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WO2015030380A1 - Procédé de criblage d'une substance insecticide - Google Patents

Procédé de criblage d'une substance insecticide Download PDF

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WO2015030380A1
WO2015030380A1 PCT/KR2014/007101 KR2014007101W WO2015030380A1 WO 2015030380 A1 WO2015030380 A1 WO 2015030380A1 KR 2014007101 W KR2014007101 W KR 2014007101W WO 2015030380 A1 WO2015030380 A1 WO 2015030380A1
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allatostatin
cells
ires
protein
cell
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Korean (ko)
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윤봉준
나준호
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Korea University Research and Business Foundation
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Korea University Research and Business Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a method for screening pesticidal substances.
  • Pests and weeds that affect crops have many kinds and have different characteristics, so there must be various kinds of pesticides, and if the same pesticides are used, pests have resistance to pesticides. This is necessary.
  • farmers need drugs that are easy to use and suitable for the growing season or cultivation method of crops.In order to prevent too many kinds of pesticides, the government selects and registers only pesticides with excellent pest control effect and safe. Pesticides with reduced effectiveness or risk are prohibited.
  • the number of pests and weeds distributed in Korea is 5,850 species (both 2,771, 2,618 pests, 461 weeds). Among them, 4,010 pests and weeds that cause crops are introduced. This increase is expected to increase the number of species.
  • Korean Patent Laid-Open Publication No. 1990-0015871 discloses a technique related to the isolation and characterization of Lepidoptera allatostatin neuropeptide, which has a LAS-type activity liberated from an aggregate pest polypeptide. There is no description of a screening method for finding an agonist that acts identically to allatostatin using the Allatostatin receptor (AlstR) protein.
  • AlstR Allatostatin receptor
  • An object of the present invention is to provide a method for screening pesticides using an allatostatin agonist for the development of next-generation pesticides that can minimize harmful effects on the human body and the environment through the development of environmentally friendly pesticides.
  • an adeno-associated virus vector comprising an Allatostatin receptor (AlstR) protein, a fluorescent protein and an internal ribosome entry site (IRES);
  • an adeno-associated virus vector that does not include an allatostatin receptor protein and comprises a fluorescent protein and an internal ribosomal entry site (IRES);
  • step c) introducing each virus prepared in step c) into one species selected from the group of animal cells capable of expressing a GIRK channel (G protein-coupled inwardly-rectifying potassium channel);
  • GIRK channel G protein-coupled inwardly-rectifying potassium channel
  • step d preparing a plasmid containing a GIRK channel for introduction into the animal cell of step d), and introducing a plasmid containing a GIRK channel into each cell by using a calcium phosphate transfection method (CaHPO 4 Transfection) to the GIRK channel. Overexpressing it; And
  • the insecticidal candidate and the voltage sensitive dye were added to each of the animal cells into which the virus including the GIRK channel was introduced in step e), and then the fluorescence change according to the difference in cell membrane potential in the cell was monitored to determine the allatostatin receptor.
  • the present invention provides a method for screening pesticide substances, the method comprising finding a substance acting as an allatostatin agonist among pesticidal candidates through fluorescence measurement of expressed animal cells and animal cells not expressing an allatostatin receptor.
  • the screening method of pesticides using the allatostatin agonist of the present invention can be used as a pesticide that inhibits the normal development of pests by finding a natural substance similar to allatostatin that inhibits the synthesis of larvae hormone expressed in insects. Since allatostatin and its receptors are specifically present in the insect system, they are expected to have no effect on mammals including humans, which is suitable for the development of environmentally friendly pesticides. In addition, the allatostatin system is widely present in the insect system, making it a useful target for the development of pesticides that act on various pests.
  • FIG. 1 illustrates an AAV system vector for introducing a desired gene into a cell using an Adeno-associated virus (AAV) Helper-Free System, wherein (a) is a vector including AlstR-IRES-mCherry. , (b) shows a vector containing IRES-mCherry as a control for (a).
  • AAV Adeno-associated virus
  • Figure 2 is a graph showing the measurement of the fluorescence change of the cells using cells not expressing the allatostatin receptor.
  • the AlstR-IRES-mCherry graph of FIG. 3 shows that the fluorescence change over time of allatostatin action was measured in the cells expressing the allatostatin receptor through the AAV system, and the IRES of (b).
  • the mCherry graph shows that the fluorescence changes were measured to confirm the expression of cells except for the allatostatin receptor as control cells of AlstR-IRES-mCherry, and (AlstR-IRES-mCherry)-(IRES-) of (c).
  • mCherry) graph shows the effect of allatostatin in cells by comparing the variation in fluorescence changes between the cells expressing and not expressing allatostatin according to the presence or absence of the allatostatin receptor protein.
  • the graph of AlstR-IRES-mCherry (AAV) + GIRK1, GIRK2 in FIG. 4 (a) shows the effect of allatostatin by transfecting plasmids containing GIRK1 and GIRK2 to cells expressing allatostatin receptors through the AAV system.
  • Figure 5 shows a vector that is the basis for producing adeno-associated virus (adeno-associated virus) vector in the embodiment of the present invention
  • A is a pAAV-sh-mCherry vector
  • B is pAlstR- An IRES-GFP vector is shown.
  • Figure 6 shows a vector that is the basis for the preparation of the GIRK2-IRES-GIRK1 vector (a) in the embodiment of the present invention, (b) shows a GIRK2 openbiosystem vector, (c) shows a GIRK1 openbiosystem vector.
  • the present invention uses an Adeno-associated virus (AAV) Helper-Free system to prepare adeno-associated virus vectors.
  • AAV Adeno-associated virus
  • an adeno-associated virus vector comprising an Allatostatin receptor (AlstR) protein, a fluorescent protein and an internal ribosome entry site (IRES);
  • an adeno-associated virus vector that does not include an allatostatin receptor protein and comprises a fluorescent protein and an internal ribosomal entry site (IRES);
  • step c) introducing each virus prepared in step c) into one species selected from the group of animal cells capable of expressing a GIRK channel (G protein-coupled inwardly-rectifying potassium channel);
  • GIRK channel G protein-coupled inwardly-rectifying potassium channel
  • step d preparing a plasmid containing a GIRK channel for introduction into the animal cell of step d), and introducing a plasmid containing a GIRK channel into each cell by using a calcium phosphate transfection method (CaHPO 4 Transfection) to the GIRK channel. Overexpressing it; And
  • the insecticidal candidate and the voltage sensitive dye were added to each of the animal cells into which the virus including the GIRK channel was introduced in step e), and then the fluorescence change according to the difference in cell membrane potential in the cell was monitored to determine the allatostatin receptor.
  • the present invention relates to a method for screening pesticides, the method comprising finding a substance acting as an allatostatin agonist among pesticidal candidates by fluorescence measurement of expressed animal cells and animal cells not expressing an allatostatin receptor.
  • Allatostatin is a neuropeptide that has the function of inhibiting the synthesis of juvenile hormone produced by the insect endocrine organ, corpora allata.
  • Larvae hormone as is widely known, has various physiological effects of insects. Is an important hormone that directly regulates the production of the hormone. In particular, the production of the hormone plays an essential role in ovulation in females and is regarded as a major target molecule in pest control or pest control. Therefore, allatostatin, which is well known to directly regulate it, has also been proposed as a highly insecticidal target molecule.
  • Adeno-associated virus (AAV) Helper-Free system is a system that injects and expresses a desired gene into a cell by using an adeno-associated virus and introduces a gene of a desired trait into a virus-making vector. When the virus is infected with a cell, the gene is expressed to produce a desired protein.
  • AAV Adeno-associated virus
  • Adeno-associated viruses were replication-deficient parvoviruses that were simultaneously infected with helper adenovirus or herpes virus for good infection efficiency.
  • the AAV Helper-Free system used in the present invention is a system for making human adeno-associated virus-2 (AAV-2) capable of infection without the use of a helper virus.
  • the adenovirus gene is transfected into the cell and expressed by the AAV Helper-Free system. Under the system, most adenovirus genes are transfected together with plasmid pHelper and human AAV-2 vector DNA to produce infectious AAV particles. At this time, the cells used are AAV-293 cells as cells derived from HEK293.
  • Certain genes inserted in the middle of the human AAV-2 vector DNA during virus production cause the virus to be expressed in the host cell when infected.
  • Adeno-associated virus (AAV) Helper-Free system enhances the expression of allatostatin receptors by using the characteristics of adeno-associated virus, and can be used for rapid and sustainable virus infection. There is this. In addition, there is an advantage of minimizing the maintenance and migration of cells by expressing the allatostatin receptor in a simple manner without the need to use a cell line expressing the allatostatin receptor.
  • AAV Adeno-associated virus
  • the allatostatin receptor (AlstR) was first expressed in the system, and it was confirmed by expressing a fluorescent protein to confirm the presence or absence of the expression of the allatostatin receptor.
  • a fluorescent protein to confirm the presence or absence of the expression of the allatostatin receptor.
  • the voltage sensitive dye used in the present invention and the mCherry fluorescent protein that do not overlap the range of the fluorescence wavelength were expressed.
  • both genes were expressed in the vector using an internal ribosome entry site (IRES) to prevent possible interference.
  • an adeno-associated virus vector was prepared comprising an Allatostatin receptor (AlstR) protein, an mCherry protein and an internal ribosomal entry site (IRES).
  • step b) of the present invention an adeno-associated virus vector containing mCherry protein and an internal ribosome inlet site (IRES) was prepared as a control that does not include an allatostatin receptor, and each vector was expressed. .
  • the test also does not include the allatostatin receptor using the negative control to determine false positives, thereby enabling more accurate screening than there is only a positive control containing the allatostatin receptor. Therefore, there is an effect of screening the desired pesticidal candidates more accurately by comparing the change in fluorescence in the cells expressing the viral vector having a difference in the presence or absence of the allatostatin receptor.
  • Figure 1 shows an AAV system vector for introducing a desired gene into a cell using the adeno-associated virus Helper-Free System prepared by the above method, (a) is a vector containing AlstR-IRES-mCherry, (b) shows a vector containing IRES-mCherry as a control for (a).
  • Step c) of the present invention is a step of packaging each of the adeno-associated virus vectors prepared in steps a) and b) with a virus, each of which is expressed in the cell adeno- The associated virus is generated and packaged in HEK293T.
  • This packaging step will be described in detail in the following examples.
  • Step d) of the present invention is a step of introducing each of the viruses prepared in step c) to one species selected from the group of animal cells expressing a GIRK channel (G protein-coupled inwardly-rectifying potassium channel), allatostatin It is the GIRK channel (G protein-coupled inwardly-rectifying potassium channel) that causes a difference in intracellular membrane potential with or without an allatostatin receptor (AlstR) protein.
  • Each of the prepared viruses was introduced into one species selected from the group of animal cells expressing the GIRK channel to enable expression of the allatostatin receptor in the cells.
  • the animal cell may be, for example, one selected from the group consisting of HEK 293T cell, HEK 293 cell, NIH3T3 cell, and Neuro2a cell, preferably HEK 293T cell.
  • an allatostatin receptor (AlstR) specifically present in insects in animal cells
  • AlstR allatostatin receptor
  • a GIRK channel is activated to cause potassium ions (K + ).
  • K + potassium ions
  • 2 is a control test which is a graph measuring the fluorescence change of cells using cells not expressing an allatostatin receptor, using potassium ions (K + ) and carbachol to confirm the expression of GIRK channels.
  • potassium ions (K + ) are introduced into cells by high concentrations of potassium ions, membrane potential changes, depolarization occurs, and the fluorescence becomes strong.
  • Carbachol is a derivative of acetylcholine, which activates the acetylcholine receptors and thereby activates Gi signal transduction in the cell, which activates the GIRK channel to open the channel and release potassium ions in the cell.
  • K + potassium ions
  • a plasmid containing a GIRK channel for introducing into the animal cell of step d) is prepared, and a plasmid containing a GIRK channel is prepared using a calcium phosphate transfection method (CaHPO 4 Transfection).
  • CaHPO 4 Transfection a calcium phosphate transfection method
  • the GIRK channel is a channel in which the difference in intracellular membrane potential changes when the allatostatin receptor reacts with the allatostatin.
  • the GIRK channel has four types of GIRK1, GIRK2, GIRK3 and GIRK4, and the double allatostatin receptor Channels affected by (AlstR) are known as GIRK1, GIRK2. Therefore, the GIRK channel is preferably one or more selected from the group consisting of GIRK1, GIRK2, GIRK3, and GIRK4, and more preferably includes GIRK1, GIRK2. It is also known that the GIRK channel of the mouse performs the most specific reaction among the GIRK channels present in various animals such as humans and mice. In the present invention, a step of overexpressing and reacting GIRK1 and GIRK2 channels was further introduced to enhance the effect of allatostatin by the virus.
  • step f) of the present invention in the method for screening pesticides using an allatostatin agonist, the standard for determining the possibility of agonists of natural pesticide candidates is a membrane potential change, and thus the membrane potential change. Is detectable by fluorescence expression of voltage sensitive dye. Voltage sensitive dyes include bis- (1,3-dibutylbarbituric acid) trimethine oxonol (Bis- (1,3-) with the characteristics of a slow-response potential-sensitive probe.
  • Dibutylbarbituric Acid Trimethine Oxonol: DiBAC4 (3)
  • bis- (1,3-diethylthiobarbituric acid) trimethine oxonol Bis- (1,3-diethylthiobarbituric acid) trimethine oxonol: DiSBAC2 (3)
  • Bis- (1,3-dibutylbarbituric acid) pentamethine oxonol Bis- (1,3-dibutylbarbituric acid) pentamethine oxonol: DiBAC4 (5)
  • Bis- (1,3-dibutylbarbituric acid) trimethine oxonol Bis- (1,3-Dibutylbarbituric Acid
  • Trimethine Oxonol: DiBAC4 (3) may be used, but is not limited thereto.
  • the voltage sensitive dye enters a cell and binds to an intracellular protein, a membrane, and the like, and has a characteristic of enhancing fluorescence.
  • the depolarization is enhanced, the dye is introduced into the cell and the fluorescence is increased, while the fluorescence is decreased during the hyperpolarization, and these characteristics are easy to confirm the expression of G protein-coupled inwardly-rectifying potassium channel. Can be used.
  • the present invention uses a Fluorescence Imaging Plate Reader (FLIPR) device for monitoring the difference in cell membrane potential in the presence or absence of an allatostatin receptor protein, wherein the fluorescence imaging plate reader is a microwell with a microplate reader or a plate reader. Samples are placed on a plate to measure biological, chemical, and physical changes, allowing for changes in fluorescence.
  • FLIPR Fluorescence Imaging Plate Reader
  • Zenyth 3100 Microplate Multimode Detector
  • the microwell plate was used to test many kinds of materials using a 96 well plate.
  • the present invention by expressing the allatostatin receptor in the cell using a viral vector, it is possible to confirm the change in the membrane potential when the allatostatin protein is treated.
  • (A) AlstR-IRES-mCherry graph of Figure 3 of the present invention shows that the fluorescence changes over time of allatostatin action in the cells expressing the allatostatin receptor through the AAV system
  • (b) The IRS-mCherry graph shows that the fluorescence change was measured to confirm the expression of cells excluding the allatostatin receptor as control cells of AlstR-IRES-mCherry
  • -mCherry) graph shows the effect of allatostatin in the cells by comparing the variation in fluorescence changes between the cells expressing and not expressing allatostatin with or without the allatostatin receptor protein.
  • AlstR-IRES-mCherry (AAV) + GIRK1, GIRK2 graph of Figure 4 of the present invention is transfected plasmid containing GIRK1, GIRK2 in the cell expressing the allatostatin receptor through the AAV system in the cell Fluorescence change over time of allatostatin action was measured, and
  • IRES-mCherry (AAV) + GIRK1, GIRK2 graph is a control cell of AlstR-IRES-mCherry except for the allatostatin receptor.
  • Fluorescence changes were measured by transfection of plasmids containing GIRK1 and GIRK2 to confirm expression, and (c) (AlstR-IRES-mCherry)-(IRES-mCherry) + GIRK1, GIRK2 graph shows GIRK1, GIRK2. According to the presence or absence of the allatostatin receptor protein according to the transfection of the plasmid containing a comparison of the variation in the fluorescence change between the allatostatin-expressing cells and the non-expressing cells confirmed that the action of allatostatin in the cells Served.
  • Pesticide candidates can be determined to act similar to allatostatin.
  • Adeno-associated virus vectors were prepared by the following method.
  • Figure 5 shows the vector as a basis for the production of adeno-associated virus (adeno-associated virus),
  • A is a pAAV-sh-mCherry vector
  • B is a pAlstR-IRES-GFP vector It was. The part with sh in (A) was removed and AlstR and IRES of (B) were used.
  • A is the vector and B's AlstR-IRES is the insert.
  • the preparation of A includes 5ul of NEB buffer2, 5ul of BSA buffer, 5ug of DNA (1ug / ul), 5ul of NheI enzyme (NEB, cat # R0131S), Autoclaved DW (autoclaved tertiary distilled water, ADW) ) Put 30ul, enzyme cut for 1 hour 30 minutes at 37 °C and heat inactivation at 65 °C 20 minutes to inhibit the activity. After that, Klenow (NEB, cat # M0210S) was treated to change the enzyme cut site to the blunt end. The mixture was treated with 2ul dNTP and 1ul of Klenow and treated for 1 hour at room temperature, followed by heat inactivation at 75 ° C. for 20 minutes.
  • B (pAlstR-IRES-GFP) was prepared by adding 5 ul of NB buffer2, 5 ul of BSA buffer, 5 ug of DNA (1 ug / ul), 5 ul of NheI, and 30 ul of ADW, followed by enzyme cut at 37 ° C. for 1 hour 30 minutes. Activity was inhibited by heat inactivation for 20 minutes at °C. Thereafter, 5ul of SmaI (NEB, cat # R0141S) was added and the enzyme cut was performed at 37 ° C. for 1 hour and 30 minutes, followed by heat inactivation at 65 ° C. for 20 minutes to inhibit activity.
  • SmaI NEB, cat # R0141S
  • the blunt end site of A and the blunt end site generated by SmaI of B were ligation, and the XbaI site of A and the NheI site of B were ligation.
  • Gel extraction was performed on A and B.
  • A was about 6 Kb in size, and B was about 1.9 Kb in size.
  • concentrations and molecular sizes of A and B were then calculated and ligation.
  • vector and insert DNA were put in proportion and 1ul of T4 DNA ligase (NEB cat # M0202S) and 2ul of 10Xligase buffer were added to make a total of 20ul. Ligation was performed for 16 hours at room temperature.
  • A is the vector and B's IRES is the insert site.
  • A (pAAV-sh-mCherry) is prepared by adding 5 ul of NEB buffer4, 5 ul of BSA buffer, 5 ug of DNA (1 ug / ul), 5 ul of XbaI enzyme, and 30 ul of ADW, followed by enzyme cut at 37 ° C for 1 hour 30 minutes. Activity was inhibited by heat inactivation at 65 ° C. for 20 minutes. After that, the enzyme cut site was changed to blunt end by Klenow treatment. The mixture was treated with 2ul dNTP and 1ul of Klenow and treated for 1 hour at room temperature, followed by heat inactivation at 75 ° C. for 20 minutes.
  • the manufacturing process of B (pAlstR-IRES-GFP) was carried out at 37 ° C. for 1 hour after adding NEB buffer EcoRI 5ul, BSA buffer 5ul, DNA (1ug / ul) 5ug, EcoRI (NEB cat # R0101S) 5ul, and ADW 30ul. After 30 minutes of enzyme cut, the activity was inhibited by heat inactivation at 65 ° C. for 20 minutes. After that, the enzyme cut site was changed to blunt end by Klenow treatment. The mixture was treated with 2ul dNTP and 1ul of Klenow, and treated for 1 hour at room temperature, followed by heat inactivation at 75 ° C. for 20 minutes.
  • the blunt end site of A and the blunt end site of B were ligation, and the NheI site of A and the XbaI site of B were ligation.
  • Gel extraction of A and B was carried out, where A was about 6Kb DNA and B was about 0.6Kb DNA. After that, the concentrations and molecular sizes of A and B were calculated and ligation.
  • Vector and Insert DNA were added in proportion and 1ul of T4 DNA ligase and 2ul of 10Xligase buffer were added to make total 20ul. Ligation was performed for 16 hours at room temperature.
  • Fluorescent protein is used to confirm whether the virus is normally produced and entered into cells.
  • mCherry protein among various fluorescent proteins was used. Fluorescence Excitation of Bis- (1,3-Dibutylbarbituric Acid) Trimethine Oxonol (DiBAC 4 (3)) Used as Voltage Sensitive Dye The wavelength was 490 nm and the emission wavelength was 516 nm, and mCherry (excitation 587 nm, emisiion 610 nm) protein among fluorescent proteins was used to minimize interference with these wavelengths.
  • the internal ribosome entry site (IRS) was used to allow the expression of the allatostatin receptor and mCherry protein independently, thereby minimizing other effects on the allatostatin receptor.
  • an adeno-associated virus is generated and packaged in HEK293T.
  • Plasmids and cells were present in the AAV Helper-Free system (Agilent, cat # 240071).
  • AAV-293 cells were incubated in a 100-mm tissue culture plate. At this time, the cells were sprayed at about 5 * 10E5 ⁇ 2 * 10E6 per plate and incubated in 37 ° C and 5% CO 2 incubator. After about two to three days, the cells have a 70-80% confluency, followed by plasmid transfection.
  • pAAV expression plasmid prepared vector pAAV-AlstR-IRES-mCherry or pAAV-IRES-mCherry
  • pAAV-RC prepared vector pAAV-AlstR-IRES-mCherry or pAAV-IRES-mCherry
  • pAAV-RC prepared vector pAAV-RC
  • pHelper three plasmids were prepared in TE buffer, PH 7.5 at a concentration of 1 ug / ul and simultaneously transfected into cells. I let you. The three kinds of plasmids were put in 10 ml of 15 ml conical tubes, and 1 ml of 0,3M CaCl 2 was added and mixed gently. The mixed solution was dropped dropwise into 1 ml of 2XHBS and gently mixed.
  • HEK 293T cells human kidney cells, were placed in 96 black well plates (SPL, Cat # -SP30296) at 1 * 104 cells / well (100 uL each well) and incubated in a 5% CO 2 incubator at 37 ° C for one day.
  • the cells cultured for one day had a confluent of about 50% to 60%. If they did not grow enough, they were further cultured. After removing the media from the wells with well-grown cells, 100ul each of the virus with the alatostatin receptor prepared using the Adeno-associated virus (AAV) Helper-Free system was mixed with the media. Treated. At this time, the control virus which does not contain an allatostatin receptor was similarly treated. 16 to 24 hours after the virus treatment, transfection of the plasmid containing the GIRK channel was performed by the following method.
  • AAV Adeno-associated virus
  • GIRK2-IRES-GIRK1 plasmid was prepared by the following method.
  • FIG. 6 shows a vector that is the basis for the preparation of the GIRK2-IRES-GIRK1 vector (a), (b) shows a GIRK2 openbiosystem vector, and (c) shows a GIRK1 openbiosystem vector.
  • Remove the AlstR portion from the pAlstR-IRES-GFP vector of FIG. 5 (b), remove the GIRK2 portion of FIG. 6 (b), remove the GFP portion of FIG. 5 (b), and the GIRK1 portion of FIG. 6 (c). was inserted to complete the GIRK2-IRES-GIRK1 vector (Fig. 6 (a)).
  • A represents pAlstR-IRES-GFP
  • B represents the GIRK2 openbiosystem vector
  • C represents the GIRK1 openbiosystem. That is, A clones the INSERT part of B first with a vector, and then clones the INSERT part of C.
  • the manufacturing process of A is as follows. After adding NEB buffer3 5ul, BSA buffer 5ul, DNA (1ug / ul) 5ug, MluI (NEB, cat # R0198S) 5ul, ADW 30ul, enzyme cut at 37 °C for 1 hour 30 minutes and heat at 80 °C for 20 minutes. Inactivation was inhibited. Thereafter, 5ul of SmaI (NEB, cat # R0141S) was added to the enzyme cut for 1 hour and 30 minutes at 37 ° C, and heat inactivation was performed at 65 ° C for 20 minutes to inhibit activity. Thereafter, Klenow (NEB, cat # M0210S) was treated to change the enzyme cut site to the blunt end.
  • the mixture was treated with 2ul dNTP and 1ul of Klenow (NEB, cat # M0210S) and treated at room temperature for 1 hour. After that, only DNA was extracted through DNA cleanup. Using the extracted DNA, NEB buffer2 5ul, BSA buffer 5ul, DNA (1ug / ul) 5ug, NheI enzyme 5ul, ADW 30ul was added, and the enzyme cut for 1 hour 30 minutes at 37 °C and heat for 20 minutes at 65 °C. Inactivation was inhibited. Thereafter, Klenow (NEB, cat # M0210S) was treated to change the enzyme cut site to the blunt end.
  • Klenow (NEB, cat # M0210S) was treated to change the enzyme cut site to the blunt end.
  • the mixture was treated with 2ul dNTP and 1ul Klenow and treated at room temperature for 1 hour, followed by heat inactivation at 75 ° C. for 20 minutes to inhibit activity.
  • the preparation of B is performed by adding 5ul of NEB buffer4, 5ul of BSA buffer, 5ug of DNA (1ug / ul), 5ul of SacII enzyme (NEB, cat # R0157S), and 30ul of Autoclaved DW (autoclaved tertiary distilled water, ADW). After enzyme cut for 1 hour and 30 minutes at 37 °C, heat inactivation was performed at 65 °C for 20 minutes to inhibit activity. Thereafter, Klenow (exo) (NEB, cat # M0212S) was treated to change the enzyme cut site to the blunt end. The mixture was treated with 2ul dNTP and 1ul Klenow and treated at room temperature for 1 hour, followed by heat inactivation at 75 ° C. for 20 minutes.
  • a and B were ligation.
  • Gel extraction was performed on A and B.
  • A was about 6.7 Kb in size
  • B was about 1.6 Kb in size.
  • concentrations and molecular sizes of A and B were then calculated and ligation.
  • the vector and the insert DNA were put in proportion, and 1ul of T4 DNA ligase (NEB cat # M0202S) and 2ul of 10Xligase buffer were added to make a total of 20ul. Ligation was performed for 16 hours at room temperature.
  • a GIRK2-IRES-GFP vector was first prepared. After removing GFP, the GIRK1 region of the GIRK1 openbiosystem vector was inserted.
  • GIRK2-IRES-GFP DNA production process NEB buffer4 5ul, BSA buffer 5ul, DNA (1ug / ul) 5ug, XbaI (NEB, cat # R0145S) 5ul, ADW 30ul was added at 37 °C After enzyme cut for 1 hour 30 minutes, heat inactivation was performed at 65 ° C. for 20 minutes to inhibit activity. After that, only DNA is extracted through the DNA cleanup process.
  • the preparation of C was performed by adding 5 ul of NEB buffer4, 5 ul of BSA buffer, 5 ug of DNA (1ug / ul), 5ul of XbaI (NEB, cat # R0145S), and 30ul of ADW, followed by enzyme cut at 37 ° C. for 1 hour 30 minutes. Activity was inhibited by heat inactivation for 20 minutes at °C. After that, only DNA was extracted through the DNA cleanup process. Thereafter, 5ul of NEB buffer3, 5ul of BSA buffer, 5ug of DNA (1ug / ul), 5ul of NotI (NEB, cat # R0189S), and 30ul of ADW were added, followed by enzyme cut at 37 ° C for 1 hour 30 minutes. Activity was inhibited by heat inactivation for 20 minutes.
  • Each XbaI enzyme cut site and NotI enzyme cut site were ligation.
  • Gel extraction was performed on A and B.
  • A was about 7.6 Kb in size
  • B was about 1.7 Kb in size.
  • the concentrations and molecular sizes of A and B were then calculated and ligation.
  • the vector and the insert DNA were put in proportion, and 1ul of T4 DNA ligase (NEB cat # M0 2 0 2 S) and 2ul of 10Xligase buffer were added to make a total of 20ul. Ligation was performed for 16 hours at room temperature.
  • Plasmid transfection was performed using Calcium Phosphate Transfection (CaHPO4 Transfection).
  • a total of 10ul of 0.3M CaCl 2 to each of the reference 1well 96well plate is 8.7ul the transfected DNA (GIRK2-IRES-GIRK1) with 0.5ug remaining capacity DW to design After mixing and mixing with 10ul of HBS (2X), a total of 20ul was treated in the well.
  • the media in the wells were treated with 20 ul of fresh media after removing the virus.
  • the difference in cell membrane potential in the presence or absence of an allatostatin receptor (AlstR) protein was monitored to determine the fluorescence difference between the cells expressing the allatostatin receptor and the cells not expressing the allatostatin receptor. Fluorescence changes in accordance with intracellular membrane potential difference, and the change in fluorescence was measured by the following method using a Zenyth 3100 (microplate Multimode Detector), a Fluorescence Imaging Plate Reader (FLIPR).
  • Zenyth 3100 microplate Multimode Detector
  • FLIPR Fluorescence Imaging Plate Reader
  • the packaged virus was introduced into human cells and prepared for measuring fluorescence after 48 hours of expression of the virus, and the presence of cell residues in the well would interfere with fluorescence measurement.
  • DiBAC 4 (3) was washed with an undissolved Tyrode's solution to remove cell residue. The media was removed from each well, 100ul of tie rod solution was added, and the operation was repeated twice.
  • 5BAM DiBAC solution was prepared by dissolving DiBAC 4 (3), which was previously dissolved in dimethyl sulfoxide (DMSO) in a tie rod solution, and treated with 180uL per well, followed by 5% CO at 37 ° C for one hour. Incubated in 2 incubators.
  • DMSO dimethyl sulfoxide
  • the material to be measured was dissolved in 5uM DiBAC solution and additionally added 20uL for each well, and the change in fluorescence over time was measured using Zenyth 3100. Fluorescence was measured at 3 minute intervals. Initially, the fluorescence was measured without any processing to determine if the instrument and the well were optimized for measurement. Was compared. More specifically, the control well was set to 0, and the increase in fluorescence was measured by comparing the well with other substances with the control.
  • the intensity of fluorescence was measured, adding 20ul of measuring substance each.
  • the difference in fluorescence was compared between the value treated with the measurement material and the change value thereafter. This is because as the amount of buffer increases in the well, the absolute value of fluorescence also changes.

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Abstract

Cette invention concerne un procédé de criblage d'une substance insecticide. Quand le procédé de criblage selon l'invention est utilisé, une substance naturelle, similaire à l'allatostatine qui supprime la synthèse de l'hormone juvénile exprimée chez les insectes, est découverte. Ladite substance peut par conséquent être utilisée comme pesticide pour entraver le développement normal des insectes nocifs. L'allatostatine et son récepteur existant spécifiquement dans le monde des insectes, ladite substance ne devrait avoir aucun effet sur l'homme et autres mammifères et présente l'avantage de se prêter à la mise au point d'un pesticide respectueux de l'environnement.
PCT/KR2014/007101 2013-08-28 2014-08-01 Procédé de criblage d'une substance insecticide Ceased WO2015030380A1 (fr)

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KR10-2013-0102331 2013-08-28
KR1020130102331A KR101461426B1 (ko) 2013-08-28 2013-08-28 살충 물질을 스크리닝 하는 방법

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060003326A (ko) * 2002-08-06 2006-01-10 파마시아 앤드 업존 캄파니 엘엘씨 드로소필라 ｇ 단백질-커플링된 수용체, 핵산 및 그와관련된 방법
WO2011036536A2 (fr) * 2009-09-25 2011-03-31 Arterra Bioscience S.R.L. Micro-organismes inactivés contenant des molécules d'arn double brin (dsarn), leur utilisation comme pesticides et leurs procédés de préparation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060003326A (ko) * 2002-08-06 2006-01-10 파마시아 앤드 업존 캄파니 엘엘씨 드로소필라 ｇ 단백질-커플링된 수용체, 핵산 및 그와관련된 방법
WO2011036536A2 (fr) * 2009-09-25 2011-03-31 Arterra Bioscience S.R.L. Micro-organismes inactivés contenant des molécules d'arn double brin (dsarn), leur utilisation comme pesticides et leurs procédés de préparation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GADE, G. ET AL.: "Review: Insect peptide hormones: a selective review of their physiology and potential application for pest control.", PEST MANAG SCI., vol. 59, 2003, pages 1063 - 1075 *
KAI, ZHEN-PENG. ET AL.: "A potential insect growth regulator: Synthesis and bioactivity of an allatostatin mimic.", PEPTIDES, vol. 30, 2009, pages 1249 - 1253 *
WOODS D. ET AL.: "Receptor-Based Discovery Strategies for Insecticides and Parasiticides: A Review.", ADV EXP MED BIOL, vol. 692, 2010, pages 1 - 9 *

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