JP2016054698A - Screening method of dopamine receptor active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and efficient screening method of a dopamine receptor active substance, and to provide cells used in the screening method.SOLUTION: A dopamine receptor active substance can be screened simply and efficiently by using cells in which a polynucleotide having: a polynucleotide region 1 having (a) a polynucleotide encoding a dopamine receptor protein, (b) a cAMP response element, and also having a promoter function; and a polynucleotide region 2 encoding a secreted reporter protein arranged at 3'-terminal side of the polynucleotide region 1 is introduced.SELECTED DRAWING: None

Description

  The present invention relates to a screening method for a substance that acts on a dopamine receptor, particularly an invertebrate-specific dopamine receptor, and a cell used for the screening method.

Dopamine is one of the biogenic amines widely present in living organisms. When dopamine binds to its receptor, dopamine receptor, G protein is activated and various intracellular information is regulated. For example, when G _s protein is activated, adenylate cyclase is activated, the activation of the increased concentration and protein kinase A in intracellular cAMP occurs. This triggers phosphorylation of various proteins or regulates gene transcription, and as a result, regulates information transmission in the nervous system. Disturbing this regulatory mechanism controls insect feeding and growth, making dopamine receptors a target for insecticides or pest control agents. Therefore, in order to create a pest-selective insecticide or control agent, it is essential to develop a method for screening dopamine receptor agonists (agonists, antagonists, etc.).

Various methods are known for screening substances that regulate information transmission in the nervous system for the purpose of creating insecticides or pest control agents. For example, a method of screening by conducting a binding experiment between a neurotransmitter receptor protein and a pesticide or a pest control agent candidate substance and evaluating its binding ability, or a cell line that stably expresses a neurotransmitter receptor. In addition, a screening method is known in which an insecticide or a pest control agent candidate substance is allowed to act and the influence on the current response is analyzed by a patch clamp method (Patent Document 1).
However, these conventional screening methods are complicated in operation due to circumstances such as disruption of cells, preparation of membrane fractions containing target proteins, use of radioactive substances, or the need for skilled assay systems. It is not suitable for processing samples quickly.

Vincenzina Reale et al. The expression of a cloned Drosophila octopaminertyramine receptor in Xenopus oocytes Brain Research 769 _1997. 309-320

  An object of the present invention is to provide a simple and efficient screening method for a dopamine receptor agonist and a cell used in the screening method.

As a result of repeated earnest research to solve the above problems, the present inventors,
Polynucleotide (a): polynucleotide encoding a dopamine receptor protein, and polynucleotide (b): polynucleotide region 1 having a cAMP response element and having a promoter function, and 3 ′ end side of polynucleotide region 1 It was found that a dopamine receptor agonist can be easily and efficiently screened by using a cell containing a polynucleotide having a polynucleotide region 2 encoding a secretory reporter protein arranged in 1.

  The principle of this screening method is as follows. When cells containing the polynucleotides (a) and (b) are brought into contact with a test substance, the test substance is expressed from the polynucleotide (a) by the contact if it is a substance that acts on a dopamine receptor. Changes in intracellular cAMP concentration occur through the dopamine receptors. Then, the change in intracellular cAMP concentration is detected by the polynucleotide region 1 having a cAMP element and having a promoter function in the polynucleotide (b), and expression of the secretory reporter protein encoded by the polynucleotide region 2 is expressed. To change. Thus, the action of the test substance on the dopamine receptor appears as a change in the expression level of the secretory reporter protein. Therefore, by comparing the amount of secretory reporter protein when the cell is brought into contact with the test substance and the amount of secretory reporter protein when the cell is not brought into contact with the test substance, the test substance becomes a dopamine receptor. It can be determined whether or not it is an agent, and a dopamine receptor agonist can be screened.

  In addition, the present inventors initially examined increasing the amount of secreted reporter protein detected by increasing the contact time between the cell and the test substance. However, quite unexpectedly, if the contact time is increased, the amount of secreted reporter protein should increase depending on the concentration of the test substance. In some cases, we found the problem of decreasing. If such a problem occurs in the screening method of the present invention, a substance originally having an action on the dopamine receptor cannot be screened, or vice versa, so that the accuracy of the screening method of the present invention is reduced. End up. For this reason, from the viewpoint of further improving the screening accuracy, it is preferable that the contact time between the cell and the test substance is shorter.

  As a result of further research based on the above findings, the present invention was completed.

  That is, the present invention includes the following aspects.

Item 1. A cell comprising the following polynucleotide (a) and the following polynucleotide (b):
(A) a polynucleotide encoding a dopamine receptor protein, and (b) a polynucleotide region 1 having a cAMP response element and having a promoter function, and a secreted form disposed on the 3 ′ end side of the polynucleotide region 1 A polynucleotide having a polynucleotide region 2 encoding a reporter protein.

  Item 2. Item 5. The cell according to any one of Items 1 to 4, wherein the polynucleotide (a) is a polynucleotide encoding an invertebrate-specific dopamine receptor protein.

  Item 3. Item 3. The cell according to Item 1 or 2, which is a mammalian cell containing the polynucleotide (a) and the polynucleotide (b).

  Item 4. Item 4. The cell according to any one of Items 1 to 3, wherein the reporter protein is secretory placental alkaline phosphatase.

  Item 5. Item 5. The cell according to any one of Items 1 to 4, which is a HEK-293 cell comprising a polynucleotide (a) and a polynucleotide (b).

Item 6. A screening method for a dopamine receptor agonist comprising the following steps (a), (b), and (c):
(A) contacting the cell according to any one of Items 1 to 5 with a test substance;
(A) After step (a), the reporter activity of the secreted reporter protein secreted from the cell is measured, and the activity value is compared with the reporter activity value when not contacting with the test substance; ) A step of selecting a test substance as a dopamine receptor agonist when the reporter activity value when contacted with the test substance is significantly different from the reporter activity value when not contacted with the test substance .

  Item 7. Item 7. The screening method according to Item 6, wherein the contact time in step (a) is less than 8 hours.

Item 8. A method for screening a dopamine receptor agonist,
In the step (c), when the reporter activity value when contacted with the test substance is significantly higher than the reporter activity value when not contacted with the test substance, the test substance is selected as a dopamine receptor agonist. Is the process of
Item 8. The screening method according to Item 6 or 7.

Item 9. A screening method for dopamine receptor antagonists, comprising:
The step (a) is a step of bringing the cell according to any one of Items 1 to 5 into contact with a dopamine receptor agonist and a test substance,
In the step (a), after the step (a), the reporter activity of the secretory reporter protein secreted from the cell is measured, and the activity value is brought into contact with the dopamine receptor agonist without contacting with the test substance. The reporter activity value in the case of
In the step (c), the reporter activity value when contacted with the dopamine receptor agonist and the test substance is more significant than the reporter activity value when contacted with the dopamine receptor agonist without contact with the test substance. The test substance is selected as a dopamine receptor antagonist,
Item 8. The screening method according to Item 6 or 7.

  According to the present invention, an agent such as a dopamine receptor agonist or antagonist can be conveniently and rapidly screened from multiple test substances.

The agonistic activity of the test substance measured by the screening method of the present invention is shown (Example 3-1). The agonistic activity of the test substance measured by the screening method of the present invention is shown (Example 3-2). The antagonistic activity of the test substance measured by the screening method of the present invention is shown (Example 3-3).

1. Cell of the Present Invention The cell of the present invention is a cell characterized by comprising polynucleotide (a) and polynucleotide (b).

  The polynucleotide (a) is a polynucleotide encoding a dopamine receptor protein. The polynucleotide (a) is contained in the cell in a state where the dopamine receptor protein can be expressed. The expressible state is a state in which the polynucleotide (a) is placed in the cell under the control of an appropriate transcriptional regulatory region, specifically, an appropriate promoter. A suitable promoter is not particularly limited as long as it has promoter activity in cells containing the polynucleotides (a) and (b), and a known promoter can be used. For example, in the case of mammalian cells, viral promoters such as CMV promoter and SV40 promoter, EF-1α promoter, UbC promoter and the like can be used.

  The dopamine receptor protein encoded by the polynucleotide (a) can be derived from various organisms and is not particularly limited. For example, dopamine receptor proteins derived from vertebrates such as mammals, dopamine receptor proteins derived from invertebrates such as insects, and the like can be employed. Among them, invertebrate-derived dopamine receptor proteins are preferable, and invertebrate-specific dopamine receptor proteins are more preferable for the purpose of creating insecticides or pest control agents. Examples of invertebrates include silkworms, Drosophila, honeybees, cockroaches, grasshoppers, anopheles, and moss.

The invertebrate-specific dopamine receptor protein is not particularly limited as long as it is a type of dopamine receptor protein that exists specifically in invertebrates. Such types include, for example, Reference 1 (Insect Biochem Mol Biol. 39 (5-6), 342-7, 2009.), Reference 2 (Archives of Insect Biochemistry and Physiology. 59, 103-117, 2005. ) Etc. Specifically, Bombyx mori DopR2 (protein consisting of the amino acid sequence shown in SEQ ID NO: 1), Drosophila melanogaster DAMB (protein consisting of the amino acid sequence shown in SEQ ID NO: 2), honey bee ( Apis mellifera ) DOP2 (protein consisting of the amino acid sequence shown in SEQ ID NO: 3) and the like, and among these, silkworm ( Bombyx mori ) DopR2 is preferable.

  The protein consisting of the amino acid sequence represented by SEQ ID NO: 1, 2, or 3 is one or more in the amino acid sequence, preferably 1 or so long as it does not impair the function of causing an increase in intracellular cAMP amount by dopamine binding. 2 to 20, more preferably 1 or 2 to 8, more preferably 1 or 2 to 5, particularly preferably 1 or 2 to 3 amino acids are substituted, deleted or inserted, Also good. As a site where an amino acid is substituted, deleted, or inserted, a site other than a known functional domain on the dopamine receptor protein is preferable. A known functional domain of a dopamine receptor can be examined by a known search method from a known database such as NCBI (http://www.ncbi.nlm.nih.gov). Further, as long as the function of the dopamine receptor is not impaired, the site where the amino acid is substituted, deleted, or inserted may be a site on a known functional domain. The presence or absence of a function that causes an increase in intracellular cAMP amount by dopamine binding can be easily determined by using dopamine as a test substance in the screening method of the present invention described later.

  The polynucleotide (b) is a polynucleotide having a polynucleotide region 1 and a polynucleotide region 2.

  The polynucleotide region 1 is a polynucleotide region having a cAMP response element and having a promoter function.

  A cAMP response element (CRE) is an element that can bind to a cAMP response element binding protein (CREB) and activate expression of a gene encoded downstream of the element. The origin of the cAMP response element is not particularly limited as long as it has such a function, and it may have a known mutation that is known not to affect the above function.

  Having a promoter function means having a function of expressing a secreted reporter protein encoded by the polynucleotide region 2 arranged on the 3 'side of the polynucleotide region 1. For example, it shows that it has a known minimum promoter region necessary for allowing RNA polymerase to bind and initiate transcription, specifically, a TATA-like promoter (TAL).

  The polynucleotide region 2 is a polynucleotide that is located 3 'to the polynucleotide region 1 and encodes a secreted reporter protein.

  The secretory reporter protein is a protein that has the property of being secreted outside the cell after expression as a protein and that can be easily quantified, and is not particularly limited as long as it has such a property. Can be mentioned. Specific examples of the secretory reporter protein include enzyme proteins that can be quantified by measuring enzyme activity, such as secretory placental alkaline phosphatase (SEAP) and secreted luciferase derived from Metridia longa.

  The polynucleotide (a) and / or (b) may contain other sequences. For example, a region encoding a sequence encoding a protein tag such as a His tag, Myc tag, HA tag, or GFP tag or a gene resistant to various antibiotics may be included. After introducing a polynucleotide (a) and / or (b) into a cell by using a polynucleotide (a) and / or (b) containing a region designed to express such a resistance gene, By culturing the cells in an antibiotic-containing medium in which the resistance gene exhibits resistance, only the cell group containing the polynucleotide (a) and / or (b) can be selected.

  The polynucleotide (a) can be prepared using a known method. For example, a sequence encoding a dopamine receptor protein is appropriately inserted into a known expression vector containing a promoter such as a CMV promoter and having a multiple cloning site for inserting a gene coding sequence on the 3 ′ side of the promoter. It can be prepared by inserting through The sequence information of the sequence encoding the protein of the dopamine receptor is published on various databases, for example, by a known search method from a database such as NCBI (http://www.ncbi.nlm.nih.gov). Can be obtained. A polynucleotide comprising the target sequence can be obtained using a known method such as PCR or DNA artificial synthesis technology.

  The polynucleotide (b) can be prepared in the same manner as the polynucleotide (a). The sequence encoding the secretory reporter protein can be determined based on known information. A commercially available product may be used as the polynucleotide (b). For example, Clontech's pCRE-SEAP Vector.

  The cell is not limited as long as it has a mechanism for converting a signal through the dopamine receptor into a change in cAMP concentration. Specific examples of such cells include eukaryotic cells, preferably vertebrate cells or invertebrate cells, more preferably mammalian cells, more preferably human or rodent cells. be able to. Moreover, from the viewpoint that the signal background caused by the intracellular adrenergic receptor can be further reduced, kidney cells, ovary-derived cells and the like are preferable, and HEK-293 cells, CHO cells and the like are more preferable. Further, from the viewpoint that functional expression and high expression of the dopamine receptor protein encoded by the polynucleotide (a) can be expected more specifically, that is, a signal derived from a test substance can be detected with higher sensitivity, kidney cells are preferably mentioned. HEK-293 cells are more preferable.

  Introduction of the polynucleotide (a) and the polynucleotide (b) into the cell can be performed using a known method. Specific examples include transfection methods using these polynucleotides with introduction reagents based on cationic lipids, polyamines, and the like, the calcium phosphate method, the electroporation method, and methods using viruses. The state of the polynucleotide (a) and / or polynucleotide (b) introduced into the cell is not particularly limited. For example, even if the polynucleotide (a) and the polynucleotide (b) are transiently present in the cell, they are integrated into the genome. It may be in a stable state in the cell. In the screening method of the present invention, from the viewpoint that detection sensitivity can be further increased and the expression level of the receptor is made constant (and thus the stability of data between experiments is increased), the polynucleotide (a) is: It is preferably stably present in the cell in a state of being integrated into the genome.

  By using the cells of the present invention in the screening method for dopamine receptor agonists described later, dopamine receptor agonists can be screened efficiently and simply.

2. Screening method of the present invention The screening method of the present invention is a dopamine receptor agonist screening method comprising the steps (a), (b) and (c).

2-1. Screening method for dopamine receptor agonist
A dopamine receptor agonist is a substance that acts on a dopamine receptor and regulates signal transduction downstream of the dopamine receptor, specifically, an effect of increasing or decreasing cAMP concentration. In this respect, the active substance includes, for example, an agonist, an antagonist, an inverse agonist, a super agonist, an allosteric ligand and the like. According to the method for screening a dopamine receptor agonist, a dopamine receptor agonist can be screened from test substances.

  Step (a) is a step of bringing the cells containing the polynucleotides (a) and (b) described in “1. Cells of the present invention” into contact with a test substance.

  The test substance is a target substance for screening whether or not it is a dopamine receptor agonist. The test substance can be widely used regardless of a naturally occurring compound or an artificially produced compound. Moreover, the composition which mixed not only the refined compound but various compounds, and the extract of animals and plants can also be used.

  The mode in which the cells containing the polynucleotides (a) and (b) are brought into contact with the test substance is not particularly limited as long as the test substance can come into contact with the cell membrane. For example, a mode in which a test substance is dissolved in a culture medium in which cells are cultured, or a mode in which the culture liquid in which cells are cultured is replaced with a culture liquid in which the test substance is dissolved in advance.

  The upper limit of the contact time between the cell and the test substance is preferably shorter from the viewpoint of further improving the screening accuracy, for example, less than 8 hours, preferably 6 hours or less, more preferably 4 hours or less, and still more preferably. It can be up to 2 hours. The lower limit of the contact time between the cell and the test substance is not particularly limited as long as the action of the test substance is sufficient to cause a change in the amount of cAMP via the dopamine receptor. For example, 5 minutes or more , Preferably 15 minutes or longer, more preferably 30 minutes or longer, and even more preferably 1 hour or longer.

  The culture medium is a medium for culturing cells. The culture solution is not particularly limited as long as cells can be cultured, and a known medium and serum such as FBS added thereto can be widely used depending on the cell type. For example, DMEM (Dulbecco's modified Eagle's medium) or Ham's F-12 medium, which is known to be suitable for culturing various types of cells, can be used for culturing by adding serum suitable for each cell. it can. Depending on the type of cell to be cultured, various components (sugar, amino acid, salt, metal component, etc.) can be added to the culture solution, and the concentration thereof is not particularly limited as long as it is suitable for cell culture.

Culturing is performed by incubation under conditions suitable for cell survival, for example, CO ₂ concentration 5%, 37 ° C. under humid conditions.

  After the step (a), it is preferable to culture the cells in the absence of the test substance, if necessary. Thereby, the time when the change of the cAMP amount is reflected in the change of the secretory reporter protein amount can be ensured more reliably. The time for culturing the cells in the absence of the test substance is not particularly limited, and can be, for example, about 1 to 48 hours, preferably 6 to 36 hours, and more preferably about 18 to 30 hours.

  Step (a) is a step of measuring the reporter activity of the secreted reporter protein secreted from the cell after step (a) and comparing the activity value with the reporter activity value when not contacting with the test substance. is there.

  For the measurement of the reporter activity, a known appropriate method may be employed depending on the type of secretory reporter protein. For example, when the secretory reporter protein is secretory placental alkaline phosphatase (SEAP), the known Phospha-Light system (Applied biosystems) is used as a SEAP measurement kit, or the principle of the kit is used. SEAP activity can be measured.

  The case where it is not brought into contact with the test substance is the case where the same or equivalent treatment as in the step (A) is carried out through the step (A) except that the test substance is not brought into contact with the test substance. . The reporter activity in this case can also be measured in the same manner as the reporter activity when brought into contact with the aforementioned test substance.

  The comparison of the activity values can be performed by a known method and is not particularly limited. For example, when the activity value appears as the intensity of light emission, the intensity of light emission may be visually judged and compared, or the intensity of light emission is measured as the light emission value and the magnitude of the light emission value is compared. May be.

  In the step (c), when the reporter activity value when contacted with the test substance is significantly different from the reporter activity value when not contacted with the test substance, the test substance is acted on the dopamine receptor. It is a process of selecting as a substance.

  Whether or not the reporter activity value when contacted with the test substance is significantly different from the reporter activity value when not contacted with the test substance is determined as a result of the comparison in step (a). The determination may be made based on a standard, for example, a statistical standard. Specifically, a method of determining a significant difference when the P value is obtained by measuring a plurality of times and the P value is not more than a certain value, for example, not more than 0.05, may be mentioned. As a result of the determination, when the difference is significant, the test substance is selected as a dopamine receptor agonist.

  The thus selected dopamine receptor agonist may be further subjected to secondary screening as an insecticide or insect control agent candidate substance.

2-2. Screening method for dopamine receptor agonist A dopamine receptor agonist is a substance that acts on a dopamine receptor and activates signal transduction downstream of the dopamine receptor, specifically, an effect of increasing cAMP concentration. is there. According to the screening method for dopamine receptor agonists, dopamine receptor agonists can be screened from test substances.

  The method for screening a dopamine receptor agonist can be performed in the same manner as the method for screening a dopamine receptor agonist comprising the steps (a), (b), and (c) described above. However, in the screening method for dopamine receptor agonists, the step (c) is the following step (d).

  In the step (d), when the reporter activity value when contacted with the test substance is significantly higher than the reporter activity value when not contacted with the test substance, the test substance is selected as a dopamine receptor agonist. It is a process.

  Whether the reporter activity value when contacted with the test substance is significantly higher than the reporter activity value when not contacted with the test substance can be determined in the same manner as in the step (c). If the result of the determination is significantly high, the test substance is selected as a dopamine receptor agonist.

  The dopamine receptor agonist thus selected may be further subjected to a secondary screening as an insecticide or insect control agent candidate substance.

2-3. Screening method for dopamine receptor antagonist A dopamine receptor antagonist is an action that acts on dopamine and / or a dopamine receptor activated by a dopamine receptor agonist and suppresses signal transduction downstream of the dopamine receptor, specifically Is a substance having the effect of reducing the cAMP concentration. According to the screening method for dopamine receptor antagonists, dopamine receptor antagonists can be screened from test substances.

  The method for screening a dopamine receptor antagonist can be performed in the same manner as the method for screening a dopamine receptor agonist comprising the steps (a), (b), and (c) described above. However, in the screening method for dopamine receptor antagonist, the step (a) is the following step (e), the step (b) is the following step (f), and the step (c) is the following step (ki). ).

  The step (e) is a step of bringing the cells into which the polynucleotides (a) and (b) have been introduced as described in “1. Cells of the present invention” into contact with a dopamine receptor agonist and a test substance.

  As the dopamine receptor agonist, a substance that acts on the dopamine receptor and activates signal transduction downstream of the dopamine receptor, specifically, has an effect of increasing cAMP concentration can be used. As specific examples, dopamine, which is an endogenous ligand of dopamine receptor, or 6,7-ADTN, apomorphine, etc., which are known dopamine receptor agonists, can be used. Moreover, the test substance selected by the screening method for the dopamine receptor agonist can also be used as a dopamine receptor agonist.

When contacting in the culture solution, the concentration of the dopamine receptor agonist in the culture solution is not particularly limited, but is, for example, 10 ⁻¹ M to ^{10 −10} M, preferably 10 ⁻³ M to ¹⁰ M. ⁻⁹ M, more preferably 10 ⁻⁵ M to 10 ⁻⁷ M.

  About others, it is the same as that of a process (a).

  In the step (f), after the step (e), the reporter activity of the secreted reporter protein secreted from the cells was measured, and the activity value was brought into contact with the dopamine receptor agonist without contacting with the test substance. It is the process of comparing with the reporter activity value in the case.

  Step (f) can be performed in the same manner as step (b).

  In the step (ki), the reporter activity value when contacted with the dopamine receptor agonist and the test substance is significantly higher than the reporter activity value when contacted with the dopamine receptor agonist without contact with the test substance. When it is low, the test substance is selected as a dopamine receptor antagonist.

  Determination of whether the reporter activity value when contacted with a dopamine receptor agonist and a test substance is significantly lower than the reporter activity value when contacted with a dopamine receptor agonist without contact with the test substance Can be performed in the same manner as in the step (c). If the determination result shows that the test substance is significantly low, the test substance is selected as a dopamine receptor antagonist.

  The dopamine receptor antagonist thus selected may be further subjected to secondary screening as a candidate insecticide or insect control agent.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1. Preparation of cells into which polynucleotides (a) and (b) have been introduced 1
First, cells into which a polynucleotide encoding a dopamine receptor protein (corresponding to the polynucleotide (a)) was introduced were prepared as follows.

The cells were cultured in DMEM (10% FBS / DMEM) containing 10% FBS, 100 unit / ml penicillin and 100 μg / ml streptomycin in an atmosphere of 37 ° C. and 5% CO ₂ concentration. Human embryonic kidney cells HEK293 on the first day, were seeded such that the 6 × 10 ⁵ / 3ml to 35mm dishes. On the second day, 3 μl GeneJuice (registered trademark) Transfection Reagent (Merck) was added to the pcDNA3 vector linked in a state in which the polynucleotide encoding the silkworm dopamine receptor 2 protein (BmDopR2) (SEQ ID NO: 1) can be expressed. And 100 μl of OPTI-MEM I serum-reducing medium (Life Technologies) mixed solution (previously mixed and incubated at room temperature for 5 minutes), and incubated at room temperature for 15 minutes. All of the mixture was added to the medium of the cells seeded on the first day while adding dropwise evenly, and the dish was gently shaken, followed by incubation at 37 ° C. for 24 hours. On the second day, the cells were subcultured into 60 mm dishes (5 ml 10% FBS / DMEM per plate), G418 50 mg / ml was added 100 μl (final concentration 1.0 mg / ml), and selection was started. Thereafter, the selection medium was changed every 3 to 5 days, and selection was performed for 3 to 4 weeks. Cells obtained after this selection were defined as cells (BmDopR2 cells) into which a polynucleotide encoding a dopamine receptor protein (polynucleotide (a)) was introduced.

  Next, a polynucleotide having a polynucleotide region 1 having a cAMP response element and having a promoter function, and a polynucleotide region 2 encoding a secretory reporter protein arranged on the 3 ′ end side of the polynucleotide region 1 ( Cells into which the polynucleotide (b)) was introduced were prepared as follows.

BmDopR2 cells, which are cells into which the polynucleotide (a) was introduced, were cultured in 10% FBS / DMEM at 37 ° C./5% CO ₂ concentration. The cells were transiently transfected with a reporter gene-containing vector pCRE-SEAP (a vector in which secreted placental alkaline phosphatase (SEAP) was linked under the control of a promoter having a cAMP response element). Transfection was performed using GeneJuice Transfection Reagent (Merck) as described above. One day after transfection, the cells (BmDopR2 (stable) -pCRESEAP cells) into which the polynucleotides (a) and (b) were introduced were used.

Example 2 Preparation of cells into which polynucleotides (a) and (b) have been introduced 2
Cells were prepared in the same manner as in Example 1 except that the polynucleotide (a) and the polynucleotide (b) were transiently transfected at the same time. The obtained cells were designated as BmDopR2 (transient) -pCRESEAP cells.

Example 3 Screening for dopamine receptor agonists using BmDopR-pCREseap cells As examples of screening for dopamine receptor agonists, Examples 3-1 and 3-2 below include screening examples for dopamine receptor agonists or antagonists. Show.

Example 3-1. Screening for dopamine receptor agonists 1
BmDopR2 (stable) -pCREseap cells into which polynucleotides (a) and (b) have been introduced are seeded in a 96-well culture plate at 2 × 10 ⁴ / well and 150 μl / well, Cultured. On the other hand, in addition to dopamine and a known dopamine receptor agonist as a test substance, a substance group to be evaluated is dissolved in DMSO, and the lysate is used as test substance concentrations of 10 ⁻⁹ , 10 ⁻⁸ , 10 ⁻⁷ , An agonist medium was prepared by adding to a serum-free medium at 10 ⁻⁶ , 10 ⁻⁵ , or 10 ⁻⁴ M, and a DMSO concentration of 1%. The medium of cells that had passed 1 day after seeding was discarded and replaced with 150 μl (/ 1 well) of the agonist medium and cultured for 4 hours. The agonist medium was removed, the cells were washed with 150 μl of 1 × DPBS, 150 μl of serum-free DMEM was added to each well, and cultured for one day in a CO ₂ incubator (37 ° C., 5% CO ₂ ). The SEAP activity of the supernatant was measured using Phospha-Light System (Applied Biosystems). Specifically, 25 μl of culture supernatant was added to each well of a white 96-well plate to which 25 μl of 1 × Dilution Buffer (/ 1 well) was added, and the medium was diluted. Thereafter, 50 μl (1 / well) of Assay Buffer was added to 50 μl (/ 1 well) of the solution, and incubated at room temperature for 5 minutes. Thereafter, 50 μl (/ 1 well) of Reaction Buffer was added and incubated at room temperature for 10 minutes. The SEAP activity was measured by measuring the amount of luminescence in each well at 1 second / well using a Wallac 1420 ARVOsx multilabel counter.

  FIG. 1 shows the mean value ± SE obtained by measuring the SEAP activity four times. In FIG. 1, the horizontal axis represents the test substance concentration, and the vertical axis represents the relative light emission amount. As a positive control, forskolin, which is an adenylate cyclase activator, was used. Relative luminescence is the relative value when the luminescence (“Ba” in FIG. 1: Basal level) is 1 measured when a serum-free medium (control) containing no dopamine is used instead of the agonist medium. It is.

From the result of FIG. 1, when dopamine which is a ligand is used as the test substance, the amount of luminescence is significantly increased in a dopamine concentration of 10 ⁻⁶ to 10 ⁻⁴ M in a concentration-dependent manner compared to the Ba value. Was seen. From this, it was shown that the screening method of the present invention can detect signal transduction by dopamine receptor ligands. From this, it was shown that the screening method of the present invention can accurately and quantitatively detect signal transduction by an agonist via a dopamine receptor.

Example 3-2. Screening for dopamine receptor agonists 2
The same procedure as in Example 3-1 was performed except that BmDopR2 (transient) -pCRESEAP cells were used instead of BmDopR2 (stable) -pCREseap cells and the culture time in the agonist medium was 1 hour. The results are shown in FIG.

  From the results of FIG. 2, it was shown that signal transduction by the ligand of dopamine receptor can be detected even when the culture time in the agonist medium is 1 hour.

Example 3-3. Screening for dopamine receptor antagonists Instead of agonist medium, antagonist medium (a solution of dopamine, a ligand dissolved in DMSO, is added to a serum-free medium to a dopamine concentration of 10 ⁻⁶ M and further known as a test substance) In addition to the dopamine receptor antagonists flupentixol and chlorpromazine, a solution in which a group of substances to be evaluated is dissolved in DMSO is added to a test substance concentration of 10 ⁻⁹ , 10 ⁻⁸ , 10 ⁻⁷ , 10 ⁻⁶ , or 10 ^{− The same} procedure as in Example 3-1 was performed except that a medium in which DMSO concentration was adjusted to 1% was added to a serum-free medium so as to be ⁵ M.

FIG. 3 shows the mean ± SE obtained by measuring SEAP activity four times. In FIG. 3, the horizontal axis indicates the concentration of chlorpromazine or yohimbine in the antagonist medium used, and the vertical axis indicates the relative luminescence amount. Relative luminescence amount is based on the luminescence amount when using control medium 1 (serum-free medium in which dopamine is added to a concentration of 10 ⁻⁶ M and the DMSO concentration is adjusted to 1%) instead of the antagonist medium. Luminescence amount minus the luminescence amount when using control medium 2 (serum-free medium with DMSO concentration adjusted to 1% without dopamine and test substance) instead of antagonist medium ("DA" in Fig. 3) Is a value with 100%.

  From the results of FIG. 3, it was shown that when chlorpromazine or yohimbine coexists with dopamine, the amount of luminescence decreases depending on the concentration of chlorpromazine or yohimbine. From this, it was shown that the screening method of the present invention can detect the action of an antagonist on the dopamine receptor, that is, the action of inhibiting the signal transduction by the ligand.

Claims (9)

A cell comprising the following polynucleotide (a) and the following polynucleotide (b):
(A) a polynucleotide encoding a dopamine receptor protein, and (b) a polynucleotide region 1 having a cAMP response element and having a promoter function, and a secreted form disposed on the 3 ′ end side of the polynucleotide region 1 A polynucleotide having a polynucleotide region 2 encoding a reporter protein.   The cell according to any one of claims 1 to 4, wherein the polynucleotide (a) is a polynucleotide encoding an invertebrate-specific dopamine receptor protein.   The cell according to claim 1 or 2, which is a mammalian cell comprising the polynucleotide (a) and the polynucleotide (b).   The cell according to any one of claims 1 to 3, wherein the secretory reporter protein is secretory placental alkaline phosphatase.   The cell according to any one of claims 1 to 4, which is a HEK-293 cell comprising a polynucleotide (a) and a polynucleotide (b). A screening method for a dopamine receptor agonist comprising the following steps (a), (b), and (c):
(A) the step of contacting the cell according to any one of claims 1 to 5 with a test substance;
(A) After step (a), the reporter activity of the secreted reporter protein secreted from the cell is measured, and the activity value is compared with the reporter activity value when not contacting with the test substance; ) A step of selecting a test substance as a dopamine receptor agonist when the reporter activity value when contacted with the test substance is significantly different from the reporter activity value when not contacted with the test substance .   The screening method according to claim 6, wherein the contact time in step (a) is less than 8 hours. A method for screening a dopamine receptor agonist,
In the step (c), when the reporter activity value when contacted with the test substance is significantly higher than the reporter activity value when not contacted with the test substance, the test substance is selected as a dopamine receptor agonist. Is the process of
The screening method according to claim 6 or 7. A screening method for dopamine receptor antagonists, comprising:
The step (a) is a step of bringing the cell according to any one of claims 1 to 5 into contact with a dopamine receptor agonist and a test substance,
In the step (a), after the step (a), the reporter activity of the secretory reporter protein secreted from the cell is measured, and the activity value is brought into contact with the dopamine receptor agonist without contacting with the test substance. The reporter activity value in the case of
In the step (c), the reporter activity value when contacted with the dopamine receptor agonist and the test substance is more significant than the reporter activity value when contacted with the dopamine receptor agonist without contact with the test substance. The test substance is selected as a dopamine receptor antagonist,
The screening method according to claim 6 or 7.

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