more, and more preferably 80% or more. It is more preferably present, and particularly preferably 90% or more.

The mutant beetle luciferase that is not easily affected by the inhibition of luminescence by sodium chloride is, for example, a group consisting of at least the following (a), (b), (c) and (d) in the amino acid sequence encoding the wild-type beetle luciferase. Examples thereof include those having one or more mutations selected from the above (see Patent Document 9).
(A) A mutation in which the amino acid corresponding to valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is isoleucine, leucine, or phenylalanine.
(B) A mutation in which the amino acid corresponding to leucine at position 376 in the amino acid sequence of wild-type North American firefly luciferase is proline.
(C) A mutation in which the amino acid corresponding to glutamic acid at position 455 in the amino acid sequence of wild-type North American firefly luciferase is valine, alanine, serine, leucine, isoleucine, or phenylalanine.
(D) A mutation in which the amino acid corresponding to glutamic acid at position 488 in the amino acid sequence of wild-type North American firefly luciferase is valine, alanine, serine, leucine, isoleucine, or phenylalanine.

When the wild-type beetle luciferase is not North American firefly luciferase, the "amino acid corresponding to the amino acid at position X of the amino acid sequence of North American firefly luciferase" in the amino acid sequence of the wild-type beetle luciferase is an amino acid sequence homology analysis software (for example). , Micro GenieTM (manufactured by Beckman)), etc., when the amino acid sequences of the wild-type beetle luciferase and North American firefly luciferase are aligned so as to have the highest homology, "the X-position of the amino acid sequence of North American firefly luciferase" It means an amino acid at a position corresponding to "amino acid".

The luciferase used in the present invention is preferably a mutant beetle luciferase having a luminescence intensity higher than that of the wild-type beetle luciferase. Among the mutant beetle luciferases, those having a luciferase activity of 110% or more, preferably 120% or more, and more preferably 150% or more of the luciferase activity of wild-type beetle luciferase are preferable. It is more preferably 180% or more, and even more preferably 200% or more. Examples of such a mutant beetle luciferase include the mutant beetle luciferase described in Patent Document 8.

In the present invention and the present specification, the "luminescence intensity" of beetle luciferase refers to the peak luminescence intensity in the luciferin-luciferase luminescence reaction, that is, the beetle luciferase is referred to as ATP, divalent metal ion and oxygen. It means the peak luminescence intensity when reacting with firefly luciferin in the presence of. It can be determined that the larger the "luminescence intensity" is, the stronger the luciferase activity of the beetle luciferase is.

Further, the reaction solution of the luciferin-luciferase luminescence reaction is a "sodium chloride-free solution", which means that the reaction solution is prepared without adding sodium chloride. The "sodium chloride-free solution" and the "0.9 mass% sodium chloride solution" in the luciferin-luciferase luminescence reaction for calculating the residual activity all have the same composition except for sodium chloride, and the reaction temperature. Reaction conditions such as time and time are also adjusted.

The sequence identity (homosphere) between the amino acid sequences is the alignment obtained by juxtaposing the two amino acid sequences with a gap in the part corresponding to the insertion and deletion so that the corresponding amino acids match most. It is determined as the ratio of matching amino acids to the entire amino acid sequence excluding the gap inside. The sequence identity between amino acid sequences can be determined by using various homology search software known in the art.

The luciferase contained in the reagent for measuring protease activity may be a protein extracted from a beetle such as a North American firefly, or a recombinant protein produced by a gene recombination technique. It may also be a protein synthesized by peptide synthesis. In the case of a recombinant protein, generally, a transformant obtained by transforming a host microorganism is cultured to produce a recombinant protein. As the luciferase used in the present invention, the recombinant protein is recovered from the culture of the transformant, and the transformant is recovered from the culture by a method well known to those skilled in the art, for example, centrifugation, and freeze-thawed thereto. It can be recovered from the transformant by treatment, ultrasonic crushing treatment, or treatment with a lytic enzyme such as lysozyme. The recombinant protein of beetle luciferase may be recovered in a solution state. Further, the recovered beetle luciferase (crude enzyme) can be obtained by, for example, performing ammonium sulfate precipitation, SDS-PAGE, gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc. individually or in combination as appropriate. Can be purified.

In the protease activity measuring method according to the present invention, the saliva used for the measurement may be collected from any animal. The animal may be a human or a non-human animal. Examples of non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs. Further, the method of collecting saliva is not particularly limited, and saliva may be collected directly in a storage container, mouthwash may be collected, and cotton or the like holding saliva by containing it in the oral cavity may be collected as it is. It may be used for measurement. In addition, the collected saliva may be used as it is for measurement, or pretreatment may be performed as long as the protease activity in saliva is not excessively impaired. Examples of the pretreatment include a dilution treatment and the like.

For the contact between saliva and the reagent for measuring protease activity, for example, a protease reaction can be carried out by preparing a reaction solution in which both are added to an appropriate solvent and incubating the obtained reaction solution for a predetermined time. Saliva may be diluted with an appropriate solvent in advance, and a reagent for measuring protease activity may be added thereto and mixed. The reagent for measuring protease activity and a solvent may be mixed in advance, and then saliva or a diluted product thereof. May be mixed with a solution containing a reagent for measuring protease activity. The solvent is not particularly limited as long as it can dissolve both saliva and a reagent for measuring protease activity and does not significantly affect the protease activity, luminescence intensity or color development intensity. For example, water, phosphate saline (PBS), HEPES buffer, Tris buffer and the like can be mentioned.

The reaction time of the reaction solution containing saliva and the reagent for measuring protease activity is preferably 1 minute to 36 hours, more preferably 5 minutes to 24 hours, still more preferably 5 minutes to 30 minutes. Further, the emission intensity or the color development intensity may be measured immediately after the reaction solution is prepared. The reaction may be carried out in a thermostat or in an environment where the temperature is not controlled (for example, room temperature). When carried out in a constant temperature device, the reaction temperature is preferably 20 to 42 ° C, more preferably 28 to 37 ° C.

After completion of the reaction, the emission intensity or color development intensity of the reaction solution is measured. The measurement can be performed using a commercially available measuring device such as a microplate reader or a biochemical luminescence measuring device. The luminescence intensity or color development intensity of the reaction solution depends on the amount of the luminescent substance or the color-developing substance produced by the protease reaction in the reaction solution, and depends on the amount of protease in the reaction solution. Therefore, based on the calibration curve showing the relationship between the amount of protease (unit / mL) in the reaction solution and the emission intensity or the color development intensity, the protease contained in the saliva is obtained from the emission intensity value or the color development intensity value of the reaction solution. The intensity of activity can be measured, and the amount of activity of microorganisms contained in saliva can be quantified. As the calibration curve, one prepared in advance may be used, and the luminescence intensity value for preparing the calibration curve may be measured at the same time as the measurement of the target saliva. The calibration curve can be prepared by using a purified enzyme having a known concentration under the same conditions as the luminescence reaction. The purified enzyme used for preparing the calibration curve may be the same as the protease for measuring the protease activity, or a general-purpose enzyme such as trypsin may be used.

[Evaluation method of oral bacterial activity]
In the method for evaluating the activity of oral bacteria according to the present invention, the protease activity of saliva collected from a test animal is measured by the above-mentioned method for measuring the protease activity of saliva, and the test animal is based on the measurement result. Evaluate the activity of bacteria in the oral cavity. The animal may be a human or a non-human animal. Examples of non-human animals include those listed above.

The saliva used for evaluation is preferably resting saliva. Resting saliva is saliva that naturally accumulates in the oral cavity in a relaxed state while sitting on a chair or the like. Above all, in order to suppress the influence of meals, it is particularly preferable to use resting saliva collected 10 to 60 minutes after brushing the teeth after meals.

It is evaluated that the greater the protease activity of saliva, the greater the activity of bacteria in the oral cavity of the animal from which the saliva was collected. Therefore, the possibility of morbidity of a disease caused by oral bacteria such as periodontal disease can be evaluated by the method for evaluating the activity amount of oral bacteria according to the present invention. For example, when the amount of protease activity in saliva collected from a test animal is equal to or higher than a preset predetermined threshold, the amount of bacterial activity in the oral cavity of the test animal is large, that is, considerably in the oral cavity. Due to the presence of a large amount of bacteria or high activity of bacteria in the oral cavity, and therefore a high amount and high activity of oral bacteria such as periodontal disease. It can be evaluated that there is a high possibility of having a disease. Conversely, if the amount of protease activity is less than a preset threshold, it is unlikely that you are suffering from a disease caused by high activity of oral bacteria, such as periodontal disease. Can be evaluated as. The threshold value can be obtained experimentally based on the measured values obtained by measuring the protease activity under the same conditions for each of a patient group suffering from periodontal disease and a healthy subject group.

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Reference example 1]
Using a luciferin derivative, ATP, and mutant beetle luciferase as a reagent for measuring protease activity, a calibration curve showing the relationship between the amount of protease activity and the luminescence intensity due to the luciferin-luciferase luminescence reaction was prepared using trypsin.

For trypsin, a commercially available purified enzyme (Lot. SLBR6334V, manufactured by SIGMA-ALDRICH) having a trypsin activity of 34818 BAEE U / mL was added to 287 μL of the trypsin stock solution and 713 μL of water for injection was added and stirred well. A solution of 10000 BAEE U / mL obtained by diluting this with water for injection 10 times was used as a protease standard solution. For calibration curve preparation, this protease standard solution was serially diluted with water for injection to an activity intensity of 0.05, 0.1, 0.3, or 0.5 BAEE U / mL.

As a reagent for measuring protease activity, a BL (bioluminescence) reagent (Lot. 2018.06.19, manufactured by DKK-TOA CORPORATION) was used. The BL reagent includes a luciferin derivative (BZ-Leu-Gly-Arg-Luciferin: BZ represents a benzoyl group) in which an oligopeptide containing a recognition site by trypsin is introduced into the carboxyl group of luciferin, ATP, and a mutant form. Includes beetle luciferase (SEQ ID NO: 5). The mutant beetle luciferase is a mutation in which the amino acid corresponding to valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is isoleucine (the mutation (a)) and the mutation at position 488 in the amino acid sequence of wild-type North American firefly luciferase. It has a mutation (the mutation (d)) in which the amino acid corresponding to glutamic acid is valine.

Put 200 μL of each trypsin dilution series into a dry heat sterilized 1.5 mL glass test tube, put the BL reagent into the test tube, incubate at 37 ° C for 6 minutes, and then biochemiluminescence measuring device (device name: Luminitz, Protease activity was measured by Toa DKK (manufactured by Toa DKK) (high pressure power supply Hi: 659.7V, high pressure power supply Low: 391.4V, number of stirrings: 5 times, data acquisition interval: 0.1 seconds). The device measures the amount of light emitted from the measurement sample. A calibration curve showing the relationship between the amount of luminescence and the protease activity (BAEE U / mL) was calculated by the software installed in the device (high voltage primary equation: 0822-Protease-single-High).

The obtained calibration curve is shown in FIG. In FIG. 1, the luminescence intensity value at 0 BAEE U / mL was not 0, which is presumed to be due to the free luciferin originally contained in the BL reagent. Protease activity and luminescence intensity values showed a high correlation.

[Example 1]
The protease activity in saliva was measured using the BL reagent used in Reference Example 1.
From two subjects (subjects A and B), teeth were brushed after lunch, and about 30 minutes later, about 1 mL of resting saliva was collected. The collected saliva was diluted 10-fold or 100-fold with water for injection.

200 μL of the prepared diluted saliva was placed in a 1.5 mL glass test tube in the same manner as in Reference Example 1, the BL reagent was placed in the test tube, incubated at 37 ° C. for 6 minutes, and then biochemiluminescent. It was measured using a measuring device. The protease activity of saliva was examined from the measured luminescence intensity value based on the calibration curve prepared in Reference Example 1. Since the amount of luminescence of saliva collected from subject B was completely shaken off with the 10-fold diluted solution, the dilution rate was increased to 100-fold and the measurement was performed again. The results are shown in Table 1.

The protease activity determined from the calibration curve in FIG. 1 was divided by the dilution rate to determine the protease activity in saliva of each subject. As a result, the protease activity in saliva of each subject was 8.74 BAEE U / mL for subject A and 151.34 BAEE U / mL for subject B. From these results, it was confirmed that the activity intensity of protease in saliva can be quantitatively measured by preparing a calibration curve using trypsin as a standard substance using BL reagent.

[Reference example 2]
The BL reagent used in Reference Example 1 and a commercially available reagent for measuring protease activity (product name: Proteasome-Glo (registered trademark) Assays, manufactured by Promega) (hereinafter, may be referred to as "Promega reagent") are used. The protease activity in saliva was measured and compared under the same conditions. The Promega reagent contains a luciferin derivative, ATP, and a mutant beetle luciferase, and is a reagent for a luciferin-luciferase luminescence reaction.

The protease standard solution prepared in Reference Example 1 was serially diluted with water for injection to an activity intensity of 0.1 or 0.5 BAEE U / mL.

200 μL of the prepared trypsin dilution series was placed in a 1.5 mL glass test tube in the same manner as in Reference Example 1, the BL reagent was placed in the test tube, incubated at 37 ° C. for 6 minutes, and then biochemiluminescent. The measurement was performed using a measuring device (device name: 4 test machines, manufactured by DKK-TOA CORPORATION).

On the other hand, 100 μL of each trypsin dilution series is put into a dry heat sterilized 1.5 mL glass test tube, 100 μL of Promega reagent is put into the test tube, lightly stirred with a vortex mixer, and immediately set in a biochemical luminescence measuring device. Protease activity measurement was started (measurement temperature: 37 ° C., data acquisition interval: 1 second).

The change over time in the amount of luminescence of each reaction solution is shown in FIG. In the figure, “BL (0.1)” and “BL (0.5)” are reaction solutions containing the BL reagent used in Reference Example 1 and trypsin of 0.1 or 0.5 BAEE U / mL, respectively. "Promega (0.1)" and "BL (0.5)" are the results of a reaction solution containing the Promega reagent and trypsin of 0.1 or 0.5 BAEE U / mL, respectively. ..

As shown in FIG. 2, in the case of a reaction solution containing trypsin with 0.5 BAEE U / mL, when the Promega reagent is used, the peak appearance time is about 9 minutes and the peak luminescence amount is 1,180. On the other hand, when the BL reagent was used, the peak appearance time was about 3 minutes, and the peak luminescence amount was 10,718. As described above, it was found that the amount of luminescence was larger when the BL reagent was used, and the magnitude of the amount of luminescence at the peak top of both reagents was about 9 times different. Both reagents are reagents for the luciferin-luciferase luminescence reaction, but the mutant beetle luciferase used is different. The mutant beetle luciferase used in the BL reagent has a shorter reaction time and a faster luminescence peak than the mutant beetle luciferase used in the Promega reagent, so that the measurement time can be shortened. Further, since the absolute value of the amount of light emitted is large, there is an advantage that the detection sensitivity is high. In addition, since the BL reagent is commercially available in a syringe-encapsulated freeze-dried product packed in individually wrapped aluminum, the reagent has a long expiration date of 11 months and can be used immediately by simply removing it from the individually wrapped aluminum pack. There was also an advantage.

Claims (5)

A saliva protease that measures the protease activity of saliva based on the intensity of luminescence or color development after contacting saliva with a protease activity measuring reagent that produces luminescence or color development by being digested by a protease. Method of measuring activity. The method for measuring saliva protease activity according to claim 1, wherein the protease activity measuring reagent comprises a luciferin derivative in which luciferin is produced by being digested by a protease, ATP, and luciferase. The method for measuring saliva protease activity according to claim 2, wherein the luciferase is a mutant beetle luciferase in which a mutation is introduced into the wild-type beetle luciferase. The method for measuring saliva protease activity according to claim 3, wherein the luciferase is a mutant luciferase having a luminescence intensity higher than that of a wild-type luciferase. The protease activity of saliva collected from a test animal is measured by the method for measuring the protease activity of saliva according to any one of claims 1 to 4, and based on the result of the measurement, the mouth of the test animal is used. A method for evaluating the amount of oral bacterial activity, which evaluates the amount of bacterial activity.

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