JPH07227285A - Method for detecting mutagen without using reagent - Google Patents

Abstract

PURPOSE:To provide a method having about ten times wider a measuring range of a genetic poison such as a mutagen than a conventional colorimetry, the minimum detection concentration reduced by <= five times and being an extremely simple operation. CONSTITUTION:A host microorganism transformed with a recombinant gene containing an SOS gene to manifest during damage of DNA, a gene to manifest luciferase activity arranged at the downstream of the gene and a gene to manifest an enzyme capable of catalyzing production of a substrate with luciferase activity is cultured in a medium containing a specimen to be tested. A method which determines existence of a genetic poison such as a mutagen in the specimen to be tested for measuring luminescence by the manifestation of the gene group to manifest luminescence activity or measures the existing amount is provided.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a luminescence-based method for detecting a mutagen.

[0002]

2. Description of the Related Art At present, many mutation test methods using microorganisms have been reported as short-term screening methods for carcinogens. Am, which is a reverse mutation test
The es test is the most widely used. However, this test method has a problem that it takes about 3 days until measurement and aseptic operation is required. Recently, unlike the Ames test for detecting the inducibility of mutation, a short-term mutagen detection method (umu test, SOS chromotest) for detecting DNA damage was reported (Oda, Nakamura et al., "Evaluation 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).

According to these methods, the SOS response induced upon DNA damage is one of the SOS genes, umuD ,
In this method , the lacZ gene is linked to the downstream of the C gene or sfiA gene to measure the expression level of β-galactosidase, and the DNA damage and mutagenicity are detected. More specifically, the gene-introduced Salmonella (umu test) and E. coli (SOS chromotest) are cultured in the presence of a test substance, and the culture solution is diluted with a predetermined buffer and then diluted with toluene or Chloroform and an aqueous solution of a sulfonate type surfactant such as sodium dodecylbenzenesulfonate are 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, was added, and the enzyme reaction was carried out at 28 ° C. Stop the reaction 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
The β-galactosidase activity is calculated based on Arbor Laboratory, 1972).

The above-mentioned umu test and SOS chromo test are characterized in that they can be detected rather easily and rapidly than the Ames test. When the operations are arranged according to steps, the first step for inducing the SOS reaction, specifically, the step of culturing a host microorganism in the presence of a test sample, and the step of expressing in the cells were performed. β
-The galactosidase activity can be roughly divided into the second step of detecting by chromogenic reaction.

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

As described above, the test method based on color detection requires complicated operation in the second step and has low detection sensitivity. Therefore, in order to compensate for this drawback, the color development time is lengthened or complicated. However, there are problems that the rapidity of detection is impaired when various microbial cell crushing processes are performed, and the addition of an expensive coloring reagent is indispensable.

[0008]

Therefore, the present invention does not particularly require the complicated operations in the above-mentioned second step, namely, the operations of dilution, cell disruption treatment, addition of substrate, progress and termination of color reaction. , SOS that is significantly simplified in operation, and 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 utilizing the DNA damaging property of a gene.

[0009]

As a result of various studies to solve the above problems, the present inventors have found that a gene expressing luciferase activity downstream of the SOS gene susceptible to DNA damage and its luciferase activity. When a microorganism transformed with a recombinant gene in which a gene that expresses an enzyme that catalyzes the production of the substrate is placed in a medium containing a genotoxic substance such as a mutagen, the expression of the SOS gene A gene having a luciferase activity and a gene expressing an enzyme that catalyzes the production of the substrate for the luciferase activity are also expressed, and as a result, the condition that the substrate for the luciferase activity is continuously supplied in the cells is achieved, and the continuous luciferase activity is achieved. Luminescence 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 a response obtained under the condition that a genotoxic substance such as a mutagenic substance is divided by a response obtained under a condition that does not contain a genotoxic substance is analyzed as an index, it is a typical mutagenic substance. (2-furyl) -3- (5-nitro-2)
In the case of the conventional colorimetric detection in the case of -furyl) acrylamide, this value increases as the content of the genotoxic substance increases and changes from 1 to around 10, whereas in the case of the luminescence detection of the present invention, We have found that it varies from 1 to around 100. As described above, in the case of the present invention, it was revealed that the dynamic range of measurement was expanded by up to about 10 times as compared with the conventional method, and the genotoxic substance could be detected and quantified with high sensitivity and high accuracy.

Further, as a criterion for determining whether a genotoxic substance is present or not, it is generally determined that genotoxicity is obtained by showing a double response to the condition that the test sample is not added, The concentration showing a double response is called the minimum detected concentration. As a result of conducting tests on various genotoxic substances, the minimum detection concentration of luminescence detection of the present invention is:
Compared with the conventional colorimetric detection, the concentration was at least about 5 times lower, which revealed that the genotoxic substance can be detected with high sensitivity.

Therefore, the present invention comprises a SOS gene expressed upon DNA damage, a gene expressing luciferase activity located downstream of the gene and a gene expressing an enzyme which catalyzes the production of a substrate for the luciferase activity. A recombinant gene consisting of The present invention also provides a host microorganism transformed with the above gene.

The present invention further provides luminescence obtained by culturing the host microorganism in a medium containing a test sample and expressing a gene group expressing an enzyme which catalyzes the production of luciferase activity and a substrate for the luciferase activity. A method for determining the presence or absence of a genotoxic substance such as a mutagenic substance in a test sample or measuring the abundance of the test substance, which is characterized by being measured.

In the present invention, the SOS gene is used as an expression control means for expressing a gene expressing an enzyme which catalyzes the luciferase activity and the production of its substrate depending on the damage of DNA. Therefore, SOS
Any gene can be used as long as it is expressed when DNA is damaged.
It may be any as long as it includes a control portion called an SOS box. Therefore, in the present invention, the SOS gene expressed at the time of DNA damage means one containing a control portion called the SOS box, which may be the SOS box itself, or any DNA fragment containing this. May be.

Examples of the SOS gene include umu gene, for example, umuC gene and umuD gene, and sfi
Examples of the gene include A gene, but are not limited thereto. Among these genes, the umuD and umuC genes are Pro.Nat
l.Acad.Sci.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
3), the umuD and umuC genes can be easily obtained from this plasmid.

The gene expressing the enzyme that catalyzes the luciferase activity and the production of its substrate may be any gene as long as both genes are expressed under the control of the SOS gene. Examples include, but are not limited to: A marine bacterium having a bioluminescence gene group having the above-mentioned properties is Vibrio (Vibrio) .
In Vibrio (Vibrio) of the genus io) group, Vibrio Harvey (V. harveyi), Vibrio fischeri (V. fische
ri ), V. Splendidu
s ), Vibrio cholera ( V. cholerae ), and in the genus Photobacterium (Photobacterium), there are Photobacterium phosphoreum ( P. phosphoreum) , Photobacterium reingnathi ( P. leiognathi ), and the like.

For example, V. fics ( V. fics
The bioluminescence gene group derived from heri ) removes the operator region of the bioluminescence gene group to facilitate the use as a reporter gene, and catalyzes the production of structural gene part (gene group expressing luciferase activity and its substrate aldehyde). Various cassette vectors containing only the gene group expressing the enzyme fatty acid reductase activity), for example, pUCD320, pUCD613, pUCD614, pUCD618, pUCD62
0, pUCD623, pUCD1111 etc. have been reported. For these cassette vectors, see Clarence I. Kado et al., Plant.
Molecular Biology Reporter, 5 , 225 (1987), and the structural gene portion of the bioluminescence gene group can be easily obtained from these vectors.

In the present invention, the luciferase substrate means a long-chain aldehyde and the like, and the enzyme that catalyzes the production of these is NAD (P) H: FMN reductase,
Means fatty acid reductase and the like. In the above example, the gene of the same origin was used as the gene expressing the luciferase activity and the gene of the enzyme that catalyzes the production of the luciferase substrate, but it is also possible to use these genes of different origins by linking them. 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, that is, SOS.
Any microorganism having a function may be used.
richia coli ), Salmonella typhimur
ium) , for example, the TA1535 strain, TA1538 strain, etc.
Yeast (Saccharomyces cerevisiae ) etc. are mentioned.

A recombinant gene comprising the SOS gene and a gene expressing an enzyme that catalyzes the production of luciferase activity and its substrate is a DNA fragment containing at least the SOS box of the SOS gene, which is downstream of the SOS box. , Luciferase activity and a group of genes expressing enzymes that catalyze the production of its substrate can be linked. This ligation can be carried out by a conventional method using DNA ligase. In order to introduce this recombinant gene into a host microorganism, the recombinant gene needs to be present in a vector, and as the plasmid, for example, pBR type plasmid, pUC type plasmid or the like can be used.

To transform a host microorganism with the expression vector, a conventional method used for transforming a microorganism such as a bacterium may be used. 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 then the transformed host microorganism is cultured in this medium. Culturing is usually performed for 1 to 3 hours. Since the culture solution containing the test sample itself emits light by culturing, the light can be detected immediately after culturing according to a conventional method. When the test sample contains a genotoxic substance such as a mutagenic substance, the amount of luminescence increases in a concentration-dependent manner, so the genotoxic substance such as a mutagenic substance in the test sample is detected or measured from this increased amount. 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 even a gas or a solid can be measured as long as it can be dissolved in the medium.

[0023]

According to the method of the present invention, the test sample and the medium containing the transformed host microorganism can be mixed and the luminescence can be detected immediately after culturing. Therefore, the complicated operation of the second step is not required. For this reason, the detection time can be greatly shortened by a simple operation, the reagent is not required, and thus the measurement can be performed at a low cost. Furthermore, since the luminescence detection is performed, the dynamic range of the measurement is up to 10 compared to the conventional colorimetric detection. It is possible to detect with high sensitivity and high accuracy the minimum detection concentration of a genotoxic substance such as a mutagenic substance at least about 5 times lower than the low concentration side.

[0024]

EXAMPLES Example 1. Construction of luminescent vector and preparation of transformant As SOS gene, Escherichia coli (CSH26 "Miller, JH, Ex
periments in Molecular Genetics, Cold Spring Harbor
Laboratory, 1972 ": F ^- ara del (lac-pro) thi). The umuD and C genes of plasmid pSK1002 were used. As a gene expressing an enzyme that catalyzes the luciferase activity and the production of its substrate, Escherichia coli (HB10
^{_{1: hsd20 (r B -,}} m B -), recA13, ara-14, proA2, lacY1, ga
plasmid pUCD620 (Clarence I. Kado et al., Plant Molecular Biol) introduced in lK2, rpsL20, xyl-5, mtl-1, supE44).
Ogy Reporter, 5 , 225 (1987)) was used.

As a 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 ^+). Luminescence measuring device
Micro Lumino Reader (MLR- manufactured by Corona Electric Co., Ltd.)
100) was used.

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

A plasmid was isolated from the obtained transformant by the alkali extraction method. This plasmid was designated Mlu I
After cleaving with T4, it was blunt-ended with T4 DNA polymerase. Acts as a stop codon for umuC , and Ba
DNA linkers (TAGGATCC with m HI site
TA) was chemically synthesized. This synthetic linker and the above 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 the 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 the plasmid pUCD620
After culturing B101 in an LB medium containing ampicillin, a large amount of pUCD620 was prepared by the alkali extraction method. Plasmid pUCD620 was cloned into 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 Bam HI- Sal I DNA fragment of about 7.4 kb were added to T4.
A luminescent vector was constructed by ligating with DNA ligase.

SJ10002 was cultured overnight in LB medium. To LB medium, 1/100 volume of the above culture solution was added,
Incubation was performed until the turbidity at 600 nm (OD ₆₀₀ ) was about 0.4. Centrifuge 5 ml of the culture broth to obtain a precipitate fraction of 5 ml.
Suspended in 30 mM CaCl ₂ aqueous solution and left in ice for 45 minutes. Centrifuge again to add 0.4 ml of 30 mM
It was suspended in a CaCl ₂ aqueous solution to prepare a competent cell of SJ10002.

TA1535 was cultured overnight in LB medium.
Add 1/100 volume of the culture solution to LB medium,
Culturing was performed until the turbidity at 0 nm (OD ₆₀₀ ) was about 0.4. Centrifuge 5 ml of the culture broth and add the precipitated fraction to 5 ml of 1
0mMCaCl _2, 10mMMnCl _2, 10mMMgCl ₂
Suspended in aqueous solution and left in ice for 45 minutes. Centrifuge again to add 0.4 ml of 10 mM CaCl ₂ , 10 mM.
Suspend in MnCl ₂ , 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 the mixture was left in ice for 30 minutes. After treating at 42 ° C. for 2 minutes, it 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 by the same method as described above.
Transformation into 1535 was performed.

Example 2. Luminescence Metabolic Activating Enzyme Obtained from rat liver, containing phenobarbital and 5,6
-S9 fraction (manufactured by Oriental Yeast Co., Ltd.) treated with benzoflavone, and Cofa as a cofactor
S9 mix in which ctor-I (manufactured by Oriental Yeast Co., Ltd.) was mixed 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 (tryptone 1%, salt 0.5%, glucose 0.2%, ampicillin 20 μg / ml) at 30 ° C. overnight.
It was cultured in. This culture solution was inoculated into a TGA medium at 1/50 volume and cultured at 30 ° C. for 1.5 hours. OD this culture
It diluted with TGA culture medium so that it might become ₆₀₀ = 0.1. Each 1.45 ml of this diluted culture solution was dispensed into a test tube, 50 μl of a test sample having a predetermined concentration was added to each, and the mixture was incubated at 30 ° C. for 2 hours. If a metabolic activation enzyme is required, 0.25 ml of S9 mix and 50 μl of test sample should be added to 1.2 ml of diluted culture medium.
Was added and the mixture was cultured at 30 ° C. for 2 hours. Immediately after the culturing, the luminescence amount of 100 μl of the culture solution was measured by a luminescence measuring device. Also,
The OD ₆₀₀ of the culture was measured.

Activity (Intensity / OD ₆₀₀ )
Was determined by measuring the amount of light emission for 10 seconds and dividing the value by the value of OD ₆₀₀ . The measurement results are shown in FIGS.
The following comparative examples were carried out in order to compare the sensitivity of the present invention and the conventional method.

Comparative Example Using Salmonella bacterium (TA1535 / pSK1002) into which plasmid pSK1002 was introduced, 0.1mo
0.7 l / l potassium phosphate buffer (pH 7.0) 11
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 solution.

TA1535 / pSK1002 was added to TGA
Incubated in medium overnight at 37 ° C. This culture solution was inoculated into a TGA medium at 1/50 volume and cultured at 37 ° C. for 1.5 hours.
This culture solution was diluted with TGA medium so that OD ₆₀₀ = 0.1. Each 1.45 ml of this diluted culture solution was dispensed into a test tube, 50 μl of a test sample having a predetermined concentration was added to each, and the mixture was incubated at 37 ° C. for 2 hours. When metabolic activation enzyme is required, S9 mix 0.25m in 1.2ml diluted culture solution
l and 50 μl of the test sample were added, and the mixture was incubated at 37 ° C. for 2 hours. The OD _{600 of} this bacterial solution was measured.

The remaining bacterial solution (0.1 ml) was added with Z-buffer solution 0.9.
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).
A 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 immediately measured. The activity (unit) was calculated by the following formula based on the Miller method.

[0039]

[Equation 1]

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

[0041]

[Table 1]

[Brief description of drawings]

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

FIG. 2 shows AF-2,4N obtained in Examples and Comparative Examples.
Value obtained by dividing the response at each concentration of QO and 1-NP by the response under the condition that the test sample was not included Ratio (0 to
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 on the condition which does 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 on the condition which does not contain a test sample.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12Q 1/66 6807-4B // (C12N 15/09 C12R 1:63) (C12N 15/09 C12R (1:01) (C12N 1/21 C12R 1:42) (C12N 1/21 C12R 1:19) C12R 1:63) (C12N 15/00 A C12R 1:01)

Claims (3)

[Claims] 1. A set comprising an SOS gene that is expressed upon DNA damage, a gene that is located downstream of the gene and that expresses a luciferase activity, and a gene that expresses an enzyme that catalyzes the production of a substrate for the luciferase activity. Replacement gene. 2. A host microorganism transformed with the recombinant gene according to claim 1. 3. A mutation in a test sample, which comprises culturing the host microorganism according to claim 2 in a medium containing the test sample, and measuring luminescence due to expression of a gene group expressing luminescence activity. A method for determining the presence or absence or determining the abundance of a genotoxic substance such as a protozoal substance.