JPH08116991A - Examination of toxicity using nematode - Google Patents

Abstract

PURPOSE: To provide a method for examining toxicity to argamisms of a chemical substance such as a heavy metal which exists in a specimen. CONSTITUTION: A thermal shock promoter is activated by existence of a chemical substance such as a heavy metal which has toxicity to organisms. Applying this property, a nematode is modified by genetic engineering technique using a plasmid ohtained by binding a firefly luciferase gene to a thermal shock promoter to prepare a transgenic nematode. The existence of a chemical substance such as heavy metal which has toxicity to organisms is detected as development of luciferase in the nematode. Luciferase in the nematode is finally detected as increase of luminous amount by luminescent reaction, by using an extracted solution of the nematode.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting toxicity of a chemical substance such as a heavy metal in a sample to a living organism in a very short time by using a transgenic nematode modified by genetic engineering.

There are a huge number of chemicals in existence today,
The number is increasing every day. Chemical substances include toxic substances such as pesticides, physiologically active substances produced by microorganisms, heavy metals, and air pollution gases. Heavy metals (lead, mercury, cadmium, chromium, etc.) are harmful to the natural environment and often have extremely strong toxicity to living organisms. It is well known that heavy metals discharged into the natural environment through the effluent of some industries and agriculture are bio-concentrated and eventually cause serious damage to humans due to the food chain. Therefore, it is desired to be able to quickly detect the toxicity of toxic substances contained in tap water or waste water, especially heavy metals. INDUSTRIAL APPLICABILITY The present invention is applied to inspection and management of water quality of tap water, industry, agriculture or domestic wastewater, and detection of toxicity of chemical substances such as heavy metals existing in the natural environment.

[0003]

2. Description of the Related Art Higher animals such as mice, rats, dogs, rabbits and monkeys have been used for detecting toxicity of chemical substances. However, the toxicity test using these higher animals requires a great deal of labor, cost and a long time, and cannot cope with a huge number of chemical substances. Therefore, conventionally, for carcinogenicity and mutagenicity, a test method using a bacterium such as Salmonella is often used. However, bacteria are unicellular organisms, no higher-order life phenomena such as morphogenesis and organ formation are observed, and the metabolic system has a large gap with animals that are multicellular organisms. Therefore, it is desired to establish a rapid and inexpensive method for detecting toxicity of a huge number of chemical substances using animals.

Thus, Miwa et al. Proposed a toxicity test method using nematodes for mutagenicity, teratogenicity and neurotoxicity of chemical substances (JP-A-55-153599). Caenorhabditis elegans (Ca
Enorhabditis elegans) is a kind of self-active soil nematode with a length of about 1 mm, and hermaphrodites can grow by self-fertilization. The time required to change generations is 25 ℃
It takes about 2 days, which is faster than other animals, and about 300 offspring are born from one hermaphroditic individual, so a large number of individuals can be obtained in a short period of time. Since Escherichia coli is fed on an agar medium for feeding, the breeding is extremely simple and low cost, and the observation of the body structure and movement of the nematode can be facilitated. Since nematodes are animals, it is possible to estimate the toxicity of chemical substances to higher animals such as humans with some degree of affinity. According to the test method of Miwa et al., Mutagenicity and teratogenicity of the test substance are detected within a relatively short period of 2 to 4 days.

[0005]

However, the method of Miwa et al. Described above involves a step of observing the nematode or its embryo in order to detect the toxicity of the chemical substance, which requires much labor and time. Further, for that purpose, a certain degree of skill and observation eye are required.

The present invention solves the above problems and makes it possible to biologically detect the influence of a chemical substance such as a heavy metal contained in a test sample on an organism in an extremely short time and by a relatively simple operation. The purpose is to

[0007]

[Means for Solving the Problems] The first invention is to cultivate a nematode modified by genetic engineering in a medium containing a test sample and to measure the induced enzyme activity.
A toxicity test method using nematodes, characterized in that the test sample is tested for toxicity to organisms.

[0008] In a second aspect of the present invention, the nematode comprises a hybrid gene (fusion gene) in which the firefly luciferase gene is linked to a heat shock promoter of the nematode, Cenolabditis elegans.
2. A transgenic nematode obtained by introducing into E. elegans), wherein the enzyme activity is the activity of a heat shock promoter that induces gene expression by the action of a substance having biotoxicity. It is a toxicity test method using nematodes of.

A third invention is the toxicity test method using nematodes according to the second invention, characterized in that the activity of the heat shock promoter is measured by increasing the expression level of luciferase.

A fourth invention is the line according to the first invention, the second invention or the third invention, characterized in that the test sample contains a heavy metal, and the toxicity of the heavy metal to organisms is tested. This is a toxicity test method using insects.

[0011]

In the present invention, a plasmid in which the firefly luciferase gene is ligated directly under the heat shock promoter of nematode is constructed, and this plasmid is microinjected into a wild strain of nematode, and the plasmid can be stably subcultured. A nematode was produced.

The promoter portion of the gene controls that the gene is properly and properly transcribed into mRNA in various tissues. The heat shock protein was discovered as a protein whose expression level increases when it is exposed to an abnormally high temperature for the organism, and is found in many organisms including not only nematodes. The heat shock promoter controls the expression of the heat shock protein, and the promoter activity is increased at high temperature to induce the synthesis of the heat shock protein. Moreover, the heat shock promoter is not only responsive to high temperatures, but also responsive to the presence of chemicals such as heavy metals if it is toxic to living organisms. The present invention utilizes this property of the heat shock promoter,
The activity of the heat shock promoter increased by a chemical substance such as a biotoxic heavy metal is measured by the increase in the expression level of the firefly luciferase gene linked to this promoter. The expression level of the firefly luciferase gene can be obtained by adding luciferin, which is a substrate, to firefly luciferase in the presence of adenosine triphosphate to decompose luciferin, and measuring the amount of luminescence emitted at that time. The amount of luminescence can be directly measured with a scintillation counter or the like. The amount of luminescence depends on the amount of luciferase added under certain conditions. Therefore, in the present invention, after breeding the transgenic nematodes in a sample, the worms are crushed to prepare an extract, and the amount of luciferase contained therein is measured as an increase in the amount of luminescence, thereby determining biotoxicity. It becomes possible to easily and quantitatively detect the presence of chemical substances such as heavy metals.

[0013]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described with reference to the drawings.

Example 1 Plasmid pLuc49.8
2) The plasmid pL in which the firefly luciferase gene was ligated downstream of the heat shock promoter of C. elegans
uc49.83 was constructed as follows. This will be described with reference to FIG. A commercially available plasmid pMAM-neo-luc in which the firefly luciferase gene was inserted was digested with restriction enzymes SalI and KpnI, and a DNA fragment of about 1.9 kb containing the luciferase gene was isolated. on the other hand,
The plasmid pPD49.83 containing the promoter of the nematode heat shock protein hsp16-41 was transformed into SmaI.
It is cleaved and cleaved, and a SalI linker is added to the resulting end by T4
SalI and KpnI after ligation with ligase
Disconnect with. The heat shock promoter hsp16-
For 41, Molecular and Cellular Biology (Molec. Cell. Biol. 3, 2)
1 (1992)). A DNA fragment containing the above luciferase gene was added to pPD49.83 treated as described above, and ligated using T4 ligase to insert the firefly luciferase gene in the forward direction downstream of the heat shock promoter. Plasmid DNA was recovered from the transformant obtained by introducing this into E. coli and designated as plasmid pLuc49.83.

Example 2 Preparation of Transgenic Nematode A transgenic nematode having the above plasmid pLuc49.83 was prepared as follows. Microinjection of plasmid DNA into nematodes is essentially
Molecular and Cellular Biology (Mol. Cell. Biol. 10, 3959, 1)
It was performed according to the method described in 991).

The glass needle used for microinjection was prepared by working a glass tube with a core having a diameter of 1 mm using a micro electro-doppler. The thickness of the tip was 1 μm or less. The plasmid DNA used for injection was prepared by cesium chloride density gradient centrifugation, and the DNA was buffered (2% polyethylene glycol 6000, 20 mM potassium phosphate, 3 m).
M potassium citrate, pH 7.5) and placed in the needle.

80% to 9% of 2% agarose dissolved in pure water
Keep at 0 ° C and place a drop on a glass slide. A thin glass agarose pad was created by covering it with a cover glass and pressing the center with your fingers. It was dried in an oven at 100 ° C for 1 hour with the agarose side facing up. A drop of paraffin oil is placed on an agarose pad and a young adult hermaphroditic nematode is placed therein. Observing with an inverted Nomarski differential interference microscope, waiting for the movement to slow down, operating a micromanipulator to insert a needle tip into the gonad, and injecting a plasmid DNA solution by pressure. Immediately after the end of the microinjection, immediately use M9 buffer (6 g Na ₂ HP in 1 L pure water).
O ₄ , 3 g KH ₂ PO ₄ , 5 g NaCl, 0.5 mM
MgSO ₄₎ was added dropwise over the insect. After the microinjection, the nematodes were taken out using a glass capillary, and NG plates on which E. coli was grown (3 g NaCl, 5 in 1 L pure water).
gBacto-peptone, 17g agar, 5mg cholesterol, 0.5mMCaCl _2, 1mM MgS
It was bred on O ₄ , 25 mM potassium phosphate [pH 6.0]).

In nematodes, DNA introduced into the gonad by microinjection becomes a large molecule in which a large number are bound regardless of whether the shape is circular or linear. It usually exists as an extrachromosomal factor without being integrated into the chromosome. It is believed that the larger the molecule produced, the more likely it is to be passed on to the next generation. Outpatient DN
When transformation is carried out using A, an appropriate amount of the marker plasmid pRF4 containing the gene roll-6 (su1006) is mixed with the target gene and microinjected in many cases. For mol-6, Molecular and Cellular Biology (Mol. Cell. Bio
l. 10, 2081 (1990)).
When DNA is bound after microinjection, microinjected DNA may be taken up randomly depending on concentration. Roll-6 is a gene encoding a kind of collagen that constitutes the cuticle of the body surface, and in the individual having the dominant mutation gene su1006, the cuticle structure is spirally formed along the body axis. Therefore, when moving forward or backward, the body rotates about the body axis and exhibits an arc-like movement, that is, a roller phenotype. Transformants are detected as roller individuals in the first generation (F1) born from hermaphrodites that received microinjection. Of these,
There are usually several tens of rollers that generate rollers in the next generation (F2).
%, And was established as a strain that continues to stably hold the plasmid DNA introduced with these.

In the present invention, the above plasmid pLuc. Four
9.83 was microinjected into wild type N2 with pRF4. The concentrations of pLuc49.83 and pRF4 were 50 μg / ml and 100 μg / ml, respectively. Microinjected nematodes were incubated at 20 ° C. Usually, about 10 F1 rollers were obtained from one microinjected hermaphroditic individual, and 10 to 30% of them produced roller individuals even after the next generation. Among the transgenic nematodes thus obtained, the TM-3 strain and the TM-4 strain were used here as representatives and a toxicity test was conducted.

Example 3 Preparation of Extract from Transgenic Nematode Treated with Heavy Metal Solution and Measurement of Luciferase Activity In this example, a heavy metal solution was used as a test sample, and whether it showed toxicity to living organisms. I'll do an examination,
The embodiment of the present invention is not limited to this, and the test sample is not limited to the heavy metal solution.

Treatment of the transgenic nematodes with heavy metals (lead and cadmium) was carried out as follows. NG agar medium containing various concentrations of heavy metals was prepared, inoculated with Escherichia coli, and cultured at 37 ° C. overnight. The transgenic nematode TM-3 strain or TM-4 strain was put into each of the 5000 individuals and cultured at 20 ° C. for 2 hours. After the treatment, the worms were washed with M9 buffer, transferred to a centrifuge tube, and centrifuged at 1000 rpm for 1 minute. After washing again with M9 buffer and centrifuging, the supernatant is discarded and a buffer for cell disruption (20 mM Tris
-Phosphoric acid [pH 7.8], 2 mM dithiothreitol,
2 mM 1,2-diamino cyclohexan
e-N, N, N ', N'-tetraacetica
cid, 10% glycerol, 1% Triton X-
Add 100 ml of 100) and stir. The suspended worms were dropped into a mortar containing liquid nitrogen, immediately frozen, and then ground until powdered. The insect powder was transferred to a test tube and dissolved on ice, and used as a nematode extract for the measurement.

The activity of luciferase contained in the nematode extract was measured as follows. Nematode extract 10 ⁵
To 20 μl of the double-diluted product, 100 μl of a luminescent substrate luciferin (manufactured by Toyo Ink Co., Ltd .: Piccagene) was added, and the amount of luminescence was measured by a scintillation counter. Since the amount of total protein contained in the extract was different for each sample, the amount of protein contained in the sample was measured, and the amount of luminescence was converted to 1 mg of protein in the sample. A protein assay kit (manufactured by Bio-Rad) was used for protein quantification, and the reaction solution of the kit was 900 μm.
Add 20 μl of nematode extract to 1 and add 595 with spectrophotometer.
It was measured by looking at the absorption at nm.

By culturing TM-3 and TM-4 in the presence of a heavy metal-containing sample, it was found that the firefly luciferase synthesis was induced in the transgenic nematode strains TM-3 and TM-4. It can be detected in a very short time of 2 to 4 hours, and it was found that the heavy metals in the sample are toxic to the organism. In addition, the amount of increase in the luminescence amount varies depending on the type of heavy metal contained in the medium, and a correlation with the amount of heavy metal was observed, so it is possible to quantitatively detect heavy metal having biotoxicity. It will be possible.

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, by using a genetically engineered transgenic nematode, it is relatively easy and highly reliable in a shorter time than before, without the need for skill or special observation eyes. Biological detection of toxicity of chemical substances such as heavy metals to living organisms is possible. To protect the environment, we have to deal with a huge number of samples.
The present invention addresses this problem and is useful for detecting and evaluating the toxicity of chemical substances such as heavy metals to the environment and organisms.

[Brief description of drawings]

FIG. 1 is a procedure for constructing a plasmid pLuc49.83 used for producing a transgenic nematode. KpnI, Sal
I and SmaI represent restriction enzymes.

[Explanation of symbols]

 None

Claims (4)

[Claims] 1. A method for testing the toxicity of a test sample to an organism by culturing a nematode modified by genetic engineering in a medium containing the test sample and measuring the induced enzyme activity. Toxicity test method using characteristic C. elegans. 2. A hybrid gene (fusion gene) in which the firefly luciferase gene is linked to a heat shock promoter of nematode, wherein Cenolabditis
Elegance (Caenorhabditis eleg)
Ans) is a transgenic nematode, wherein the enzyme activity is the activity of a heat shock promoter that induces gene expression by the action of a substance having biotoxicity. Toxicity test method using nematodes. 3. The activity of the heat shock promoter is
The toxicity test method using a nematode according to claim 2, wherein the measurement is performed by increasing the expression level of the luciferase. 4. The test sample contains a heavy metal, and the toxicity of the heavy metal to an organism is tested.
Alternatively, a toxicity test method using the nematode according to claim 2 or claim 3.