CN112680498A

CN112680498A - High-throughput screening method for genotoxic substances

Info

Publication number: CN112680498A
Application number: CN202011581900.1A
Authority: CN
Inventors: 卓敏; 赵伟斌; 李爽
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2021-04-20

Abstract

The invention discloses a high-throughput screening method for genotoxic substances, and relates to the technical field of genotoxicity high-throughput screening. The method comprises the steps of inserting a coding nitroreductase gene, a coding O-acetyltransferase gene, a DNA damage detection response element and a phage lysis gene into an escherichia coli expression vector to obtain a recombinant vector; transferring the recombinant vector into escherichia coli to obtain recombinant escherichia coli; the recombinant Escherichia coli is applied to high-throughput detection of genotoxic substances. The operation object of the screening method is escherichia coli, no pathogenic risk exists, and the operation is simple and easy; the test period is short, and the completion can be detected within 2 h; the detection sensitivity is high; therefore, the method has the advantages of convenient operation, short time consumption, high detection efficiency, no need of adding additional chemical reagents, no interference of pigments, low cost, time and labor saving, and easy realization of high-throughput sample detection.

Description

High-throughput screening method for genotoxic substances

Technical Field

The invention relates to the technical field of high-throughput screening of genotoxicity, in particular to a high-throughput screening method of genotoxicity substances.

Background

In recent years it has been found that in the production of pharmaceuticals, pesticides and dyes, uncontrolled release of certain amounts of genotoxic compounds such as Nitro Polycyclic Aromatic Hydrocarbons (NPAH), nitro containing pesticides, phenols etc. into the air, soil and water poses potential risks for the ecological environment and human health. Some of the nitro-containing compounds undergo metabolism in the human body by various enzyme systems to activate their biological activities and cause irreversible genetic damage to human cells. Therefore, there is also an increasing need for the detection of such stress factors.

Currently, the main methods available for screening the genetic toxicity of nitro-containing compounds include the Salmonella typhimurium back mutation test (also known as Ames test) and the SOS/umu test. However, salmonella typhimurium is a pathogenic bacterium which is harmful to human and livestock, and can cause diseases such as acute gastroenteritis, enterocolitis, typhoid fever, paratyphoid fever, septicemia and the like, so that the experimental safety is poor. The detection method generally has the factors which are not beneficial to popularization, such as complicated operation, long detection time, incapability of realizing high flux, need of strict sterile operation and the like. And due to the reasons of patent protection and the like, individual detection methods are not favorable for domestic popularization, and the wide application of the detection methods is limited.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a high-throughput screening method for genotoxic substances. The screening method is convenient to operate, short in time consumption, free of additional chemical reagent addition, free of pigment interference and easy to realize high-throughput sample detection.

The purpose of the invention is realized by the following technical scheme:

the invention provides a high-throughput screening method for genotoxic substances, which comprises the following steps:

the method comprises the following steps: inserting a coding nitroreductase gene, a coding O-acetyltransferase gene, a DNA damage detection response element and a phage lysis gene into an escherichia coli expression vector to obtain a recombinant vector; transferring the recombinant vector into escherichia coli to obtain recombinant escherichia coli;

step two: preparing an escherichia coli detection solution by using the recombinant escherichia coli, incubating the escherichia coli detection solution and a sample to be detected in a microporous plate, and cracking escherichia coli cells; simultaneously adding a pure solvent with the same volume of escherichia coli detection solution as a control; calculating the survival rate of the thalli by absorbance to realize high-throughput detection;

alternatively, the first and second electrodes may be,

step three: calculating the relative survival rate through the survival rate of escherichia coli and the survival rate of recombinant escherichia coli, and realizing high-throughput detection;

relative survival (%) ═ B/a 100%; wherein, the survival rate of A-escherichia coli and the survival rate of B-recombinant escherichia coli.

The recombinant vector has the structural elements shown in figure 1.

In the recombinant vector, a coding nitroreductase gene, a coding O-acetyltransferase gene, a DNA damage detection response element and a phage lysis gene are sequentially arranged from 5 'to 3'.

The encoding nitroreductase gene can be any encoding nitroreductase gene, preferably an encoding nitroreductase gene nfSB of SGSC1412 salmonella typhimurium, and the gene sequence is SEQ ID NO. 1; the amino acid sequence of the nitroreductase is SEQ ID NO. 5.

The coding O-acetyltransferase gene can be any coding O-acetyltransferase gene, preferably a coding O-acetyltransferase gene nhoA of SGSC1412 salmonella typhimurium, and the gene sequence is SEQ ID NO. 2; the amino acid sequence of the O-acetyltransferase is SEQ ID NO. 6.

Preferably, the nucleotide sequence of the DNA damage detection response element is SEQ ID NO. 3.

The phage lysis gene can be any phage lysis gene, preferably the lysis gene SRrz of lambda phage, and the gene sequence is SEQ ID NO. 4.

In the second step, the microplate can be selected from 96-well plates, 48-well plates, 24-well plates and the like.

In the second step, the incubation time is preferably 15-60 min; further 20 min.

In the second step, the absorbance is at 600nmYield, which can be recorded as OD₆₀₀。

In the second step, the method specifically comprises the following steps:

(1) preparing an escherichia coli detection solution by using the recombinant escherichia coli;

(2) mixing a sample to be detected and the escherichia coli detection solution in a microporous plate, and adding the escherichia coli detection solution with the same volume of pure solvent as a control; continuously culturing the two groups of samples for 15-60 min;

(3) respectively measuring OD of the Escherichia coli detection solution mixed with the sample to be measured and the Escherichia coli detection solution of the control group₆₀₀；

(4) And (5) calculating the survival rate, and calculating the genotoxicity degree of the sample to be detected according to the survival rate standard curve.

The preparation steps of the escherichia coli detection solution are as follows:

1) culturing the recombinant escherichia coli stock with an LB solid culture medium to recover and activate the recombinant escherichia coli stock;

2) inoculating the obtained activated escherichia coli single colony into an LB liquid culture medium for shake culture to the late stage of logarithmic phase to obtain saturated bacterial liquid;

3) inoculating the obtained saturated bacterial liquid into a fresh LB liquid culture medium in a volume ratio of 1: 100, and culturing until bacterial liquid OD₆₀₀0.1-0.4, adding IPTG to a final concentration of 0.01-0.2 mM, and performing induction culture to obtain an escherichia coli detection solution.

In the step 3), the induction culture time is preferably 1-1.5 h.

The Escherichia coli expression vector can be any Escherichia coli vector, such as pBluescript, pUC18, pUC19, pET series vectors (such as pET30a (+)) and the like. pUC18 is used as starting vector, and the constructed Escherichia coli cracking vector is pUSTBA.

The Escherichia coli is E.coli BL21(DE3), E.coli DH5a, E.coli XL1-blue or E.coli HB 101.

The genetic toxic substance can be a nitro-containing compound, specifically at least one of 1-nitronaphthalene (1-NN), 2-nitrofluorene (2-NF), 5-nitroacenaphthene (5-NA) and the like;

the standard curve of the thallus survival rate is as follows:

1-NN，Y＝54.37+(97.81-54.37)/{1+10^{(17.74-log X)×(-0.1095)}}，R²＝0.99(2≤X≤170)；

2-NF，Y＝66.13+(100.2-66.13)/{1+10^{(1.117-log X)×(-3.676)}}，R²＝0.99(0.5≤X≤2.5)；

5-NA，Y＝65.39+(100.8-65.39)/{1+10^{(2.586-log X)×(-0.5784)}}，R²＝0.99(0.125≤X≤15)。

wherein X represents the concentration (. mu. mol/L) of a genotoxic substance, Y represents the cell survival rate (%), and R²Representing curve fit correlation coefficients.

The genetic toxic substance can also be a common pesticide, and is at least one of organic phosphate, aniline (except iprodione), amide, formate, tert-butyl sulfide, phenol, microorganism pesticides, and the like; the method specifically comprises the following steps: at least one of trichlorphon, dichlorvos-cypermethrin, glufosinate-ammonium, imidacloprid, cyromazine, pendimethalin, biphenyl-thiamethoxam, pythium-thiram, carfentrazone-zineb, carbendazim, emamectin benzoate, pyridaben, abamectin-diafenthiuron, compound sodium nitrophenolate and abamectin.

Recombinant Escherichia coli containing the recombinant vector also belongs to the protection scope of the invention.

Compared with the prior art, the invention has the following advantages and effects:

(1) the operation object is escherichia coli, no pathogenic risk exists, and the operation is simple and easy.

(2) The test period is short, and the test can be completed within 2 h.

(3) The detection process does not need to add additional reagents (such as enzyme substrates and the like), and the cost is low.

(4) The detection sensitivity is high, and the sensitivity concentration ranges of the detection to 1-NN, 2-NF and 5-NA are respectively 2-170, 0.5-2.5 and 0.125-15 mu mol/L.

(5) High-flux screening can be realized, the detection efficiency is high, and time and labor are saved.

Drawings

FIG. 1 is a schematic diagram of the construction of the genotoxicity test vector in example 1.

FIG. 2 is a graph showing the cleavage response of the recombinant E.coli (E.coli BL21(DE3)/pUSTBA) in example 2 to nitro-containing compounds having genetic toxicity to 1-NN, 2-NF, and 5-NA.

FIG. 3 is a schematic diagram of the experimental protocol of the conventional pesticide in 96-well plate in example 3.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

Example 1 genetic toxicity test vector construction

And constructing a genetic toxicity detection vector pUST by taking pUC18 as a starting vector. The specific construction method comprises the following steps:

1. synthetic DNA Damage detection response element P_umu(SEQ ID NO.3) and the T7 terminator sequence, at P_umuXbaI site is added at the upstream, and SpeI enzyme cutting site is added at the downstream; an EcoRI site was added upstream of the T7 terminator and a SmaI cleavage site was added downstream, and the sequence was designated as T7T.

2. The SRrz gene (SEQ ID NO.4) was amplified using lambda phage genomic DNA as a template. SpeI and EcoRI restriction sites are added to the upstream and downstream of the SRrz gene.

3. Adopting a biological building block (Biofricks) method, adopting an enzyme digestion and connection method to make each element according to a promoter P_umuThe terminator sequence of the split gene SRrz-T7 is ligated and inserted into the pUC18 vector to obtain the genotoxic response vector pUST.

4. Synthesizing a T7 promoter and a T7 terminator sequence, adding a KpnI restriction site at the upstream of a T7 promoter, and adding a BamHI restriction site at the downstream, wherein the KpnI restriction site is marked as T7-1; SmaI restriction enzyme cutting site is added at the upstream of another T7 promoter, and PstI restriction enzyme cutting site is added at the downstream, which is marked as T7-2; an XhoI cleavage site was added upstream of the T7 terminator and an NdeI cleavage site was added downstream, as indicated by T7T-1.

5. SGSC1412 Salmonella typhimurium genome DNA is used as a template to amplify the nfSB gene (SEQ ID NO.1) and the nhoA gene (SEQ ID NO.2) respectively. A BamHI cleavage site was added upstream of the nfSB gene, a SmaI cleavage site was added downstream, a PstI cleavage site was added upstream of the nhoA gene, and an XhoI cleavage site was added downstream.

6. And (3) connecting the elements according to the sequence of a promoter-nfSB-promoter-nhoA-terminator by adopting a biological building block (Biofricks) method and adopting an enzyme digestion and connection method, and inserting the elements into a pUST vector to obtain a genetic toxicity detection vector pUSTBA. A schematic of the vector construction is shown in FIG. 1.

7. Transferring the vector pUSTBA or pUST into E.coli BL21(DE3) competent cells to obtain recombinant Escherichia coli E.coli BL21(DE3)/pUSTBA or E.coli BL21(DE3)/pUST for detecting genotoxic substances.

EXAMPLE 2 Nitro Compound genotoxicity test

Calculation of survival

20min after the Escherichia coli detection liquid contacts the sample to be detected, respectively determining the light absorption value (OD) of each test bacterial liquid at 600nm₆₀₀)。

Survival rate (%). B/A100%

Wherein, A-bacterial liquid OD added with dimethyl sulfoxide₆₀₀

B bacterial liquid OD added with sample to be detected₆₀₀。

1. Resuscitation and activation of recombinant escherichia coli

Recombinant E.coli BL21(DE3)/pUSTBA was taken out from a-80 ℃ refrigerator, streaked on LB plate medium, and cultured at 37 ℃ for 14 hours. And (3) selecting a single colony, inoculating the single colony into an LB culture solution, and culturing for 12-16 h at 37 ℃ and 250rpm to obtain a saturated bacterial solution.

2. Preparation of Escherichia coli detection solution

Inoculating the revived and activated saturated bacterial liquid to a fresh LB culture medium according to the volume ratio of 1: 100, adding 190 mu L of the saturated bacterial liquid into a 96-hole culture plate, and culturing the saturated bacterial liquid to OD under the conditions of 35-38 ℃ and 600-1000 rpm₆₀₀Adding IPTG to a final concentration of 0.1-0.401-0.2 mM, and inducing culture for about 1-1.5 h to obtain Escherichia coli detection solution.

3. Contact with the sample to be tested

Preparing a solution with a certain concentration gradient and containing a nitro compound (1-NN, 2-NF or 5-NA) as a sample to be detected; wherein the concentration gradient of 1-NN in the solution containing 1-NN is 2, 6, 20, 58 and 170 mu mol/L; the concentration gradient of 2-NF in the solution containing 2-NF is 0.5, 0.75, 1, 1.5, 2 and 2.5 mu mol/L; the 5-NA concentration gradient in the 5-NA-containing solution was 0.125, 0.63, 1.25, 3.5, 15. mu. mol/L.

And adding 10 mu L of sample to be detected into the 96-well plate, continuously culturing for 20 minutes at 35-38 ℃ and 800rpm, and setting 3 parallel groups. The addition of the same volume of pure solvent (dimethyl sulfoxide), and the same volume of sample at the same concentration to the recombinant e.coli BL21(DE 3)/psust strain and the wild-type e.coli BL21(DE3) strain were set as negative controls at the same time.

4. Experimental results show that under the saturation concentration conditions (170 mu mol/L1-NN, 2.5 mu mol/L2-NF and 15 mu mol/L5-NA), a recombinant E.coli BL21(DE3)/pUST strain which is lack of a nitroreductase gene (nfSB) and an O-acetyltransferase gene (nhoA) in a genotoxic response vector pUST and a wild E.coli BL21(DE3) strain which does not contain a genotoxic response vector grow normally, and the thalli are not cracked. Coli BL21(DE 3)/reustba strain, however, exhibited different degrees of lysis. In this example, the detection result is shown in fig. 2, and the standard curve of each nitro compound detection corresponds to:

The results show that the sample concentration and the thallus survival rate have dose correlation within the detection concentration range. Therefore, the strain E.coli BL21(DE3)/pUSTBA can be used for detecting whether the sample containing the nitro compound has genetic toxicity or not.

Example 3 high throughput screening of genotoxicity of commonly used pesticides

In this example, the genetic toxicity screening is performed by using the conventional pesticide, and the survival rate of the thallus is used as a standard index. The corresponding degree of genotoxicity of the pesticide can be detected and reflected as the relative survival rate.

Relative survival (%) ═ B/A100%

Wherein, the survival rate of A-wild type E.coli BL21(DE3) strain

Coli BL21(DE3)/pUSTBA survival rate

1. An E.coli BL21(DE3)/pUSTBA test medium, an E.coli BL21(DE3)/pUST test medium, and a wild-type E.coli BL21(DE3) test medium were prepared in accordance with the procedure 1-2 of example 2.

2. The pesticides to be tested are respectively dissolved in dimethyl sulfoxide according to a certain proportion, and the dilution proportion is shown in table 1. Referring to example 2, in a 96-well plate, a pesticide to be detected was mixed with an escherichia coli detection solution, and an escherichia coli detection solution of the same volume of a pure solvent (dimethyl sulfoxide) was added as a control; both samples were incubated for 20 min. The pesticides are all purchased from a pesticide store in Dongguan.

3. The experimental protocol is shown in figure 3.

4. Respectively measuring OD of the Escherichia coli detection solution mixed with the pesticide to be measured and the Escherichia coli detection solution of the control group₆₀₀。

5. The relative survival rates were calculated and the results are shown in table 1.

TABLE 1 relative survival rates of conventional pesticides

The results in Table 1 show that the screened pesticide containing the nitro compound has certain genetic toxicity, and the relative survival rates of E.coli BL21(DE3)/pUSTBA induced by the pesticide of the cinnabar, the pendimethalin, the biphenyl-thiamethoxam and the compound sodium nitrophenolate are 53-89%, and are lower than the relative survival rates of E.coli BL21(DE3)/pUST without the codes of nitroreductase (nfSB) and O-acetyltransferase (nhoA). The pesticides of organic phosphates, anilines, amides (except iprodione), formate, tert-butyl sulfenyl, phenols and microorganisms all contain certain genetic toxicity, wherein the relative survival rate of trichlorfon, glufosinate-ammonium, pythium-thiram, pyridaben and abamectin induced E.coli BL21(DE3)/pUSTBA is only below 70%, and the relative survival rate of E.coli BL21(DE3)/pUST is above 90%.

The detection result shows that the method for screening the genetic toxicity containing the nitro compound detects the substance to be detected within the response concentration range, finally expresses the genetic toxicity content of the sample to be detected by the relative cell survival rate, and can realize high-throughput screening in a 96-well plate. The method has the advantages of no risk of disease, simple operation, high detection sensitivity, time and labor saving and easy popularization.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

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Claims

1. A method for high throughput screening of genotoxic materials, comprising: the method comprises the following steps:

alternatively, the first and second electrodes may be,

relative survival (%) ═ B/a 100%; wherein, A is the survival rate of Escherichia coli, B is the survival rate of recombinant Escherichia coli;

the genetic toxic substance is a nitro-containing compound, and specifically is at least one of 1-nitronaphthalene, 2-nitrofluorene and 5-nitroacenaphthene;

or the genetic toxic substance is at least one of organic phosphate, anilines except iprodione, amides, formate, tert-butyl sulfenyl, phenols and microbial pesticides.

2. The high throughput screening method of claim 1, wherein:

in the recombinant vector, a coding nitroreductase gene, a coding O-acetyltransferase gene, a DNA damage detection response element and a phage lysis gene are sequentially arranged from 5 'to 3';

the amino acid sequence of the nitroreductase is SEQ ID NO. 5;

the amino acid sequence of the O-acetyltransferase is SEQ ID NO. 6.

3. High throughput screening method according to claim 1 or 2, characterized in that:

the sequence of the coding nitroreductase gene is SEQ ID NO. 1;

the gene sequence of the coding O-acetyltransferase is SEQ ID NO. 2;

the nucleotide sequence of the DNA damage detection response element is SEQ ID NO. 3;

the sequence of the phage lytic gene is SEQ ID NO. 4.

4. High throughput screening method according to claim 1 or 2, characterized in that:

in the second step, the micro-porous plate is selected from a 96-well plate, a 48-well plate or a 24-well plate;

in the second step, the incubation time is 15-60 min;

in the second step, the absorbance is the light absorption value at 600nm and is recorded as OD₆₀₀。

5. High throughput screening method according to claim 4, characterized in that:

in the second step, the method specifically comprises the following steps:

6. High throughput screening method according to claim 1 or 2 or 5, characterized in that:

3) the obtained saturated bacterial liquid was mixed in a ratio of 1: inoculating into fresh LB liquid culture medium at a volume ratio of 100, and culturing to obtain bacterial liquid OD₆₀₀0.1-0.4, adding IPTG to a final concentration of 0.01-0.2 mM, and performing induction culture to obtain an escherichia coli detection solution.

7. High throughput screening method according to claim 1 or 2, characterized in that:

the genotoxic substance is at least one of trichlorphon, dichlorvos-cypermethrin, glufosinate-ammonium, imidacloprid, cyromazine, pendimethalin, biphenyl-thiamethoxam, pythium-thiram, azol ether-zineb, carbendazim, emamectin benzoate, pyridaben, abamectin-diafenthiuron, compound sodium nitrophenolate and abamectin.

8. High throughput screening method according to claim 1 or 2, characterized in that:

the standard curve of the thallus survival rate is as follows:

1-nitronaphthalene, Y ═ 54.37+ (97.81-54.37)/{1+10^{(17.74-logX)×(-0.1095)}}，R²＝0.99，2≤X≤170；

2-nitrofluorene, Y ═ 66.13+ (100.2-66.13)/{1+10^{(1.117-logX)×(-3.676)}}，R²＝0.99，0.5≤X≤2.5；

5-nitroacenaphthylene, Y-65.39 + (100.8-65.39)/{1+10 +^{(2.586-logX)×(-0.5784)}}，R²＝0.99，0.125≤X≤15；

Wherein X represents the concentration of genotoxic substances, mu mol/L; y represents the survival rate of the cells,% R²Representing curve fit correlation coefficients.

9. High throughput screening method according to claim 1 or 2, characterized in that:

the Escherichia coli expression vector is pBluescript, pUC18, pUC19 or pET series vector;

10. The recombinant Escherichia coli used in the high-throughput screening method according to any one of claims 1 to 3.