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WO2018133513A1 - Vecteur de détection de substance génotoxique et procédé de détection correspondant - Google Patents

Vecteur de détection de substance génotoxique et procédé de détection correspondant Download PDF

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Publication number
WO2018133513A1
WO2018133513A1 PCT/CN2017/110897 CN2017110897W WO2018133513A1 WO 2018133513 A1 WO2018133513 A1 WO 2018133513A1 CN 2017110897 W CN2017110897 W CN 2017110897W WO 2018133513 A1 WO2018133513 A1 WO 2018133513A1
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genotoxic
escherichia coli
vector
coli
detecting
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PCT/CN2017/110897
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Chinese (zh)
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李爽
卓敏
袁鹏飞
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华南理工大学
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Priority to AU2017393714A priority Critical patent/AU2017393714B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • 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.
  • 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.
  • cytogenetic indicators usually using biological cells such as plants, mammals, and microorganisms to monitor the genotoxicity of residues.
  • 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.
  • 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 SOS chromotest
  • Prophage induction assay etc.
  • 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.
  • 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).
  • the researchers considered that most of the substances that were positive for Ames test were also positive for the SOS test.
  • 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).
  • 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.
  • 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.
  • 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.
  • ⁇ 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.
  • 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.
  • the efficiency of bacterial cell lysis the amount of pollutants can be quantitatively detected within a certain range.
  • 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 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 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:
  • 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:
  • X represents the concentration of genotoxic compound (mg/L)
  • Y represents the rate of lysis (%)
  • R 2 represents the correlation coefficient of the fitted curve.
  • E. coli containing the Escherichia coli genotoxic substance response vector is also within the scope of the present invention.
  • the operating object is Escherichia coli, which has no risk of disease and is easy to operate.
  • 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 .
  • the detection process does not need to add additional reagents (such as enzyme substrate, etc.), the cost is low.
  • 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.
  • 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.
  • This method can provide technical support for sudden water pollution and routine testing of water quality in water plants.
  • 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.
  • pUC18 a genotoxic response vector pUST was constructed.
  • the specific construction method is as follows:
  • Synthetic genetic response promoter P umu SEQ ID No. 1
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ l, and cultured at an OD 600 to 0.15-0.25 at 35-37 ° C, 800 rpm.
  • test standard curves of the corresponding genotoxic substances are:
  • X represents the concentration of genotoxic compound (mg/L)
  • Y represents the rate of lysis (%)
  • R 2 represents the correlation coefficient of the fitted curve.
  • a rapid detection method for genotoxic substances is based on 4-NQO as a standard toxic substance.
  • 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.).
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne un vecteur de détection de substance génotoxique et un procédé de détection correspondant. Le vecteur est un vecteur d'expression d'Escherichia coli présentant séquentiellement, d'une extrémité 5' à une extrémité 3', un promoteur de réponse génotoxique, un gène de lyse de bactériophage et un terminateur d'Escherichia coli. Le procédé de détection comprend l'introduction du vecteur de réponse à une substance génotoxique dans Escherichia coli pour obtenir des bactéries recombinantes, l'incubation des bactéries recombinantes et de substances génotoxiques et la lyse des Escherichia coli. Les Escherichia coli recombinants présentent le vecteur de réponse génotoxique et une lyse autocellulaire est initiée lorsque les bactéries recombinantes entrent en contact avec les substances génotoxiques. Le procédé de détection quantifie des substances génotoxiques au moyen de l'efficacité de lyse. Le procédé prend peu de temps, présente une sensibilité de détection élevée, est facile à mettre en œuvre, peu coûteux et facile à promouvoir.
PCT/CN2017/110897 2017-01-18 2017-11-14 Vecteur de détection de substance génotoxique et procédé de détection correspondant WO2018133513A1 (fr)

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CN106636164A (zh) * 2017-01-18 2017-05-10 华南理工大学 一种遗传毒性物质检测载体及检测方法
CN107478795A (zh) * 2017-08-24 2017-12-15 山东省城市供排水水质监测中心 一种城市饮用水水体遗传毒性的检测方法
CN110873790B (zh) * 2018-09-03 2021-01-29 华南理工大学 检测水溶性样品中重金属离子的全细胞生物传感器及其构建与应用
EP4010496A4 (fr) * 2019-08-09 2023-09-06 Nantomics, LLC Procédés automatiques pour déterminer une toxicité de charge utile de néo-épitope
CN112680498A (zh) * 2020-12-28 2021-04-20 华南理工大学 一种遗传毒性物质的高通量筛查方法

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