WO2018133513A1 - Genotoxic substance detection vector and detection method thereof - Google Patents

Abstract

A genotoxic substance detection vector and a detection method thereof are provided. The vector is an Escherichia coli expression vector sequentially having, from a 5' end to a 3' end, a genotoxic response promoter, a bacteriophage lysis gene, and an Escherichia coli terminator. The detection method includes introducing the genotoxic substance response vector into Escherichia coli to obtain recombinant bacteria, incubating the recombinant bacteria and genotoxic substances, and lysing the Escherichia coli. The recombinant Escherichia coli has the genotoxic response vector, and a self-cell lysis is initiated when the recombinant bacteria contacts the genotoxic substances. The detection method quantitates genotoxic substances by means of lysis efficiency. The method consumes little time, has a high detection sensitivity, is easy to implement, low cost, and easy to promote.

Description

Genetic toxicity substance detection carrier and detection method Technical field

The invention relates to the technical field of detecting genotoxic substances in the environment, in particular to detecting genotoxic substances in the environment by using recombinant E. coli carrying a reporter gene.

Background technique

Methods for the detection of genotoxic substances are divided into long-term tests and short-term tests. The long-term test method is time-consuming and labor-intensive, and the long-term maintenance cost of experimental animals is high, and has been gradually replaced by a short-term screening method that is fast and low-cost. Short-term testing is the screening of chemical mutagenizing factors by cytogenetic indicators, usually using biological cells such as plants, mammals, and microorganisms to monitor the genotoxicity of residues. At present, there are comet assays, sister chromatid exchange tests, SOS chromogenic assays, extraprogrammed DNA synthesis assays, bacterial back mutations, and prophage induction assays. These detection methods often have factors that are unreasonable for promotion, such as complicated operation, long detection time, and strict aseptic operation. And due to patent protection and other reasons, individual testing methods are not conducive to domestic promotion, which limits its widespread use.

The genotoxicity test refers to in vitro and in vivo tests for detecting a test substance that directly or indirectly induces genetic damage by different mechanisms, which can detect DNA damage. This DNA damage is one of the links in the development of malignant tumors. In recent years, some short-term rapid in vitro genotoxicity test methods have been established to detect DNA damage. Traditional methods for detecting genotoxic substances are gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry or high pressure liquid chromatography-mass spectrometry. These technologies enable accurate and quantitative detection of hundreds of these chemicals with trace levels of accuracy. However, these methods require large-scale precision instruments for testing, and these methods have problems such as long detection period, cumbersome operation, high cost, and high analytical performance required by analysts, which cannot meet the requirements of on-site inspection. Therefore, the development of a simple, fast and economical means of detection can meet the needs of on-site testing. More than 200 short-term genotoxicity tests have been established. Among them, the genotoxicity test methods that have been studied and applied are: Coment assay, also known as Single Cell gel electrophoresis (SCGE), Sister chromatid exchange (SCE). , extra-procedural DNA synthesis (also known as DNA repair synthesis, Unscheduled DNA synthesis, UDS), Salmonella typhimurium reverse mutation test (The reverse mutation test of salmonella Typhi munine, also known as Ames test), SOS chromotest, Prophage induction assay, etc. Among the above methods, the Ames test is the most conventional method in genotoxicity analysis. McCann's test of 300 chemicals using the Ames test showed that most carcinogens are mutagens with a correlation of more than 80%. The advantage of the Ames test is that the method is sensitive and the detection rate is high; the method is simple, easy, and does not require special equipment, and is easy to promote. The disadvantages are: (1) the DNA repair system of the microorganism is simpler than the mammal, the gene is not as large as the mammal, and cannot fully represent the actual situation of the mammal; (2) the sample containing histidine, glycine or lactose cannot be detected; The workload is large, the detection time is long, and the strain is not easy to store. Despite this, due to the above advantages, it is currently the most important test method in mutagenicity testing. Many countries, such as Canada, the United States, and Japan, have placed the Ames test at the preferred location in the mutagenesis test system. Combined with cytogenetic in vitro or in vivo tests, it can be used as a first-stage screening method.

The SOS chromogenic assay was designed based on the genotoxic substance-induced SOS repair initiation and expression of the umuC gene (G. Reifferscheid, J. Heil, Y. Oda and RKZahn, et al. A microplate version of the SOS/umu -test for rapid detection of genotoxins and genotoxic potentials of environmental samples. Mutation Research, 253 (1991) 215-222). In 1982, Quillardet et al. first used the SOS reaction principle to detect genetic toxicants. They constructed the plasmid containing the sifA-LacA fusion gene, Quillardet, P., Huisman, O., D'Ari, R. et al. SOS chromotest, a Direct assay of induction of an SOS function in Escherichia coli K-12to measure genotoxicity. Proceedings of the National Academy of Sciences of the United States of America 79, 5971-5975, 1982). In 1985, based on the test results of 83 different compounds, the researchers considered that most of the substances that were positive for Ames test were also positive for the SOS test. In 1985, Oda et al. merged with umuC and LacA, and the umu test was officially proposed (Oda, Y. Induction of SOS responses in Escherichia coli by 5-fluorouracil. Mutation research 183, 103-108, 1987). In 1986, IARC Publication No. 83 listed the law as one of a series of methods for carcinogens: a critical appraisal. Reports of an ad-hoc working group. Lyons 2-6December 1985. IARC scientific publications, 1-564, 1986.). In 1991, Reifferscheid et al. further improved the umu test by using a 96-well microplate instead of a test tube, combined with computer technology, to enable automated, high-throughput screening of genetic toxicants. In Germany, the umu test is the first standardized test for the detection of genotoxicity of wastewater (DIN 38415-3) (Reifferscheid, G., Heil, J., Oda, Y. et al. A microplate version of The SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples. Mutation research 253, 215-222, 1991.). The existing SOS/umu-based genotoxicity detection and detection system has a long detection time and is cumbersome. Even after the detection bacteria have been activated, the entire detection process still takes about 6 hours, and the bacteria are required to be lysed to release beta-galactosidase, and the beta-galactosidase enzyme activity is determined by the enzyme activity.

Genotoxic pollutants are widely distributed in China's environment, and their types and quantities are large. They are representative of environmental pollutants and are harmful to humans. It is very important to establish a rapid, effective, convenient and convenient detection method.

Summary of the invention

The invention aims at the deficiencies of the prior art, and provides a convenient operation, high sensitivity, good biosafety, no need for external reagents to lyse cells, no pigment interference, short time-consuming, low cost, and easy to achieve high-throughput screening. Rapid detection method for environmental genotoxic substances.

When microbial DNA is damaged (such as ultraviolet radiation, genotoxic substances), it will cause a large number of repair genes to be expressed. The large expression of these genes is dependent on the activation of the recA protein under the action of damage, and the hydrolysis of the repressor protein LexA, which promotes the expression of the repair gene downstream of the repressor protein. These repair reactions are called SOS reactions. SOS reactions are widely present in prokaryotes and eukaryotes and are instinct for organisms to protect themselves in adverse environments. The binding region between LexA and the upstream of the repair gene is called SOS box, and the coding gene is located in the promoter region -35 to -10 region of the repair gene. This study is to use the SOS box response region to regulate the expression of downstream reporter proteins and achieve qualitative and quantitative detection of genotoxic substances in the environment.

λ phage of Escherichia coli is one of the most well-known and widely used phage. The genes causing cell lysis in phage include S, R and Rz genes, wherein the R gene encodes a water-soluble transglycosylase, which can cause hydrolysis of peptide bonds and decompose peptides of cell walls. sugar. The product of the Rz gene may be an endopidase that cleaves the linkage between the peptidoglycan and the oligosaccharide and/or between the peptidoglycan and the outer membrane of the cell wall. The function of the R and Rz gene products is to degrade the cell wall, and the role of the S gene product is to change the permeability of the plasma membrane, forming a porous structure on the plasma membrane, so that the products of the R and Rz genes pass through the plasma membrane. Acting on the cell wall, breaking the cell wall and releasing intracellular substances.

The present invention utilizes recombinant Escherichia coli as a genotoxic substance detecting bacterium. The strain is ligated to a phage cleavage-protein SRRz gene sequence by a specific genotoxic substance-responsive promoter sequence, ligated into a plasmid vector, and introduced into Escherichia coli to form Escherichia coli carrying the phage cleavage protein SRRz, ie, It is a recombinant Escherichia coli used in the present invention. When the recombinant strain encounters a genotoxic substance that causes DNA damage, the expression of the cleavage gene SRRz is initiated, eventually leading to the rupture of E. coli. By detecting the efficiency of bacterial cell lysis, the amount of pollutants can be quantitatively detected within a certain range.

SUMMARY OF THE INVENTION An object of the present invention is to provide a genotoxic substance detecting vector which is an Escherichia coli expression vector in which a genotoxic response promoter, a phage lysing gene and an Escherichia coli terminator are ligated in sequence from the 5' to the 3' end.

The genotoxic substance responsive element may be any element of the SOS response, preferably the nucleotide sequence of SEQ ID No. 1 in the Sequence Listing.

The phage lysing gene may be any phage cleavage gene, preferably a cleavage gene SRRz of lambda phage, having the nucleotide sequence of SEQ ID No. 2 in the Sequence Listing.

The E. coli terminator can be any E. coli terminator, preferably a T7 terminator.

The starting vector for constructing the vector may be any one of E. coli vectors, preferably pBluescript, pUC18, pUC19, pET series vectors. Using pUC18 as a starting vector, the E. coli lytic vector constructed was pUST.

A second object of the present invention is to provide a method for detecting genotoxic substances.

The method for detecting genotoxic substances provided by the present invention is to introduce the genotoxic substance response vector into Escherichia coli to obtain a recombinant bacteria, and then incubated the recombinant bacteria with the genotoxic substance, and lyse the Escherichia coli cells. The recombinant Escherichia coli carrying a genotoxic response vector, the recombinant bacteria inducing self-cell lysis when exposed to genotoxic chemicals, and the method for detecting genotoxic substances by the cleavage efficiency.

The Escherichia coli is preferably E. coli BL21, E. coli DH5a, E. coli XL1-blue or E. coli HB101.

The above method for detecting genotoxic substances includes the following steps:

(1) preparing an Escherichia coli detection solution;

(2) mixing the sample to be tested with the E. coli detection solution, and adding the Escherichia coli test solution of the same volume of pure solvent as a control; both samples are further cultured for 0.3-1 h;

(3) measuring the OD ₆₀₀ of the Escherichia coli test solution mixed with the sample to be tested and the E. coli test solution of the control group;

(4) Calculate the cracking efficiency, and calculate the concentration of genotoxic substances in the sample to be tested according to the standard curve of the cracking efficiency.

Preparation of the E. coli detection solution:

a) culturing the recombinant E. coli stock with LB solid medium to resuscitate and activate;

b) the obtained activated single-colony of the Escherichia coli is connected to the LB liquid medium for shaking culture to the late logarithmic growth phase;

c) The obtained saturated bacterial solution was inoculated into fresh LB medium at a volume ratio of 1:100, and cultured until the OD ₆₀₀ of the bacterial solution was 0.15-0.25, and an Escherichia coli detection solution was obtained.

The genotoxic substances include methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4-NQO), mitomycin C (MMC), 2-aminopurine (2-AA) And benzopyrene (BaP).

The cracking efficiency standard curve is as follows:

4-NQO, Y=-1.78+10.95X, R ² =0.99 (1<X<5)

MMS, Y=-16.98+0.57X, R ² =0.98 (40<X<100)

MMC, Y=-19.04+4.11X, R ² =0.98 (5<X<20)

2-AA, Y=-8.32+86.24X, R ² =0.99 (0.2<X<0.8)

BaP, Y=4.03+89.10X, R ² =0.98 (0.1<X<0.8)

Wherein X represents the concentration of genotoxic compound (mg/L), Y represents the rate of lysis (%), and R ² represents the correlation coefficient of the fitted curve.

Recombinant E. coli containing the Escherichia coli genotoxic substance response vector is also within the scope of the present invention.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. The operating object is Escherichia coli, which has no risk of disease and is easy to operate.

2, the test cycle is short, can be detected within 3h (2h detection of bacterial solution preparation, 0.5 hour bacterial solution and test sample contact culture, 0.5h determination of bacterial liquid OD ₆₀₀ value), greatly shortening the time required for the detection of genotoxic substances in the past .

3, the detection process does not need to add additional reagents (such as enzyme substrate, etc.), the cost is low.

4. The detection sensitivity is high. The sensitive concentration ranges for 4-NQO, MMS, MMC, 2-AA and BaP are in the range of 1-5, 40-100, 5-20, 0.2-0.8, 0.1-0.8 mg/L, respectively. Sensitive.

5. The content of the genotoxic substance detected in the water sample can be converted into the equivalent concentration of 4-NQO, so that the result is more intuitive and uniform.

6. This method can provide technical support for sudden water pollution and routine testing of water quality in water plants.

DRAWINGS

Figure 1 is a schematic diagram of the construction of the vector.

Figure 2 shows wild-type Escherichia coli (E. coli BL21/pUC18) and recombinant bacteria (E. coli BL21/pUST) A plot of the lysis efficiency of 0.5 h in contact with different genotoxic substances.

detailed description

The present invention will be further described in detail below with reference to specific embodiments, but the embodiments of the present invention are not limited thereto, and the process parameters not specifically noted may be referred to conventional techniques.

Example 1

Construction of genotoxic response vector

Using pUC18 as a starting vector, a genotoxic response vector pUST was constructed. The specific construction method is as follows:

a) Synthetic genetic response promoter P _umu (SEQ ID No. 1) and T7 terminator sequences were added with EcoRI and XbaI sites upstream of the promoter and T7 terminator, and SpeI and PstI restriction sites were added downstream.

b) Amplification of the SRRz gene using lambda phage genomic DNA as a template. EcoRI and XbaI, SpeI and PstI restriction sites were also added upstream and downstream of the SRRz gene.

c) Using the Biobricks method, each element was ligated according to the promoter-cleavage gene-terminator sequence by enzyme digestion and ligation, and inserted into the pUC18 vector to obtain a genotoxic response vector pUST.

d) The vector pUST was transferred to E. coli BL21 competent cells to obtain recombinant Escherichia coli E. coli BL21/pUST for detection of genotoxic substances.

Example 2

Cracking efficiency calculation

The light absorption value (OD ₆₀₀ ) of each test bacterial solution at 600 nm was measured 0.5 h after the E. coli test solution was exposed to the sample to be tested.

Cracking rate (%) = (A-B) / A * 100%

Among them, A—the methanol solution OD ₆₀₀ added with methanol

B—Add the test solution to the bacterial solution OD ₆₀₀ .

Example 3

Genetic toxicity test

1. Recovery and activation of recombinant E. coli

(1) Wild type Escherichia coli (E. coli BL21/pUC18) and recombinant bacteria (E. coli BL21/pUST) were streaked from the -80° refrigerator in LB plate medium, and cultured at 37 ° C. 14h.

(2) Picking single colonies, inoculated separately into LB medium, and culturing at 37 ° C, 250 rpm 12-16h.

2. Preparation of E. coli test solution

The resuscitation-activated bacterial solution was inoculated to fresh LB medium at a volume ratio of 1:100, added to a 96-well culture plate in a volume of 190 μl, and cultured at an OD ₆₀₀ to 0.15-0.25 at 35-37 ° C, 800 rpm.

3. Contact with the test sample

10 μl of methanol and different concentrations of 4-NQO, MMS, MMC, 2-AA and BaP were separately added to a 96-well plate, and culture was continued for 0.5 h at 35-37 ° C and 800 rpm, and three parallel groups were set.

4. The experimental results showed (Fig. 2) that the original strain E. coli BL21/pUC18 grew normally in the tested concentration range, and no bacterial lysis was observed. The recombinant strain E.coli BL21/pUST containing the genotoxic response vector has different degrees of lysis.

According to the testFig. 2, the test standard curves of the corresponding genotoxic substances are:

4-NQO, Y=-1.78+10.95X, R ² =0.99 (1<X<5)

MMS, Y=-16.98+0.57X, R ² =0.98 (40<X<100)

MMC, Y=-19.04+4.11X, R ² =0.98 (5<X<20)

2-AA, Y=-8.32+86.24X, R ² =0.99 (0.2<X<0.8)

BaP, Y=4.03+89.10X, R ² =0.98 (0.1<X<0.8)

Wherein X represents the concentration of genotoxic compound (mg/L), Y represents the rate of lysis (%), and R ² represents the correlation coefficient of the fitted curve.

Example 4

Detection of genotoxic substances in water samples

In the present embodiment, a rapid detection method for genotoxic substances is based on 4-NQO as a standard toxic substance. According to the literature, the concentration of 4-NQO in drinking water for healthy adults should not exceed 80 ng/L (based on 2 L/day of drinking water) (Martijn, BJ; Van Rompay, AR et al., Development of a4- NQO toxic equivalency factor (TEF) approach to enable a preliminary risk assessment of unknown genotoxic compounds detected by the Ames II test in UV/H2O2 water treatment samples. Chemosphere 2016, 144, 338-345.).

This embodiment includes the following steps:

(1) An Escherichia coli test solution was prepared in accordance with the method of Example 3.

(2) The water sample to be tested was taken up, adsorbed with activated resin at a rate of 40 mL/min, and then eluted with ethyl acetate. The eluent is removed by centrifugal freeze-drying method and dissolved in a certain volume of distilled water. Sample to the required volume. The water sample to be tested was mixed with the Escherichia coli test solution, and the Escherichia coli test solution of the same volume of pure solvent was added as a control; both samples were further cultured for 1 h. The water samples to be tested are taken from daily urban water (laboratory faucet discharge), urban water plant water source, a chemical plant wastewater A, and a chemical plant wastewater B.

(3) The OD ₆₀₀ was measured by separately measuring the Escherichia coli test solution mixed with the water sample to be tested and the E. coli test solution of the control group.

(4) Calculate the cracking efficiency, and calculate the 4-NQO equivalent concentration of the genotoxic substance in the sample to be tested according to the standard curve of the cracking efficiency. The results obtained are shown in Table 1.

The results in Table 1 show that the equivalent concentration of 4-NQO for genotoxic substances in daily urban water and urban waterworks is less than 25 ng/L, while the 4-NQO equivalent concentration of chemical plant wastewater A is about 460-470 ng/L. The 4-NQO equivalent concentration of the wastewater from the wastewater of the plant is about 290-300 ng/L.

The above test results indicate that the genetic toxicity-reactive recombinant Escherichia coli of the present invention can detect genotoxic compounds of the test substance within the response concentration range. The method is short in time, easy to detect and easy to promote.

Claims (10)

1. A genotoxic substance detecting vector characterized in that the vector is an Escherichia coli expression vector in which a genotoxic response promoter, a phage cleavage gene and an E. coli terminator are ligated in sequence from the 5' to the 3' end.
2. The genotoxic substance detecting vector according to claim 1, wherein the sequence of the genotoxic response promoter is as shown in SEQ ID No. 1.
3. The genotoxic substance detecting vector according to claim 2, wherein the base sequence of the phage cleavage gene is as shown in SEQ ID No. 2.
4. The genotoxic substance detecting vector according to claim 3, wherein the terminator is a T7 terminator.
5. The genotoxic substance detecting vector according to claim 1 or 2 or 3 or 4, wherein the starting vector for constructing the vector is pUC18, pUC19, pBluscript or pET30a.
6. The recombinant Escherichia coli obtained by transferring the vector according to any one of claims 1 to 5 into Escherichia coli.
7. A method for detecting genotoxic substances, comprising the steps of:

   (1) preparing an Escherichia coli detecting solution using the recombinant Escherichia coli according to claim 6;

   (2) mixing the sample to be tested with the E. coli detection solution, and adding the Escherichia coli test solution of the same volume of pure solvent as a control; both samples are further cultured for 0.3-1 h;

   (3) measuring the OD ₆₀₀ of the Escherichia coli test solution mixed with the sample to be tested and the E. coli test solution of the control group;

   (4) Calculate the cracking efficiency, and calculate the concentration of genotoxic substances in the sample to be tested according to the standard curve of the cracking efficiency.
8. The method for detecting a genotoxic substance according to claim 7, wherein the preparation of the Escherichia coli detecting solution is:

   a) culturing the recombinant E. coli stock with LB solid medium to resuscitate and activate;

   b) the obtained activated single-colony of the Escherichia coli is connected to the LB liquid medium for shaking culture to the late logarithmic growth phase;

   c) The obtained saturated bacterial solution was inoculated into fresh LB medium at a volume ratio of 1:100, and cultured until the OD ₆₀₀ of the bacterial solution was 0.15-0.25, and an Escherichia coli detection solution was obtained.
9. The detection method according to claim 6 or 7 or 8, wherein the genotoxic substance comprises methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4-NQO), Mitomycin C (MMC), 2-aminoindole (2-AA) and benzopyrene (BaP).
10. The detecting method according to claim 9, wherein the cracking efficiency standard curve is as follows:

    4-NQO, Y=-1.78+10.95X, R ² =0.99 (1<X<5)

    MMS, Y=-16.98+0.57X, R ² =0.98 (40<X<100)

    MMC, Y=-19.04+4.11X, R ² =0.98 (5<X<20)

    2-AA, Y=-8.32+86.24X, R ² =0.99 (0.2<X<0.8)

    BaP, Y=4.03+89.10X, R ² =0.98 (0.1<X<0.8)

    Wherein X represents the concentration of genotoxic compound (mg/L), Y represents the rate of lysis (%), and R ² represents the correlation coefficient of the fitted curve.

Images