JP3277426B2 - Reagent-free mutagen detection method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a luminescent mutagen detection method.

[0002]

2. Description of the Related Art At present, many mutation test methods using microorganisms have been reported as short-term screening methods for carcinogenic substances. Among them, the reverse mutation test method, Am
The es test is the most widely used. However, this test method has a problem that it takes about 3 days to measure and requires aseptic operation. Recently, short-term mutagen detection methods (umu test, SOS chromo test) for detecting DNA damage, unlike the Ames test for detecting mutagenicity, have been reported (Oda, Nakamura et al., "Evalution of
the new system (umu-test) for the detection of envir
onmental mutagens and carcinogens ", Mutation Resear
ch, 147 , 219-229, 1985, Japanese Patent Publication No. 4-12118).

[0003] In these methods, the SOS response induced at the time of DNA damage is determined by using one of the SOS genes, umuD ,
This is a method for detecting DNA damage and mutagenicity by measuring the expression level of β-galactosidase by ligating the lacZ gene downstream of the C gene or sfiA gene. More specifically, Salmonella (umu test) and Escherichia coli (SOS chromotest) into which the above-described genes have been introduced are cultured in the presence of a test substance, and this culture solution is diluted with a predetermined buffer, and then diluted with toluene or An aqueous solution of a sulfonate type surfactant such as chloroform and sodium dodecylbenzenesulfonate is added to perform cell disruption treatment.

Then, for the purpose of measuring β-galactosidase activity, an aqueous solution of 2-nitrophenyl-β-D-galactopyranoside, which is a substrate of β-galactosidase, is added, and the enzyme reaction is carried out at 28 ° C. The reaction is stopped with sodium carbonate. The optical density of the enzyme reaction solution was measured at two wavelengths (420, 550 nm), and the mirror method (Miller.J.
H., Experiments in molecular genetics, Cold Spring H
β-galactosidase activity is calculated based on the Arbor Laboratory, 1972).

The above-mentioned umu test and SOS chromo test have a feature that they can be detected much more easily and quickly than the Ames test. When the various operations are organized by step, the first step for inducing the SOS reaction, specifically, the step of culturing the host microorganism in the presence of the test sample, and the step expressed in the cells by this step, β
-The second step of detecting galactosidase activity by a color reaction can be roughly classified into two.

As described above, the operation of the second step is more complicated than that of the first step, such as dilution, cell disruption, addition of a substrate, progress and stop of the color reaction,
Various operations such as measurement of absorbance are required. In addition, colorimetric detection has a problem that detection sensitivity is low.
In order to increase the detection sensitivity, cell disruption treatment is performed to increase the reactivity between β-galactosidase expressed in the cells and the chromogenic substrate added from the outside, but this operation cannot help but complicate the test. .

[0007] As described above, the test method based on color detection requires complicated operations in the second step and has low detection sensitivity. Therefore, in order to compensate for this drawback, it is necessary to lengthen the color development time or to perform complicated operations. However, there are problems such as that the rapid detection is impaired when a complicated cell crushing treatment is performed, and that the addition of an expensive coloring reagent is indispensable.

[0008]

Accordingly, the present invention does not require any complicated operations in the second step, that is, various operations such as dilution, cell disruption, addition of a substrate, and progress and stop of the color reaction. The operation is significantly simplified, and the SOS is superior in terms of speed, detection sensitivity, and economy.
An object of the present invention is to provide a method for detecting a genotoxic substance such as a mutagenic substance using the DNA damaging property of a gene.

[0009]

Means for Solving the Problems As a result of various studies to solve the above problems, the present inventors have found that a gene expressing luciferase activity downstream of an SOS gene susceptible to DNA damage and its luciferase activity By culturing a microorganism transformed with a recombinant gene in which a gene that expresses an enzyme that catalyzes the production of a substrate of the present invention is placed in a medium containing a genotoxic substance such as a mutagenic substance, the expression of the SOS gene and A gene having a luciferase activity and a gene expressing an enzyme that catalyzes the production of a substrate for the luciferase activity are also expressed. As a result, the conditions under which the substrate for the luciferase activity is continuously supplied in the cells are achieved. A luminescent reaction occurs, and the complicated operation of the second step is particularly performed by measuring this luminescence. Found that it is possible to detect or measure genetic toxicology of such rapidly mutagen no necessity, the present invention has been completed.

When an analysis is performed using the value obtained by dividing the response under the condition containing a genotoxic substance such as a mutagenic substance by the response under the condition not containing a genotoxic substance as an index, it is a typical mutagenic substance. (2-furyl) -3- (5-nitro-2)
In the case of (furyl) acrylamide, in the case of the conventional colorimetric detection, this value increases as the content of the genotoxic substance increases and changes from 1 to about 10, whereas in the case of the luminescence detection of the present invention, It has been found that it varies from 1 to around 100. As described above, in the case of the present invention, it has been revealed that the dynamic range of measurement is up to about 10 times wider than the conventional method, and that genotoxic substances can be detected and quantified with high sensitivity and high accuracy.

As a criterion for judging whether or not a substance is a genotoxic substance, it is generally performed to indicate that the substance is genotoxic by showing a double response to the condition in which no test sample is added. The concentration showing a double response is called the minimum detection concentration. As a result of conducting tests on various genotoxicants, the minimum detection concentration of the luminescence detection of the present invention is as follows.
The concentration was at least about 5 times lower than that of the conventional colorimetric detection, and it became clear that genotoxic substances could be detected with high sensitivity.

Accordingly, the present invention includes the SOS gene expressed at the time of DNA damage, a gene arranged downstream of the gene and expressing a luciferase activity, and a gene expressing an enzyme that catalyzes the production of a substrate for the luciferase activity. And a recombinant gene comprising: The present invention also provides a host microorganism transformed with the gene.

The present invention further provides a method for culturing the host microorganism in a medium containing a test sample to obtain luminescence obtained by expression of genes that express luciferase activity and an enzyme that catalyzes the production of a substrate for the luciferase activity. Provided is a method for determining the presence or absence of a genotoxic substance such as a mutagenic substance in a test sample or measuring the amount of the genotoxic substance in the test sample.

In the present invention, in order to express a gene expressing an enzyme that catalyzes luciferase activity and its substrate production depending on DNA damage, the SOS gene is used as an expression control means thereof. Therefore, SOS
The gene only needs to be expressed at the time of DNA damage,
What is necessary is just to contain the control part called SOS box. Therefore, the SOS gene expressed at the time of DNA damage in the present invention means a gene containing a control part called the SOS box, and may be the SOS box itself or any DNA fragment containing the same. You may.

Examples of the SOS gene, umu gene, examples <br/> example, if umuC gene and umuD gene, and sfi
A gene and the like are included, but not limited thereto. Of these genes, the umuD and umuC genes are Pro.
USA, Vol. 82 , 4336-4340 (1985), and can be easily obtained based on these descriptions. Also, plasmid pSK1002 containing the fusion gene with a part and the lacZ gene of umuD gene and umuC genes, H.Shinagawa et, Gene, 23, 167 (198
The umuD and umuC genes can be easily obtained from this plasmid.

Luciferase activity and production of its substrate
Genes that express enzymes that catalyze SOS
If both genes are expressed under the control of the offspring
Well, for example, mention the bioluminescent genes of marine bacteria
But can be, but not limited to. Organism with the above properties
As a marine bacterium having a luminescent gene group, Vibrio (Vibr
io) Vibrio of the group (VibrioIn the genus, Vibrio have
I(V. harveyi), Vibrio Fisherie (V. fische
ri), Vibrio Splendidus (V. Splendidu
s), Vibrio Cholera (V. cholerae)
Photobacterium in the genus Photobacterium
Um Phosphorium (P. phosphoreum), Photo Bacte
Lum Reinokunashi (P. leiognathi) Etc.
You.

For example, V. fics
heri ) -derived bioluminescent genes remove the operator region of the bioluminescent genes to facilitate their use as reporter genes, and catalyze the production of structural genes (genes expressing luciferase activity and its substrate, aldehyde) Various cassette vectors containing only the enzymes that express the activity of the enzyme fatty acid reductase), for example, pUCD320, pUCD613, pUCD614, pUCD618, pUCD62
0, pUCD623, pUCD1111 and the like have been reported. See Clarence I. Kado et al., Plant
Molecular Biology Reporter, 5 , 225 (1987), and the structural gene portion of the bioluminescent gene group can be easily obtained from these vectors.

In the present invention, the term "luciferase substrate" means a long-chain aldehyde and the like, and the enzyme which catalyzes the production thereof includes NAD (P) H: FMN reductase,
It means fatty acid reductase and the like. In the above example, the genes expressing the luciferase activity and the gene of the enzyme that catalyzes the production of the luciferase substrate are of the same origin, but these genes of different origins may be used in combination. it can.

The host microorganism is a microorganism capable of expressing the SOS gene when its DNA is damaged by a genotoxic substance such as a mutagenic substance, ie, SOS.
Any microorganism having a function may be used, for example, Escherichia coli (Esche
richia coli ), Salmonella typhimur
ium) , for example, the TA1535 strain, the TA1538 strain, etc.
Yeast (Saccharomyces cerevisiae ) and the like.

A recombinant gene comprising the SOS gene and a gene expressing an enzyme that catalyzes luciferase activity and production of its substrate is located downstream of the SOS box of a DNA fragment containing at least the SOS box of the SOS gene. And a gene group expressing an enzyme that catalyzes luciferase activity and its substrate production. This ligation can be performed using DNA ligase according to a conventional method. In order to introduce this recombinant gene into a host microorganism, the recombinant gene must be present in a vector. For example, a pBR plasmid, a pUC plasmid, or the like can be used as a plasmid.

Transformation of a host microorganism with an expression vector may be performed by a conventional method used for transforming a microorganism, for example, a bacterium. According to the present invention, in order to detect or measure a genotoxic substance such as a mutagenic substance in a test sample, the test sample is mixed with a medium, and the transformed host microorganism is cultured in the medium. The culture is usually performed for 1 to 3 hours. Since the culture solution itself containing the test sample emits light by performing the culture, the light can be detected according to a conventional method immediately after the culture. When the test sample contains a genotoxic substance such as a mutagenic substance, the amount of luminescence increases in a concentration-dependent manner. Based on this increase, a genotoxic substance such as a mutagenic substance in the test sample is detected or measured. be able to.

According to the method of the present invention, all genotoxic substances such as mutagenic substances can be detected. A genotoxic substance such as a mutagenic substance is finally introduced into a medium as a solution, but it can be measured even if it is a gas or a solid that can be dissolved in the medium.

[0023]

According to the method of the present invention, a test sample and a medium containing a transformed host microorganism can be mixed and luminescence can be detected immediately after culturing, so that the complicated operation of the second step is not required. For this reason, the detection time is greatly reduced by simple operation, the measurement can be performed at a low cost because no reagent is required, and the luminescence detection is used. It is possible to detect the minimum detection concentration of a genotoxic substance such as a mutagenic substance at a low concentration side at least about 5 times with high sensitivity and high accuracy.

[0024]

[Embodiment 1 ] Construction of luminescence vector and preparation of transformant Escherichia coli (CSH26 "Miller, JH, Ex.
periments in Molecular Genetics, Cold Spring Harbor
Laboratory, 1972 ": UmuD, C gene of plasmid pSK1002 introduced into F ^- ara del (lac-pro) thi) was used. Genes that express enzymes that catalyze luciferase activity and its substrate production include E. coli (HB10
^{_{1: hsd20 (r B -,}} m B -), recA13, ara-14, proA2, lacY1, ga
1K2, rpsL20, xyl-5, mtl-1, supE44) introduced plasmid pUCD620 (Clarence I. Kado et al., Plant Molecular Biol
ogy Reporter, 5 , 225 (1987)).

As the microorganism to be introduced, Salmonella (TA)
1535 `` Ames, BN, J. McCann, E. Yamasaki, Mutation Res.,
31 , 347, 1975 '': hisG46, Δgal, Δchl, Δbio, Δuvr B, r
fa ^-, SJ10002: r ^-, using the m ^+). The luminescence measuring device
Microlumino Reader manufactured by Corona Electric Co., Ltd. (MLR-
100) was used.

E. coli C containing plasmid pSK1002
SH26 is LB medium containing ampicillin (Bacto tryptone 1%, Bacto yeast extract 0.5%, NaC
11%), and the alkaline extraction method "Birnboim, HC, Do
ly, J., Nucl. Acids Res., 11 , 1513, 1979, pS
K1002 was prepared in large quantities. Plasmid pSK100
2 was cut with Sal I and Sma I to obtain a Sal I- Sal I DNA fragment of about 7.5 kb containing the umuD , C gene. This DNA fragment was ligated with T4 DNA ligase, and Escherichia coli DH5 was transformed according to a conventional method.

A plasmid was isolated from the obtained transformant by an alkali extraction method. This plasmid was transferred to Mlu I
, And blunt-ended with T4 DNA polymerase. Acts as a codon for umuC and Ba
DNA linkers (TAGGATCC with m HI site
TA) was chemically synthesized. This synthetic linker and the Mlu
The IDNA fragment was ligated with T4 DNA ligase and transformed into Escherichia coli DH5 according to a conventional method. A plasmid was extracted from the obtained transformant by an alkali extraction method. This plasmid was cut with Bam HI and Sal I, to obtain a Bam HI- Sal IDNA fragment of about 7.4 kb.

E. coli H containing plasmid pUCD620
After culturing B101 in an LB medium containing ampicillin, pUCD620 was prepared in a large amount by alkali extraction. The plasmid pUCD620 Bam HI and Sa
It was cut with l I, about 7.5kb of Bam including a luminescent genes
An HI- Sal I DNA fragment was obtained. This DNA fragment and the above-mentioned Bam HI- Sal I DNA fragment of about 7.4 kb were digested with T4
By ligating with DNA ligase, a luminescent vector was constructed.

[0029] SJ10002 was cultured overnight in LB medium. To the LB medium, 1/100 volume of the culture solution was added,
The cells were cultured until the turbidity at 600 nm (OD ₆₀₀ ) was about 0.4. 5 ml of the culture solution is centrifuged, and the precipitate fraction is
Was suspended in a 30 mM CaCl ₂ aqueous solution and left on ice for 45 minutes. Centrifugation was performed again, and the cells were added to 0.4 ml of 30 mM
The suspension was suspended in a CaCl ₂ aqueous solution to prepare a competent cell of SJ10002.

TA1535 was cultured overnight in LB medium.
1/100 volume of the culture solution was added to LB medium,
The cells were cultured until the turbidity at 0 nm (OD ₆₀₀ ) was about 0.4. 5 ml of the culture solution was centrifuged, and the sediment fraction was added to 5 ml of 1 ml.
0mMCaCl _2, 10mMMnCl _2, 10mMMgCl ₂
Suspended in an aqueous solution and left on ice for 45 minutes. After centrifugation again, the cells were clarified with 0.4 ml of 10 mM CaCl ₂ , 10 mM.
MnCl ₂ , suspended in 10 mM MgCl ₂ aqueous solution, TA1
535 competent cells were prepared.

Competent cell 100 of SJ10002
About 200 ng of the luminescent vector was added to μl, and left on ice for 30 minutes. After treatment at 42 ° C. for 2 minutes, the mixture was left at room temperature for 5 minutes. 1 ml of LB medium was added and incubated at 37 ° C. for 1 hour. This solution was seeded on an LB plate containing 50 μg / ml of ampicillin. A luminescent vector was prepared from the thus obtained transformant introduced into SJ10002 by an alkali extraction method, and TA was prepared in the same manner as described above.
Transformation into 1535 was performed.

Embodiment 2 FIG . Luminometric Metabolic Activating Enzyme Obtained from rat liver, phenobarbital and 5,6
Benzoflavone-treated S9 fraction (manufactured by Oriental Yeast Co., Ltd.)
An S9 mix mixed with ctor-I (manufactured by Oriental Yeast Co., Ltd.) was used.

Test sample 2- (2-furyl) -3- (5-nitro-2-furyl)
Acrylamide (hereinafter abbreviated as AF-2), 4-nitroquinoline 1-oxide (hereinafter abbreviated as 4NQO), 1-nitropyrene (hereinafter abbreviated as 1-NP), 2-aminoanthracene (hereinafter abbreviated as 2-AA), benzo [A] Pyrene (hereinafter abbreviated as Bap) was used.

The TA1535 luminescent vector transformant was placed in a TGA medium (1% tryptone, 0.5% salt, 0.2% glucose, 20 μg / ml ampicillin) at 30 ° C. overnight.
And cultured. This culture solution was inoculated into a TGA medium at a volume of 1/50 and cultured at 30 ° C. for 1.5 hours. OD of this culture
_It was diluted with TGA medium so that ₆₀₀ = 0.1. 1.45 ml of the diluted culture solution was dispensed into test tubes, and 50 μl of a test sample having a predetermined concentration was added to each of the tubes, followed by culturing at 30 ° C. for 2 hours. If a metabolic activating enzyme is required, 0.25 ml of S9 mix and 50 μl of test sample are added to 1.2 ml of the diluted culture solution.
And cultured at 30 ° C. for 2 hours. Immediately after the culture, the amount of luminescence of 100 μl of the culture solution was measured with a luminescence measuring device. Also,
The OD ₆₀₀ of the culture was measured.

Activity (Intensity / OD ₆₀₀ )
Was determined by measuring the amount of luminescence for 10 seconds and dividing the value by the value of OD ₆₀₀ . The measurement results are shown in FIGS.
The comparative examples shown below were performed to compare the sensitivity of the present invention with that of the conventional method.

COMPARATIVE EXAMPLE 0.1 mol of a Salmonella bacterium (TA1535 / pSK1002) into which the plasmid pSK1002 was introduced was used.
l / l potassium phosphate buffer (pH 7.0)
5 g potassium chloride, 0.246 g magnesium sulfate,
2 ml of 0.05 mol / l 2-mercaptoethanol was added and dissolved to prepare a Z-buffer.

The TA1535 / pSK1002 was replaced with TGA
The cells were cultured overnight at 37 ° C. in the medium. This culture solution was inoculated in a 1/50 volume in a TGA medium and cultured at 37 ° C. for 1.5 hours.
This culture was diluted with TGA medium so that OD ₆₀₀ = 0.1. 1.45 ml of the diluted culture solution was dispensed into test tubes, and 50 μl of a test sample having a predetermined concentration was added to each, and the mixture was cultured at 37 ° C. for 2 hours. If metabolic activating enzyme is required, S9 mix 0.25m
l, 50 μl of a test sample was added, and the mixture was cultured at 37 ° C. for 2 hours. The OD _{600 of} this bacterial solution was measured.

To the remaining 0.1 ml of the bacterial solution, add 0.9 parts of Z-buffer.
ml, 0.1% SDS (50 μl) and chloroform (10 μl) were added, and the mixture was stirred for 5 seconds. Next, 4 mg / ml o-nitrophenyl- dissolved in 0.1 M phosphate buffer (pH 7.0) was used.
β-D-galactopyranoside (ONPG) aqueous solution was added to 0.
After adding 2 ml and reacting at 28 ° C. for 10 minutes, 1 mol / l
The reaction was stopped by adding 0.5 ml of sodium carbonate solution. Then, the absorbance at 420 nm and 550 nm was measured immediately. The activity (unit) was determined by the following equation based on the Miller method.

[0039]

(Equation 1)

As shown in FIGS. 1 and 3, when the luminescence measurement method of the present invention was used, the dynamic range of each test sample was increased up to about 10 times compared with the conventional colorimetric detection.
Figures 2 and 4 and Table. As shown in FIG. 1, when the luminescence measurement method of the present invention was used, the minimum detection concentration of each test sample was at least about 5 times lower than that of the conventional colorimetric detection.

[0041]

[Table 1]

[Brief description of the drawings]

FIG. 1 shows AF-2,4N obtained in Examples and Comparative Examples
A value obtained by dividing the response at each concentration of QO and 1-NP by the response under the condition not including the test sample, Ratio (0 to 0)
It is a graph which shows 100).

FIG. 2 shows AF-2,4N obtained in Examples and Comparative Examples
A value obtained by dividing the response at each concentration of QO and 1-NP by the response under the condition not including the test sample, Ratio (0 to 0)
It is a graph which shows 10).

FIG. 3 shows 2-AA and Ba obtained in Examples and Comparative Examples.
It is a graph which shows the value Ratio (0-80) which divided the response in each density | concentration of p by the response under the conditions which do not contain a test sample.

FIG. 4 shows 2-AA and Ba obtained in Examples and Comparative Examples.
It is a graph which shows the value Ratio (0-10) which divided the response in each density | concentration of p by the response under the conditions which do not contain a test sample.

────────────────────────────────────────────────── (5) Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:42) C12N 15/00 A (56) References JP-A-5-317043 (JP, A) JP-A-4-12118 (JP, B2) “The New Chemistry Experiment Course 14 Development, Differentiation, and Aging” edited by The Biochemical Society of Japan, Tokyo Chemical Industry Co., Ltd., p. 322-3 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00-15/70 C12Q 1/66

Claims (2)

(57) [Claims] 1. A set comprising a SOS gene expressed at the time of DNA damage, a gene arranged downstream of the gene and expressing a luciferase activity, and a gene expressing an enzyme that catalyzes the production of a substrate for the luciferase activity. Transgene
Salmonella transformed by E. coli . 2. The method according to claim 1 , wherein the Salmonella bacterium according to claim 1 is cultured in a medium containing a test sample, and luminescence due to expression of a group of genes that express luminescence activity is measured.
A method for determining the presence or absence of a genotoxic substance such as a mutagenic substance or the like in a test sample.