WO2018062201A1 - Odorant receptor complex and cells expressing same - Google Patents

Odorant receptor complex and cells expressing same Download PDF

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WO2018062201A1
WO2018062201A1 PCT/JP2017/034788 JP2017034788W WO2018062201A1 WO 2018062201 A1 WO2018062201 A1 WO 2018062201A1 JP 2017034788 W JP2017034788 W JP 2017034788W WO 2018062201 A1 WO2018062201 A1 WO 2018062201A1
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olfactory receptor
receptor
olfactory
seq
amino acid
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Japanese (ja)
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康彦 高橋
朋之 高久
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住友化学株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to an olfactory receptor complex, specifically, an olfactory receptor complex formed from an olfactory receptor derived from an insect and an olfactory receptor co-receptor derived from another insect, and a cell expressing the same About.
  • Detecting odorous substances may use organisms such as dogs and nematodes.
  • organisms such as dogs and nematodes.
  • the method using living organisms can detect odorous substances with high sensitivity, it is not stable due to variations in physical condition and individuals, and it is difficult to use for a long time because the duration of concentration is limited. There was also a side.
  • insects olfactory function of the organism is considered to have high recognition specificity for odorous substances, and its use for olfactory sensors and the like is being sought.
  • Insect olfactory organs include an olfactory receptor that recognizes odorants (sometimes referred to as “OR”) and an olfactory receptor co-receptor (hereinafter referred to as “ORCO” or “co-receptor”).
  • OR olfactory receptor complex formed by the odorant is present, and when activated by an odor substance, the complex exhibits ion channel activity, thereby detecting the odor substance.
  • an olfactory sensor used for detecting an odor substance is required to have high sensitivity.
  • the present invention has been made in view of the above circumstances, and an insect olfactory receptor complex formed from an insect olfactory receptor and an olfactory receptor co-receptor that exhibits excellent detection sensitivity to odor substances.
  • the purpose is to provide.
  • the present inventor It has been found that detection sensitivity to odorous substances is superior to an olfactory receptor complex formed from a co-receptor and the olfactory receptor.
  • the present invention is based on these findings.
  • the present invention relates to the following [1] to [6], for example.
  • An olfactory receptor having a ligand selected from compounds selected from the group consisting of 1-octen-3-ol, benzaldehyde, 2,4,5-trimethylthiazole, cis-jasmon, and pentyl acetate.
  • the olfactory receptor complex according to [1] or [2].
  • the olfactory receptor is OR8 or OR10 of Aedes aegypti, OR10 or OR28 of Anopheles gambiae, OR56 of Bombyx mori, or Drosophila or 47 Among these, an olfactory receptor having an amino acid sequence having 90% or more identity with the entire amino acid sequence of the olfactory receptor, wherein the olfactory receptor co-receptor is Apis melifera or its close relationship
  • the olfactory receptor complex according to any one of [1] to [3], which is a species insect olfactory receptor co-receptor.
  • the olfactory receptor is OR8 or OR10 of Aedes aegypti, OR10 or OR28 of Anopheles gambiae, OR56 of Bombyx mori, or 47 or 47 of Drosophila ago
  • an olfactory receptor having an amino acid sequence having 90% or more identity with the entire amino acid sequence of the olfactory receptor, and the olfactory receptor co-receptor is an amino acid sequence represented by SEQ ID NO: 1 and 90
  • the protein according to any one of [1] to [3], which is a protein having an amino acid sequence having an identity of at least% and having an ability to allow a cation to flow into a cell by coupling with an insect olfactory receptor Olfactory receptor complex.
  • an olfactory receptor complex formed of an insect olfactory receptor and an olfactory receptor co-receptor that exhibits excellent detection sensitivity for odor substances.
  • the relative values of the amount of luminescence when 1-octen-3-ol is added to cells expressing a complex of AaOR8 and AaORCO and cells expressing a complex of AaOR8 and AmORCO are shown.
  • the relative value of the light-emission quantity at the time of adding benzaldehyde with respect to the cell which expresses the complex of AaOR10 and AaORCO, and the cell which expresses the complex of AaOR10 and AmORCO is shown.
  • the relative value of the amount of luminescence when benzaldehyde is added to a cell expressing a complex of AgOR10 and AgORCO and a cell expressing a complex of AgOR10 and AmORCO is shown.
  • the relative value of the amount of luminescence when 2,4,5-trimethylthiazole is added to a cell expressing a complex of AgOR28 and AgORCO and a cell expressing a complex of AgOR28 and AmORCO is shown.
  • the relative values of the amount of luminescence when cis-jasmon is added to cells expressing a complex of BmOR56 and BmORCO and cells expressing a complex of BmOR56 and AmORCO are shown.
  • the relative value of the amount of luminescence when pentyl acetate is added to a cell expressing a complex of DmOR47a and DmORCO and a cell expressing a complex of DmOR47a and AmORCO is shown.
  • the present embodiment a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail.
  • this invention is not limited to the following embodiment.
  • Insect olfactory receptor complexes are olfactory receptors and olfactory receptor co-receptors that form a heterocomplex.
  • An insect olfactory receptor is a kind of G protein-coupled receptor having a seven-transmembrane structure, and has high recognition specificity for the odorant.
  • odorant-specific olfactory receptors are known.
  • the olfactory receptor co-receptor of insects is a membrane protein having a 7-transmembrane structure like the olfactory receptor, but does not recognize odorants and forms a hetero complex with the olfactory receptor. And function.
  • the olfactory receptor co-receptor known as the Orco family is conserved in all insects with relatively high homology.
  • the olfactory receptor complex has an ion channel activity activated by an odor substance. When activated, a cation such as sodium ion (Na + ) or calcium ion (Ca 2+ ) flows into the cell. .
  • the olfactory receptor complex of this embodiment is formed from an olfactory receptor of an insect that does not belong to the honey bee family and an olfactory receptor co-receptor of the honey bee family.
  • Examples of olfactory receptors of insects that do not belong to the honey bee family include insect olfactory receptors selected from the group consisting of mosquitoes, silkworms, and fruit flies.
  • Examples of the mosquito insects include Gambier anopheles (Anopheles gambiae), Aedes aegypti, Aedes squid (Culeque quinquefacciatus) and the like.
  • Examples of the insects of the Bombycidae include Bombyx mori, mulberry (Bombyx mandarina), and fig cassan (Trilocha varians).
  • Examples of Drosophila insects include Drosophila melanogaster, Drosophila pseudobscura, Drosophila virillis, and the like.
  • the type of olfactory receptor can be appropriately selected according to the target ligand.
  • the olfactory receptor may be an olfactory receptor having a compound selected from the group consisting of 1-octen-3-ol, benzaldehyde, 2,4,5-trimethylthiazole, cis-jasmon, and pentyl acetate as a ligand. Good.
  • the olfactory receptors are specifically OR8 or OR10 of Aedes aegypti, OR10 or OR28 of Anopheles gambiae, OR56 of Bombyx mori, or 47 of Drosophila. There may be.
  • the olfactory receptor may be an olfactory receptor having an amino acid sequence having 90% or more identity with the whole amino acid sequence of any of the above olfactory receptors, and has an amino acid sequence having 95% or more identity. It may be an olfactory receptor, an olfactory receptor having an amino acid sequence having 98% or more identity, or an olfactory receptor having an amino acid sequence having 99% or more identity . If it is an olfactory receptor having an amino acid sequence having an identity of 90% or more when compared with the amino acid sequence of any of the above olfactory receptors, a ligand comparable to the original olfactory receptor compared with the amino acid sequence Specificity and detection sensitivity tend to be obtained.
  • olfactory receptor co-receptors derived from honey bee insects include Apis mellifera, Apis florea, Apis dorsata, Bombus terrestris, etc. The thing derived from is mentioned. According to the complex of the honey bee olfactory receptor co-receptor and the insect olfactory receptor not belonging to the honey bee family, the olfactory receptor co-receptor and the olfactory receptor It is superior in detection sensitivity to odorous substances than the olfactory receptor complex formed from the above.
  • the olfactory receptor co-receptor may be an olfactory receptor co-receptor derived from a honey bee or its related species.
  • the term “relative species of honey bees” refers to bees of Apini, or Euglesini, bumblebee bumble bee (Bombini), or bee stingless bee (Melip) bee (Melip) It is. Bees of these families have a pollen basket corbicular (pollen basket) on the outside of the hind leg shin joint.
  • related species of honey bees include honey bees belonging to honey bees and honey bees; and honey bees belonging to bumble bees.
  • Such closely related olfactory receptor co-receptors have high amino acid sequence identity with honey bee olfactory receptor co-receptors and tend to have equivalent functions.
  • the olfactory receptor co-receptor has an amino acid sequence having 90% or more identity with the amino acid sequence of the honey bee olfactory receptor co-receptor (amino acid sequence described in SEQ ID NO: 1), and insect olfactory receptor It may be a protein that has the ability to conjugated with the body and cause cations to flow into the cell, has an amino acid sequence having 95% or more identity, and is conjugated with insect olfactory receptors It may be a protein having the ability to flow into cells, has an amino acid sequence having 98% or more identity, and has the ability to couple cations into cells by coupling with insect olfactory receptors A protein having an amino acid sequence with 99% or more identity and having the ability to allow a cation to flow into a cell by coupling with an insect olfactory receptor It may be.
  • Any protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having the ability to couple a cation into the cell in combination with an insect olfactory receptor will be used. Similar to the bee olfactory receptor co-receptor, by forming a complex with an olfactory receptor of an insect that does not belong to the honey bee family, an olfactory receptor co-receptor of the same kind of insect as the above olfactory receptor It tends to be more sensitive to detection of odorous substances than olfactory receptor complexes formed from olfactory receptors.
  • Examples of the protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having an ability to allow a cation to flow into a cell in combination with an insect olfactory receptor include: The honey bee olfactory receptor co-receptor (99% identity), the bee olfactory receptor co-receptor (identity 98%), the bee olfactory receptor co-receptor (identity 91%), etc. Can be mentioned.
  • the olfactory receptor and the olfactory receptor co-receptor interact at a ratio of 1: 1 to form a complex.
  • the olfactory receptor and the olfactory receptor co-receptor may each form a complex, may each form a complex from two, or may form a complex from four each. May be.
  • the olfactory receptor complex of the present embodiment is a method in which an olfactory receptor and an olfactory receptor co-receptor are simultaneously expressed in the cell membrane of the same cell and purified in some cases, or an olfactory receptor and an olfactory receptor co-receptor It can be prepared by a method in which each cell is expressed in different cells, purified and then mixed.
  • the method for expressing the olfactory receptor and olfactory receptor co-receptor in cells is not particularly limited, and a known method can be used.
  • an expression plasmid incorporating a DNA encoding the olfactory receptor and an expression plasmid incorporating a DNA encoding the olfactory receptor co-receptor are introduced into a cell. There is a method of expressing. At this time, the expression plasmid incorporating the DNA encoding the olfactory receptor and the expression plasmid incorporating the DNA encoding the olfactory receptor co-receptor are introduced into the same cell, thereby producing an olfactory receptor complex. It can also be prepared directly as a body. The introduction of various plasmids into the cells can be confirmed by a method such as PCR.
  • the olfactory receptor, olfactory receptor co-receptor, or olfactory receptor complex expressed in cells can be purified by solubilizing the cell membrane and applying it to various purification columns.
  • the olfactory receptor complex of the present embodiment formed from the olfactory receptor co-receptor of the same kind of insect as the olfactory receptor constituting the complex and the olfactory receptor. Excellent detection sensitivity to odorous substances.
  • the olfactory receptor complex having such characteristics is useful as an olfactory sensor capable of detecting a lower concentration of odor substance.
  • the cell of this embodiment expresses the olfactory receptor complex. That is, the cell of the present embodiment expresses an olfactory receptor of an insect that does not belong to the honey bee family and an olfactory co-receptor of the honey bee family in the cell, and thereby the olfactory receptor of the present embodiment on the cell membrane. Express the complex.
  • the cells according to the present embodiment include an expression plasmid incorporating a DNA encoding an olfactory receptor and an expression plasmid incorporating a DNA encoding an olfactory receptor co-receptor, as in the method for preparing an olfactory receptor complex.
  • the cell into which the expression plasmid is introduced can be appropriately selected according to the type of expression plasmid.
  • Examples of cells include Escherichia coli K12 and other Escherichia coli, Bacillus subtilis MI114 and other Bacillus bacteria, Saccharomyces cerevisiae AH22 and other species, Spodoptera frugiperda-derived Sf cells derived from Sponostic frugiperda and the like. And animal cells such as cells. Animal cells are preferably cultured cells derived from mammals, specifically, COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells and the like. It is done.
  • insects described in this example are as follows.
  • Anopheles gambiae hereinafter also referred to as “Anopheles gambiae” or “Ag”
  • Aedes aegypti hereinafter sometimes referred to as “Shimaka” or “Aa”
  • Drosophila melanogaster hereinafter sometimes referred to as “Drosophila” or “Dm”
  • Apis melifera hereinafter, also referred to as “bee” or “Am” Bombyx mori (hereinafter, also referred to as “silkworm” or “Bm”)
  • RNA purification kit (RNeasy Mini Kit; manufactured by QIAGEN) according to the instructions attached to the kit.
  • the PCR reaction was carried out at (1) 94 ° C for 5 minutes, (2) 98 ° C for 10 seconds, (3) 60 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles.
  • the obtained PCR product was subjected to agarose gel electrophoresis, and then about 1.4 kb of DNA detected on the gel was recovered.
  • the recovered DNA was introduced into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-AgOrco.
  • Drosophila co-receptor Using Drosophila melanogaster adult RNA (manufactured by TAKARA) as a template, Super Script III reverse transcriptase (manufactured by Invitrogen) was added, A reverse transcription reaction was performed by incubating at 55 ° C. for 50 minutes and then at 75 ° C. for 15 minutes to obtain cDNA.
  • PCR product was subjected to agarose gel electrophoresis, and then about 1.5 kb of DNA detected on the gel was recovered.
  • the recovered DNA was introduced into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-DmOrco.
  • 4 ⁇ L of pENTR-DmOrco, 1 ⁇ L of pcDNA6.2V5-DEST and 2 ⁇ L of LR clonase II (Invitrogen) were mixed and kept at room temperature for 1 hour.
  • the resulting mixture was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA6.2-DmOrco.
  • nucleotide sequence of the expression plasmid pcDNA6.2-DmOrco was analyzed by a DNA sequencer, it was found to contain the nucleotide sequence represented by SEQ ID NO: 8.
  • the base sequence represented by SEQ ID NO: 8 encodes the amino acid sequence represented by SEQ ID NO: 9.
  • RNA purification kit RNeasy Mini Kit; manufactured by QIAGEN
  • Super Script III reverse transcriptase manufactured by Invitrogen was added, and the mixture was incubated at 55 ° C. for 50 minutes and then at 75 ° C.
  • the PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles.
  • the obtained PCR product was ligated to pcDNA3.1 (manufactured by Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Then, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-AaOrco.
  • Analysis of the base sequence of expression plasmid pcDNA3.1-AaOrco using a DNA sequencer revealed that it contained the base sequence represented by SEQ ID NO: 12.
  • the base sequence represented by SEQ ID NO: 12 encodes the amino acid sequence represented by SEQ ID NO: 13.
  • Expression plasmid of honeybee co-receptor (hereinafter sometimes referred to as AmORCO) A double-stranded DNA having the base sequence shown in SEQ ID NO: 14 was synthesized in one of the DNA strands. 100 ⁇ g of the obtained DNA was used as a template, 10 ⁇ M forward primer AmOrco-5 ′ (5′-tggaattctgcagatcaccatgaagttcaagcaacaagggctaa; SEQ ID NO: 15) PCR was performed using 1 ⁇ L of Plus neo DNA polymerase (Toyobo).
  • the PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles.
  • the obtained PCR product was ligated to pcDNA3.1 (manufactured by Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Subsequently, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-AmOrco.
  • Analysis of the base sequence of expression plasmid pcDNA3.1-AmOrco using a DNA sequencer revealed that it contained the base sequence represented by SEQ ID NO: 14.
  • the base sequence represented by SEQ ID NO: 14 encodes the amino acid sequence represented by SEQ ID NO: 1.
  • Double-stranded DNA having a base sequence represented by SEQ ID NO: 17 was synthesized on either DNA strand.
  • 10 ⁇ M forward primer BmOrco-5 ′ (5′-tggaattctgcagatcaccatgatgaccaaggtcaagacgcagggcct; SEQ ID NO: 18) 1 ⁇ L
  • 10 ⁇ M reverse primer BmOrco-3 ′ (5′-gccactgtgctggatctactataccatgatggatctactataccatgatggatctacttaccatgatggatctacttaccatgatggatctacttaccatggatctacttaccat PCR was performed using 1 ⁇ L of Plus neo DNA polymerase (Toyobo).
  • the PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles.
  • the obtained PCR product was ligated to pcDNA3.1 (Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Thereafter, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-BmOrco.
  • SEQ ID NO: 17 encodes the amino acid sequence represented by SEQ ID NO: 20.
  • AaOR8 An expression plasmid for the mosquito olfactory receptor OR8 (hereinafter sometimes referred to as AaOR8) 10 ⁇ M of the forward primer AaOR8-5 ′ (5′-tggaattctgcagatcaccatgaacgacctggtgaagtttgagt; sequence) No.
  • PCR was performed using 1 ⁇ L of 1 ⁇ L and 10 ⁇ M of reverse primer AaOR8-3 ′ (5′-gccactgtgctggattcacttctgacttggttcatagatggt; SEQ ID NO: 22) and 1 ⁇ L of KOD Plus neoDNA polymerase (manufactured by Toyobo).
  • the PCR reaction was performed (1) 94 ° C. for 2 minutes, (2) 98 ° C. for 10 seconds, (3) 68 ° C. for 1 minute, and steps (2) to (4) were repeated 30 cycles.
  • the obtained PCR product was ligated to pcDNA3.1 (manufactured by Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Thereafter, this was introduced into Escherichia coli and cultured to obtain an expression plasmid named pcDNA3.1-AaOR8.
  • Analysis of the nucleotide sequence of expression plasmid pcDNA3.1-AaOR8 using a DNA sequencer revealed that it contained the nucleotide sequence represented by SEQ ID NO: 23.
  • the base sequence represented by SEQ ID NO: 23 encodes the amino acid sequence represented by SEQ ID NO: 24.
  • An expression plasmid for the mosquito olfactory receptor OR10 (hereinafter sometimes referred to as AaOR10) 10 ⁇ M of the forward primer AaOR10-5 ′ (5′-tggaattctgcagatcaccatggcaagcattcttgattgcccg; sequence) No. 25) PCR was performed using 1 ⁇ L of 1 ⁇ L, 10 ⁇ M of reverse primer AaOR10-3 ′ (5′-gccactgtgctggatttaattataaacccgacgcagcag; SEQ ID NO: 26) and 1 ⁇ L of KOD Plus neoDNA polymerase (manufactured by Toyobo).
  • the PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles.
  • the obtained PCR product was ligated to pcDNA3.1 (manufactured by Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Thereafter, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-AaOR10. Analysis of the base sequence of expression plasmid pcDNA3.1-AaOR10 using a DNA sequencer revealed that it contained the base sequence represented by SEQ ID NO: 27.
  • the base sequence represented by SEQ ID NO: 27 encodes the amino acid sequence represented by SEQ ID NO: 28.
  • Anopheles olfactory receptor OR10 (hereinafter, sometimes referred to as AgOR10) expression plasmid 10 ⁇ M of forward primer AgOR10-5 ′ (5′-caccatggaggtcctcaactgtccgctact; sequence) No. 29) PCR was performed using 1 ⁇ L of 1 ⁇ L, 10 ⁇ M reverse primer AgOR10-3 ′ (5′-ttaattgtaaactcttctcagaagcgtaa; SEQ ID NO: 30) and 1 ⁇ L of KOD Plus DNA polymerase (manufactured by Toyobo).
  • the PCR reaction was carried out at (1) 94 ° C for 5 minutes, (2) 98 ° C for 10 seconds, (3) 60 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles.
  • the obtained PCR product was subjected to agarose gel electrophoresis, and then about 1.1 kb of DNA detected on the gel was recovered.
  • the recovered DNA was introduced into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-AgOR10.
  • Anopheles olfactory receptor OR28 (hereinafter, sometimes referred to as AgOR28) expression plasmid 1 ⁇ L of an anopheles head-derived cDNA prepared by the above method was used as a template.
  • 10 ⁇ M forward primer AgOR28-5 ′ (5′-caccatggcccgtttggtactgcacgaggt; SEQ ID NO: 33) 1 ⁇ L
  • 10 ⁇ M reverse primer AgOR28-3 ′ (5′-ttattgctgattgatggtttgcagtaagg; SEQ ID NO: 34) 1 ⁇ L
  • KOD Plus DNA polymerase (1 made by Toyobo
  • the PCR reaction was carried out at (1) 94 ° C for 5 minutes, (2) 98 ° C for 10 seconds, (3) 60 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles.
  • the obtained PCR product was subjected to agarose gel electrophoresis, and then about 1.3 kb of DNA detected on the gel was recovered.
  • the recovered DNA was introduced into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-AgOR28.
  • Expression plasmid of the silkworm olfactory receptor OR56 (hereinafter sometimes referred to as BmOR56) A double-stranded DNA having the base sequence represented by SEQ ID NO: 37 was synthesized in one of the DNA strands. 10 ⁇ M of forward primer BmOR56-5 ′ (5′-tggaattctgcagatcaccatgaagctcctggagaagctag; SEQ ID NO: 38) 1 ⁇ L, 10 ⁇ M of reverse primer BmOR56-3 ′ (5 PCR was performed using 1 ⁇ L of '-gccactgtgctggattcatgttttattcattttgcgactgac; SEQ ID NO: 39) and 1 ⁇ L of KOD Plus neo DNA polymerase (Toyobo).
  • the PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles.
  • the obtained PCR product was ligated to pcDNA3.1 (Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Thereafter, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-BmOR56.
  • Analysis of the base sequence of expression plasmid pcDNA3.1-BmOR56 using a DNA sequencer revealed that it contained the base sequence represented by SEQ ID NO: 37.
  • the base sequence represented by SEQ ID NO: 37 encodes the amino acid sequence represented by SEQ ID NO: 40.
  • DmOR47a Expression plasmid of Drosophila olfactory receptor OR47a (hereinafter sometimes referred to as DmOR47a) Using 1 ⁇ L of Drosophila-derived cDNA prepared by the above method as a template, 10 ⁇ M forward primer DmOR47a-5 ′ (5′-caccatggacagttttctgcaagtacagaaga; SEQ ID NO: 41 ) PCR was performed using 1 ⁇ L of 1 ⁇ L and 10 ⁇ M reverse primer DmOR47a-3 ′ (5′-ttaggagaatgatctcagcattgtgatgta; SEQ ID NO: 42) and 1 ⁇ L of KOD Plus neo DNA polymerase (Toyobo).
  • PCR reaction was performed at (1) 94 ° C. for 2 minutes, (2) 98 ° C. for 10 seconds, (3) 68 ° C. for 1.5 minutes, and steps (2) to (3) were repeated 35 cycles. .
  • the obtained PCR product was subjected to agarose gel electrophoresis, and then about 1.5 kb of DNA detected on the gel was recovered.
  • the recovered DNA was cloned into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-DmOR47a.
  • ⁇ Preparation of Aequorin Expression Plasmid> Using 50 ng of a plasmid (pMD19-AEQ) in which the nucleotide sequence represented by SEQ ID NO: 45 encoding Aequorin was cloned in the pMD19 vector as a template, 10 ⁇ M forward primer AEQ-5 ′ (5′-caccatgacaagcaaacaatactc; SEQ ID NO: 46) 1 ⁇ L PCR was performed using 1 ⁇ L of 10 ⁇ M reverse primer AEQ-3 ′ (5′-ttaggggacagctccaccgtag; SEQ ID NO: 47) and 1 ⁇ L of KOD Plus neo DNA polymerase (Toyobo).
  • the PCR reaction was performed at (1) 95 ° C. for 3 minutes, (2) 95 ° C. for 30 seconds, (3) 60 ° C. for 30 seconds, (4) 68 ° C. for 1 minute, and (2) to (4) The process was repeated 35 cycles.
  • the obtained PCR product was subjected to agarose gel electrophoresis, and then about 600 bp of DNA detected on the gel was recovered.
  • the recovered DNA was introduced into a pENTR / D-TOPO vector (manufactured by Invitrogen) to obtain a plasmid named pENTR-AEQ.
  • the base sequence represented by SEQ ID NO: 45 encodes the amino acid sequence represented by SEQ ID NO: 48.
  • PCR was performed using 1 ⁇ L and 1 ⁇ L of KOD Plus neo DNA polymerase (manufactured by Toyobo). The PCR reaction was performed at (1) 95 ° C. for 3 minutes, (2) 95 ° C. for 30 seconds, (3) 60 ° C.
  • HEK293FT cells purchased from Invitrogen
  • DMEM medium manufactured by Nacalai Tesque
  • Aequorin is activated when bound to calcium ions, and oxidizes a substrate such as coelenterazine to emit light. Therefore, since the increase in intracellular calcium ion concentration in cells expressing aequorin directly appears as an increase in luminescence, whether or not the olfactory receptor complex functions as an ion channel by adding a test substance It can be measured from the change in the amount of luminescence.
  • the culture solution of the transformed cells was removed and replaced with Assay buffer (Hanks-HEPES (20 mM pH 7.4) containing 0.5 ⁇ M coelenterazine h (Promega) and 0.3% BSA), and The mixture was allowed to stand at room temperature for 4 hours.
  • a test substance corresponding to the olfactory receptor was added to the cell culture medium, and the amount of luminescence of the cells was measured.
  • the amount of luminescence in cells to which dimethyl sulfoxide was added as a control substance was taken as 1, and the amount of luminescence in cells to which a test substance was added was calculated as a relative value.
  • Example 1 Expression plasmid of olfactory receptor AaOR8, The amount of luminescence was measured as described above using cells having either the co-receptor AaORCO or AmORCO expression plasmid and the Aequorin expression plasmid.
  • 1-octen-3-ol was used as the test substance, and the culture solution was at a concentration of 10 ⁇ 4 M, 10 ⁇ 5 M, 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, or 10 ⁇ 9 M. Added to. The result is shown in FIG. It is shown that cells expressing the complex of the mosquito AaOR8 and the bee AmORCO respond more strongly to 1-octen-3-ol than cells expressing the complex of the mosquito AaOR8 and AaORCO. It was done.
  • Example 2 Expression plasmid of olfactory receptor AaOR10, The amount of luminescence was measured as described above using cells having either the co-receptor AaORCO or AmORCO expression plasmid and the Aequorin expression plasmid.
  • Benzaldehyde was used as a test substance and added to the culture solution at a concentration of 10 ⁇ 3 M, 10 ⁇ 4 M, 10 ⁇ 5 M, 10 ⁇ 6 M, or 10 ⁇ 7 M. The result is shown in FIG. It has been shown that cells expressing the complex of the mosquito AaOR10 and the bee AmORCO respond more strongly to benzaldehyde than cells expressing the complex of the mosquito AaOR10 and AaORCO.
  • Example 3 Expression plasmid of olfactory receptor AgOR10, The amount of luminescence was measured as described above using cells containing either the co-receptor AgORCO or AmORCO expression plasmid and the Aequorin expression plasmid.
  • Benzaldehyde was used as a test substance and added to the culture solution at a concentration of 10 ⁇ 3 M, 10 ⁇ 4 M, 10 ⁇ 5 M, 10 ⁇ 6 M, or 10 ⁇ 7 M. The result is shown in FIG. It was shown that cells expressing the complex of Anopheles AgOR10 and honey bee AmORCO respond more strongly to benzaldehyde than cells expressing the complex of Anopheles AgOR10 and AgORCO.
  • Example 4 Expression plasmid of olfactory receptor AgOR28, The amount of luminescence was measured as described above using cells containing either the co-receptor AgORCO or AmORCO expression plasmid and the Aequorin expression plasmid. 2,4,5-Trimethylthiazole is used as a test substance and cultured at a concentration of 10 ⁇ 3 M, 10 ⁇ 4 M, 10 ⁇ 5 M, 10 ⁇ 6 M, 10 ⁇ 7 M, or 10 ⁇ 8 M. Added to the liquid. The result is shown in FIG. Cells expressing the complex of Anopheles AgOR28 and honey bee AmORCO may respond more strongly to 2,4,5-trimethylthiazole than cells expressing the complex of Anopheles AgOR28 and AgORCO Indicated.
  • Example 5 Expression plasmid for olfactory receptor BmOR56, The amount of luminescence was measured as described above using cells containing either the co-receptor BmORCO or AmORCO expression plasmid and the Aequorin expression plasmid. As a test substance, cis-jasmon was used and added to the culture solution at a concentration of 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M. The result is shown in FIG. It was shown that cells expressing the complex of silkworm BmOR56 and honey bee AmORCO responded more strongly to cis-jasmon than cells expressing the complex of silkworm BmOR56 and BmORCO.
  • Example 6 Expression plasmid of olfactory receptor DmOR47a
  • the amount of luminescence was measured as described above using cells containing either the co-receptor DmORCO or AmORCO expression plasmid and the Aequorin expression plasmid.
  • pentyl acetate is used as a test substance. 10 ⁇ 4 M, 5 ⁇ 10 ⁇ 4 M, 10 ⁇ 5 M, 5 ⁇ 10 ⁇ 5 M, 10 ⁇ 6 M, 5 ⁇ 10 ⁇ 6 M, 10 ⁇ 7 M Or at a concentration of 5 ⁇ 10 ⁇ 7 M.
  • the result is shown in FIG. It was shown that cells expressing the Drosophila DmOR47a and honey bee AmORCO complex responded more strongly to pentyl acetate than cells expressing the Drosophila DmOR47a and DmORCO complex.

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Abstract

The present invention provides an odorant receptor complex formed from an insect odorant receptor and an insect odorant receptor co-receptor, and which exhibits excellent detection sensitivity to odorous substances. This odorant receptor complex is formed of an odorant receptor from an insect that does not belong to the family Apidae and of an odorant receptor co-receptor from an Apidae insect.

Description

嗅覚受容体複合体及びそれを発現する細胞Olfactory receptor complex and cell expressing the same
　本発明は、嗅覚受容体複合体、具体的には、昆虫由来の嗅覚受容体と別の昆虫由来の嗅覚受容体共受容体とから形成される嗅覚受容体複合体、及びそれを発現する細胞に関する。 The present invention relates to an olfactory receptor complex, specifically, an olfactory receptor complex formed from an olfactory receptor derived from an insect and an olfactory receptor co-receptor derived from another insect, and a cell expressing the same About.
　近年、揮発性化学物質（匂い物質）を測定する様々な技術が知られている。このような技術は、癌診断等の診断・ヘルスケア分野、人探索、麻薬等の検出等の防災・セキュリティ分野、有害物質の検出等の環境分野等への応用が期待されている。 In recent years, various techniques for measuring volatile chemical substances (odor substances) are known. Such a technique is expected to be applied to the diagnosis / healthcare field such as cancer diagnosis, disaster prevention / security field such as human search, detection of narcotics, and environmental field such as detection of harmful substances.
　匂い物質の検出には犬や線虫等の生物を利用することがある。しかしながら、生物を利用する方法は高感度に匂い物質を検知できるものの、体調や個体によってバラツキがあるため安定性に欠け、また、集中力の持続時間に限界があるため長時間の利用が難しいといった側面もあった。 Detecting odorous substances may use organisms such as dogs and nematodes. However, although the method using living organisms can detect odorous substances with high sensitivity, it is not stable due to variations in physical condition and individuals, and it is difficult to use for a long time because the duration of concentration is limited. There was also a side.
　そのため、生物そのものではなく、生物が備える嗅覚機能が注目されている。特に昆虫の嗅覚機能は匂い物質の認識特異性が高いとされており、嗅覚センサー等への利用が模索されている。昆虫の嗅覚器には、匂い物質を認識する嗅覚受容体（ｏｄｏｒａｎｔ　ｒｅｃｅｐｔｏｒ、以下「ＯＲ」という場合もある。）と嗅覚受容体共受容体（ｏｄｏｒａｎｔ　ｒｅｃｅｐｔｏｒ　ｃｏ－ｒｅｃｅｐｔｏｒ、以下「ＯＲＣＯ」又は「共受容体」という場合もある。）とが形成した嗅覚受容体複合体が存在しており、匂い物質により活性化されると、上記複合体がイオンチャネル活性を示し、それにより匂い物質が検知される（Ｕ．Ｂｅｎｊａｍｉｎ　Ｋａｕｐｐ，Ｎａｔｕｒｅ　Ｒｅｖｉｅｗｓ　Ｎｅｕｒｏｓｃｉｅｎｃｅ，ｖｏｌ　１１（２０１０），ｐ１８８－２００）。 Therefore, attention is paid to the olfactory function of the organism, not the organism itself. In particular, the olfactory function of insects is considered to have high recognition specificity for odorous substances, and its use for olfactory sensors and the like is being sought. Insect olfactory organs include an olfactory receptor that recognizes odorants (sometimes referred to as “OR”) and an olfactory receptor co-receptor (hereinafter referred to as “ORCO” or “co-receptor”). The olfactory receptor complex formed by the odorant is present, and when activated by an odor substance, the complex exhibits ion channel activity, thereby detecting the odor substance. (U. Benjamin Kaupp, Nature Reviews Neuroscience, vol 11 (2010), p188-200).
　しかしながら、匂い物質を現場で検出しようとする場合、その濃度は非常に低い場合がある。そのため、匂い物質を検出するために用いられる嗅覚センサーは、高感度であることが求められる。 However, when trying to detect odorous substances on site, the concentration may be very low. Therefore, an olfactory sensor used for detecting an odor substance is required to have high sensitivity.
　本発明は上記事情に鑑みてなされたものであり、匂い物質に対して優れた検出感度を発揮する、昆虫の嗅覚受容体と嗅覚受容体共受容体とから形成される昆虫嗅覚受容体複合体を提供することを目的とする。 The present invention has been made in view of the above circumstances, and an insect olfactory receptor complex formed from an insect olfactory receptor and an olfactory receptor co-receptor that exhibits excellent detection sensitivity to odor substances. The purpose is to provide.
　本発明者らは、ミツバチ科昆虫の嗅覚受容体共受容体とミツバチ科に属さない昆虫の嗅覚受容体とから形成される複合体によれば、上記嗅覚受容体と同種の昆虫の嗅覚受容体共受容体と上記嗅覚受容体とから形成される嗅覚受容体複合体よりも、匂い物質に対する検出感度に優れることを見出した。本発明は、これらの知見に基づくものである。 According to a complex formed by an olfactory receptor co-receptor of a honey bee insect and an olfactory receptor of an insect that does not belong to the honey bee family, the present inventor It has been found that detection sensitivity to odorous substances is superior to an olfactory receptor complex formed from a co-receptor and the olfactory receptor. The present invention is based on these findings.
　すなわち、本発明は、例えば、以下の［１］～［６］に関する。
［１］ミツバチ科に属さない昆虫の嗅覚受容体と、ミツバチ科昆虫の嗅覚受容体共受容体とから形成される、嗅覚受容体複合体。
［２］嗅覚受容体が、カ科昆虫、カイコガ科昆虫、及びショウジョウバエ科昆虫からなる群から選択される昆虫の嗅覚受容体である、［１］に記載の嗅覚受容体複合体。
［３］嗅覚受容体が、１－オクテン－３－オール、ベンズアルデヒド、２，４，５－トリメチルチアゾール、ｃｉｓ－ジャスモン、及びペンチルアセテートからなる群から選択される化合物をリガンドとする嗅覚受容体である、［１］又は［２］に記載の嗅覚受容体複合体。
［４］嗅覚受容体が、ネッタイシマカ（Ａｅｄｅｓ　ａｅｇｙｐｔｉ）のＯＲ８若しくはＯＲ１０、ガンビエハマダラカ（Ａｎｏｐｈｅｌｅｓ　ｇａｍｂｉａｅ）のＯＲ１０若しくはＯＲ２８、カイコガ（Ｂｏｍｂｙｘ　ｍｏｒｉ）のＯＲ５６、又はキイロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ）のＯＲ４７ａ、或いは、これらのうちいずれかの嗅覚受容体全体のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を有する嗅覚受容体であり、嗅覚受容体共受容体が、セイヨウミツバチ（Ａｐｉｓ　ｍｅｌｌｉｆｅｒａ）又はその近縁種昆虫の嗅覚受容体共受容体である、［１］～［３］のいずれかに記載の嗅覚受容体複合体。
［５］嗅覚受容体が、ネッタイシマカ（Ａｅｄｅｓ　ａｅｇｙｐｔｉ）のＯＲ８若しくはＯＲ１０、ガンビエハマダラカ（Ａｎｏｐｈｅｌｅｓ　ｇａｍｂｉａｅ）のＯＲ１０若しくはＯＲ２８、カイコガ（Ｂｏｍｂｙｘ　ｍｏｒｉ）のＯＲ５６、又はキイロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ）のＯＲ４７ａ、或いは、これらのうちいずれかの嗅覚受容体全体のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を有する嗅覚受容体であり、嗅覚受容体共受容体が、配列番号１に記載のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を有し、かつ、昆虫嗅覚受容体と共役して陽イオンを細胞内へ流入させる能力を有するタンパク質である、［１］～［３］のいずれかに記載の嗅覚受容体複合体。
［６］［１］～［５］のいずれかに記載の嗅覚受容体複合体を発現する細胞。 That is, the present invention relates to the following [1] to [6], for example.
[1] An olfactory receptor complex formed from an olfactory receptor of an insect not belonging to the honeybee family and an olfactory receptor co-receptor of an honeybee insect.
[2] The olfactory receptor complex according to [1], wherein the olfactory receptor is an olfactory receptor of an insect selected from the group consisting of mosquitoes, Bombycidae, and Drosophila insects.
[3] An olfactory receptor having a ligand selected from compounds selected from the group consisting of 1-octen-3-ol, benzaldehyde, 2,4,5-trimethylthiazole, cis-jasmon, and pentyl acetate. The olfactory receptor complex according to [1] or [2].
[4] The olfactory receptor is OR8 or OR10 of Aedes aegypti, OR10 or OR28 of Anopheles gambiae, OR56 of Bombyx mori, or Drosophila or 47 Among these, an olfactory receptor having an amino acid sequence having 90% or more identity with the entire amino acid sequence of the olfactory receptor, wherein the olfactory receptor co-receptor is Apis melifera or its close relationship The olfactory receptor complex according to any one of [1] to [3], which is a species insect olfactory receptor co-receptor.
[5] The olfactory receptor is OR8 or OR10 of Aedes aegypti, OR10 or OR28 of Anopheles gambiae, OR56 of Bombyx mori, or 47 or 47 of Drosophila ago Among these, an olfactory receptor having an amino acid sequence having 90% or more identity with the entire amino acid sequence of the olfactory receptor, and the olfactory receptor co-receptor is an amino acid sequence represented by SEQ ID NO: 1 and 90 The protein according to any one of [1] to [3], which is a protein having an amino acid sequence having an identity of at least% and having an ability to allow a cation to flow into a cell by coupling with an insect olfactory receptor Olfactory receptor complex.
[6] A cell that expresses the olfactory receptor complex according to any one of [1] to [5].
　本発明によれば、匂い物質に対して優れた検出感度を発揮する、昆虫の嗅覚受容体と嗅覚受容体共受容体とから形成される嗅覚受容体複合体を提供することができる。 According to the present invention, it is possible to provide an olfactory receptor complex formed of an insect olfactory receptor and an olfactory receptor co-receptor that exhibits excellent detection sensitivity for odor substances.
ＡａＯＲ８とＡａＯＲＣＯとの複合体を発現する細胞、及びＡａＯＲ８とＡｍＯＲＣＯとの複合体を発現する細胞に対し、１－オクテン－３－オールを添加した際の発光量の相対値を示す。The relative values of the amount of luminescence when 1-octen-3-ol is added to cells expressing a complex of AaOR8 and AaORCO and cells expressing a complex of AaOR8 and AmORCO are shown. ＡａＯＲ１０とＡａＯＲＣＯとの複合体を発現する細胞、及びＡａＯＲ１０とＡｍＯＲＣＯとの複合体を発現する細胞に対し、ベンズアルデヒドを添加した際の発光量の相対値を示す。The relative value of the light-emission quantity at the time of adding benzaldehyde with respect to the cell which expresses the complex of AaOR10 and AaORCO, and the cell which expresses the complex of AaOR10 and AmORCO is shown. ＡｇＯＲ１０とＡｇＯＲＣＯとの複合体を発現する細胞、及びＡｇＯＲ１０とＡｍＯＲＣＯとの複合体を発現する細胞に対し、ベンズアルデヒドを添加した際の発光量の相対値を示す。The relative value of the amount of luminescence when benzaldehyde is added to a cell expressing a complex of AgOR10 and AgORCO and a cell expressing a complex of AgOR10 and AmORCO is shown. ＡｇＯＲ２８とＡｇＯＲＣＯとの複合体を発現する細胞、及びＡｇＯＲ２８とＡｍＯＲＣＯとの複合体を発現する細胞に対し、２，４，５－トリメチルチアゾールを添加した際の発光量の相対値を示す。The relative value of the amount of luminescence when 2,4,5-trimethylthiazole is added to a cell expressing a complex of AgOR28 and AgORCO and a cell expressing a complex of AgOR28 and AmORCO is shown. ＢｍＯＲ５６とＢｍＯＲＣＯとの複合体を発現する細胞、及びＢｍＯＲ５６とＡｍＯＲＣＯとの複合体を発現する細胞に対し、ｃｉｓ－ジャスモンを添加した際の発光量の相対値を示す。The relative values of the amount of luminescence when cis-jasmon is added to cells expressing a complex of BmOR56 and BmORCO and cells expressing a complex of BmOR56 and AmORCO are shown. ＤｍＯＲ４７ａとＤｍＯＲＣＯとの複合体を発現する細胞、及びＤｍＯＲ４７ａとＡｍＯＲＣＯとの複合体を発現する細胞に対し、ペンチルアセテートを添加した際の発光量の相対値を示す。The relative value of the amount of luminescence when pentyl acetate is added to a cell expressing a complex of DmOR47a and DmORCO and a cell expressing a complex of DmOR47a and AmORCO is shown.
　以下、本発明を実施するための形態（以下、「本実施形態」という。）について詳細に説明する。なお、本発明は、以下の実施形態に限定されるものではない。 Hereinafter, a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail. In addition, this invention is not limited to the following embodiment.
　昆虫の嗅覚受容体複合体は、嗅覚受容体と嗅覚受容体共受容体とがヘテロ複合体を形成したものである。昆虫の嗅覚受容体は、７回膜貫通構造を有するＧタンパク質共役型受容体の一種であり、その匂い物質の認識特異性が高い。いくつかの匂い物質特異的な嗅覚受容体が知られている。一方、昆虫の嗅覚受容体共受容体は、嗅覚受容体と同様に７回膜貫通構造を有する膜タンパク質であるものの、それ自身は匂い物質を認識せず、嗅覚受容体とヘテロ複合体を形成して機能する。Ｏｒｃｏファミリーとして知られている上記嗅覚受容体共受容体は、比較的高い相同性で全ての昆虫に保存されている。嗅覚受容体複合体は、匂い物質で活性化されるイオンチャンネル活性が備わっており、活性化されるとナトリウムイオン（Ｎａ＋）、カルシウムイオン（Ｃａ２＋）等の陽イオンを細胞内に流入させる。 Insect olfactory receptor complexes are olfactory receptors and olfactory receptor co-receptors that form a heterocomplex. An insect olfactory receptor is a kind of G protein-coupled receptor having a seven-transmembrane structure, and has high recognition specificity for the odorant. Several odorant-specific olfactory receptors are known. On the other hand, the olfactory receptor co-receptor of insects is a membrane protein having a 7-transmembrane structure like the olfactory receptor, but does not recognize odorants and forms a hetero complex with the olfactory receptor. And function. The olfactory receptor co-receptor known as the Orco family is conserved in all insects with relatively high homology. The olfactory receptor complex has an ion channel activity activated by an odor substance. When activated, a cation such as sodium ion (Na + ) or calcium ion (Ca 2+ ) flows into the cell. .
　本実施形態の嗅覚受容体複合体は、ミツバチ科に属さない昆虫の嗅覚受容体と、ミツバチ科昆虫の嗅覚受容体共受容体とから形成される。 The olfactory receptor complex of this embodiment is formed from an olfactory receptor of an insect that does not belong to the honey bee family and an olfactory receptor co-receptor of the honey bee family.
　ミツバチ科に属さない昆虫の嗅覚受容体としては、例えば、カ科昆虫、カイコガ科昆虫、及びショウジョウバエ科昆虫からなる群から選択される昆虫の嗅覚受容体が挙げられる。カ科の昆虫としては、例えば、ガンビエハマダラカ（Ａｎｏｐｈｅｌｅｓ　ｇａｍｂｉａｅ）、ネッタイシマカ（Ａｅｄｅｓ　ａｅｇｙｐｔｉ）、ネッタイイエカ（Ｃｕｌｅｘ　ｑｕｉｎｑｕｅｆａｓｃｉａｔｕｓ）等が挙げられる。カイコガ科の昆虫としては、例えば、カイコガ（Ｂｏｍｂｙｘ　ｍｏｒｉ）、クワコ（Ｂｏｍｂｙｘ　ｍａｎｄａｒｉｎａ）、イチジクカサン（Ｔｒｉｌｏｃｈａ　ｖａｒｉａｎｓ）等が挙げられる。ショウジョウバエ科の昆虫としては、例えば、キイロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ）、ウスグロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｐｓｅｕｄｏｏｂｓｃｕｒａ）、クロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｖｉｒｉｌｌｉｓ）等が挙げられる。 Examples of olfactory receptors of insects that do not belong to the honey bee family include insect olfactory receptors selected from the group consisting of mosquitoes, silkworms, and fruit flies. Examples of the mosquito insects include Gambier anopheles (Anopheles gambiae), Aedes aegypti, Aedes squid (Culeque quinquefacciatus) and the like. Examples of the insects of the Bombycidae include Bombyx mori, mulberry (Bombyx mandarina), and fig cassan (Trilocha varians). Examples of Drosophila insects include Drosophila melanogaster, Drosophila pseudobscura, Drosophila virillis, and the like.
　嗅覚受容体の種類は、標的とするリガンドに応じて適宜選択することができる。嗅覚受容体は、１－オクテン－３－オール、ベンズアルデヒド、２，４，５－トリメチルチアゾール、ｃｉｓ－ジャスモン、及びペンチルアセテートからなる群から選択される化合物をリガンドとする嗅覚受容体であってもよい。嗅覚受容体は、具体的には、ネッタイシマカ（Ａｅｄｅｓ　ａｅｇｙｐｔｉ）のＯＲ８若しくはＯＲ１０、ガンビエハマダラカ（Ａｎｏｐｈｅｌｅｓ　ｇａｍｂｉａｅ）のＯＲ１０若しくはＯＲ２８、カイコガ（Ｂｏｍｂｙｘ　ｍｏｒｉ）のＯＲ５６、又はキイロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ）のＯＲ４７ａであってもよい。嗅覚受容体は、上記いずれかの嗅覚受容体全体のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を有する嗅覚受容体であってもよく、９５％以上の同一性を有するアミノ酸配列を有する嗅覚受容体であってもよく、９８％以上の同一性を有するアミノ酸配列を有する嗅覚受容体であってもよく、９９％以上の同一性を有するアミノ酸配列を有する嗅覚受容体であってもよい。上記いずれかの嗅覚受容体全体のアミノ酸配列と比較した場合に同一性が９０％以上であるアミノ酸配列を有する嗅覚受容体であれば、アミノ酸配列を比較した元の嗅覚受容体と同程度のリガンド特異性及び検出感度が得られる傾向にある。 The type of olfactory receptor can be appropriately selected according to the target ligand. The olfactory receptor may be an olfactory receptor having a compound selected from the group consisting of 1-octen-3-ol, benzaldehyde, 2,4,5-trimethylthiazole, cis-jasmon, and pentyl acetate as a ligand. Good. The olfactory receptors are specifically OR8 or OR10 of Aedes aegypti, OR10 or OR28 of Anopheles gambiae, OR56 of Bombyx mori, or 47 of Drosophila. There may be. The olfactory receptor may be an olfactory receptor having an amino acid sequence having 90% or more identity with the whole amino acid sequence of any of the above olfactory receptors, and has an amino acid sequence having 95% or more identity. It may be an olfactory receptor, an olfactory receptor having an amino acid sequence having 98% or more identity, or an olfactory receptor having an amino acid sequence having 99% or more identity . If it is an olfactory receptor having an amino acid sequence having an identity of 90% or more when compared with the amino acid sequence of any of the above olfactory receptors, a ligand comparable to the original olfactory receptor compared with the amino acid sequence Specificity and detection sensitivity tend to be obtained.
　ミツバチ科昆虫に由来する嗅覚受容体共受容体としては、例えば、セイヨウミツバチ（Ａｐｉｓ　ｍｅｌｌｉｆｅｒａ）、ヒメミツバチ（Ａｐｉｓ　ｆｌｏｒｅａ）、オオミツバチ（Ａｐｉｓ　ｄｏｒｓａｔａ）、セイヨウオオマルハナバチ（Ｂｏｍｂｕｓ　ｔｅｒｒｅｓｔｒｉｓ）等のミツバチ科昆虫に由来するものが挙げられる。ミツバチ科の嗅覚受容体共受容体とミツバチ科に属さない昆虫の嗅覚受容体と組み合わせた複合体によれば、上記嗅覚受容体と同種の昆虫の嗅覚受容体共受容体と上記嗅覚受容体とから形成される嗅覚受容体複合体よりも、匂い物質に対する検出感度に優れるものとなる。 Examples of olfactory receptor co-receptors derived from honey bee insects include Apis mellifera, Apis florea, Apis dorsata, Bombus terrestris, etc. The thing derived from is mentioned. According to the complex of the honey bee olfactory receptor co-receptor and the insect olfactory receptor not belonging to the honey bee family, the olfactory receptor co-receptor and the olfactory receptor It is superior in detection sensitivity to odorous substances than the olfactory receptor complex formed from the above.
　嗅覚受容体共受容体は、セイヨウミツバチ又はその近縁種に由来する嗅覚受容体共受容体であってもよい。本明細書において「セイヨウミツバチの近縁種」とは、ミツバチ族（Ａｐｉｎｉ）のハチ、又は、シタバチ族（Ｅｕｇｌｏｓｓｉｎｉ）、マルハナバチ族　ｂｕｍｂｌｅ　ｂｅｅ（Ｂｏｍｂｉｎｉ）、もしくは、ハリナシバチ族　ｓｔｉｎｇｌｅｓｓ　ｂｅｅ（Ｍｅｌｉｐｏｎｉｎｉ）のハチである。これらの族のハチは、後脚脛節の外側に花粉かご　ｃｏｒｂｉｃｕｌａ（ｐｏｌｌｅｎ　ｂａｓｋｅｔ）を有する。セイヨウミツバチの近縁種としては、例えば、ミツバチ族に属するヒメミツバチ、及びオオミツバチ；並びに、マルハナバチ族に属するセイヨウオオマルハナバチ等が挙げられる。このような近縁種の嗅覚受容体共受容体であれば、セイヨウミツバチの嗅覚受容体共受容体とのアミノ酸配列同一性が高く、同等の機能を備える傾向にある。 The olfactory receptor co-receptor may be an olfactory receptor co-receptor derived from a honey bee or its related species. In the present specification, the term “relative species of honey bees” refers to bees of Apini, or Euglesini, bumblebee bumble bee (Bombini), or bee stingless bee (Melip) bee (Melip) It is. Bees of these families have a pollen basket corbicular (pollen basket) on the outside of the hind leg shin joint. Examples of related species of honey bees include honey bees belonging to honey bees and honey bees; and honey bees belonging to bumble bees. Such closely related olfactory receptor co-receptors have high amino acid sequence identity with honey bee olfactory receptor co-receptors and tend to have equivalent functions.
　嗅覚受容体共受容体は、セイヨウミツバチの嗅覚受容体共受容体のアミノ酸配列（配列番号１に記載のアミノ酸配列）と９０％以上の同一性を有するアミノ酸配列を有し、かつ、昆虫嗅覚受容体と共役して陽イオンを細胞内へ流入させる能力を有するタンパク質であってもよく、９５％以上の同一性を有するアミノ酸配列を有し、かつ、昆虫嗅覚受容体と共役して陽イオンを細胞内へ流入させる能力を有するタンパク質であってもよく、９８％以上の同一性を有するアミノ酸配列を有し、かつ、昆虫嗅覚受容体と共役して陽イオンを細胞内へ流入させる能力を有するタンパク質であってもよく、９９％以上の同一性を有するアミノ酸配列を有し、かつ、昆虫嗅覚受容体と共役して陽イオンを細胞内へ流入させる能力を有するタンパク質であってもよい。配列番号１に記載のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を有し、かつ、昆虫嗅覚受容体と共役して陽イオンを細胞内へ流入させる能力を有するタンパク質であれば、セイヨウミツバチの嗅覚受容体共受容体と同様に、ミツバチ科に属さない昆虫の嗅覚受容体と組み合わせて複合体を形成させることで、上記嗅覚受容体と同種の昆虫の嗅覚受容体共受容体と上記嗅覚受容体とから形成される嗅覚受容体複合体よりも、匂い物質に対する検出感度に優れるものとなる傾向にある。 The olfactory receptor co-receptor has an amino acid sequence having 90% or more identity with the amino acid sequence of the honey bee olfactory receptor co-receptor (amino acid sequence described in SEQ ID NO: 1), and insect olfactory receptor It may be a protein that has the ability to conjugated with the body and cause cations to flow into the cell, has an amino acid sequence having 95% or more identity, and is conjugated with insect olfactory receptors It may be a protein having the ability to flow into cells, has an amino acid sequence having 98% or more identity, and has the ability to couple cations into cells by coupling with insect olfactory receptors A protein having an amino acid sequence with 99% or more identity and having the ability to allow a cation to flow into a cell by coupling with an insect olfactory receptor It may be. Any protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having the ability to couple a cation into the cell in combination with an insect olfactory receptor will be used. Similar to the bee olfactory receptor co-receptor, by forming a complex with an olfactory receptor of an insect that does not belong to the honey bee family, an olfactory receptor co-receptor of the same kind of insect as the above olfactory receptor It tends to be more sensitive to detection of odorous substances than olfactory receptor complexes formed from olfactory receptors.
　配列番号１に記載のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を有し、かつ、昆虫嗅覚受容体と共役して陽イオンを細胞内へ流入させる能力を有するタンパク質としては、例えば、ヒメミツバチの嗅覚受容体共受容体（同一性９９％）、オオミツバチの嗅覚受容体共受容体（同一性９８％）、セイヨウオオマルハナバチの嗅覚受容体共受容体（同一性９１％）等が挙げられる。 Examples of the protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having an ability to allow a cation to flow into a cell in combination with an insect olfactory receptor include: The honey bee olfactory receptor co-receptor (99% identity), the bee olfactory receptor co-receptor (identity 98%), the bee olfactory receptor co-receptor (identity 91%), etc. Can be mentioned.
　本実施形態の嗅覚受容体複合体は、嗅覚受容体と嗅覚受容体共受容体とが１：１の割合で相互作用して複合体を形成している。嗅覚受容体及び嗅覚受容体共受容体は、それぞれ１つずつから複合体が形成されてもよく、それぞれ２つずつから複合体が形成されてもよく、それぞれ４つずつから複合体が形成されてもよい。 In the olfactory receptor complex of the present embodiment, the olfactory receptor and the olfactory receptor co-receptor interact at a ratio of 1: 1 to form a complex. The olfactory receptor and the olfactory receptor co-receptor may each form a complex, may each form a complex from two, or may form a complex from four each. May be.
　本実施形態の嗅覚受容体複合体は、嗅覚受容体及び嗅覚受容体共受容体を同じ細胞の細胞膜に同時に発現させ、場合によって精製する方法、又は、嗅覚受容体及び嗅覚受容体共受容体をそれぞれ別の細胞で発現させ、精製した後に混合する方法等により調製することができる。嗅覚受容体及び嗅覚受容体共受容体を細胞で発現させる方法は、特に限定されるものではなく公知の方法を用いることができる。このような方法としては、例えば、上記嗅覚受容体をコードするＤＮＡを組み入れた発現用プラスミドと、上記嗅覚受容体共受容体をコードするＤＮＡを組み入れた発現用プラスミドとを細胞に導入してこれらを発現させる方法がある。この際、上記嗅覚受容体をコードするＤＮＡを組み入れた発現用プラスミドと、上記嗅覚受容体共受容体をコードするＤＮＡを組み入れた発現用プラスミドとを同じ細胞に導入することで、嗅覚受容体複合体として直接調製することもできる。細胞に各種プラスミドが導入されたことは、ＰＣＲ等の方法により確認することができる。細胞で発現させた嗅覚受容体、嗅覚受容体共受容体、又は嗅覚受容体複合体は、細胞膜を可溶化した後、各種精製用カラムに供することにより精製することができる。 The olfactory receptor complex of the present embodiment is a method in which an olfactory receptor and an olfactory receptor co-receptor are simultaneously expressed in the cell membrane of the same cell and purified in some cases, or an olfactory receptor and an olfactory receptor co-receptor It can be prepared by a method in which each cell is expressed in different cells, purified and then mixed. The method for expressing the olfactory receptor and olfactory receptor co-receptor in cells is not particularly limited, and a known method can be used. As such a method, for example, an expression plasmid incorporating a DNA encoding the olfactory receptor and an expression plasmid incorporating a DNA encoding the olfactory receptor co-receptor are introduced into a cell. There is a method of expressing. At this time, the expression plasmid incorporating the DNA encoding the olfactory receptor and the expression plasmid incorporating the DNA encoding the olfactory receptor co-receptor are introduced into the same cell, thereby producing an olfactory receptor complex. It can also be prepared directly as a body. The introduction of various plasmids into the cells can be confirmed by a method such as PCR. The olfactory receptor, olfactory receptor co-receptor, or olfactory receptor complex expressed in cells can be purified by solubilizing the cell membrane and applying it to various purification columns.
　本実施形態の嗅覚受容体複合体によれば、当該複合体を構成する嗅覚受容体と同種の昆虫の嗅覚受容体共受容体と上記嗅覚受容体とから形成される嗅覚受容体複合体よりも匂い物質に対する検出感度に優れる。このような特性を有する嗅覚受容体複合体は、より低濃度の匂い物質を検出することができる嗅覚センサーとして有用である。 According to the olfactory receptor complex of the present embodiment, the olfactory receptor complex formed from the olfactory receptor co-receptor of the same kind of insect as the olfactory receptor constituting the complex and the olfactory receptor. Excellent detection sensitivity to odorous substances. The olfactory receptor complex having such characteristics is useful as an olfactory sensor capable of detecting a lower concentration of odor substance.
　本実施形態の細胞は、上記嗅覚受容体複合体を発現する。すなわち、本実施形態の細胞は、その細胞内において、ミツバチ科に属さない昆虫の嗅覚受容体とミツバチ科昆虫の嗅覚共受容体とを発現し、それにより細胞膜上において本実施形態の嗅覚受容体複合体を発現する。 The cell of this embodiment expresses the olfactory receptor complex. That is, the cell of the present embodiment expresses an olfactory receptor of an insect that does not belong to the honey bee family and an olfactory co-receptor of the honey bee family in the cell, and thereby the olfactory receptor of the present embodiment on the cell membrane. Express the complex.
　本実施形態の細胞は、嗅覚受容体複合体の調製方法と同様に、嗅覚受容体をコードするＤＮＡを組み入れた発現用プラスミドと、嗅覚受容体共受容体をコードするＤＮＡを組み入れた発現用プラスミドとを同じ細胞に導入することで調製することができる。発現プラスミドを導入する細胞としては、発現プラスミドの種類に応じて適宜選択することができる。細胞としては、例えば、Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　Ｋ１２等の大腸菌、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＭＩ１１４等のバチルス属細菌、Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　ＡＨ２２等の酵母、Ｓｐｏｄｏｐｔｅｒａ　ｆｒｕｇｉｐｅｒｄａ　由来の　Ｓｆ細胞系もしくはＴｒｉｃｈｏｐｌｕｓｉａ　ｎｉ由来のＨｉｇｈＦｉｖｅ細胞系等の昆虫細胞、ＣＯＳ７細胞等の動物細胞等を挙げることができる。動物細胞としては、好ましくは、哺乳動物由来の培養細胞、具体的には、ＣＯＳ７細胞、ＣＨＯ細胞、ＨＥＫ２９３細胞、ＨＥＫ２９３ＦＴ細胞、Ｈｅｌａ細胞、ＰＣ１２細胞、Ｎ１Ｅ－１１５細胞、ＳＨ－ＳＹ５Ｙ細胞等が挙げられる。 The cells according to the present embodiment include an expression plasmid incorporating a DNA encoding an olfactory receptor and an expression plasmid incorporating a DNA encoding an olfactory receptor co-receptor, as in the method for preparing an olfactory receptor complex. Can be prepared by introducing into the same cell. The cell into which the expression plasmid is introduced can be appropriately selected according to the type of expression plasmid. Examples of cells include Escherichia coli K12 and other Escherichia coli, Bacillus subtilis MI114 and other Bacillus bacteria, Saccharomyces cerevisiae AH22 and other species, Spodoptera frugiperda-derived Sf cells derived from Spodachtera frugiperda and the like. And animal cells such as cells. Animal cells are preferably cultured cells derived from mammals, specifically, COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells and the like. It is done.
　以下、実施例に基づき本発明を具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.
　本実施例に記載の昆虫は以下のとおりである。
ガンビエハマダラカ（Ａｎｏｐｈｅｌｅｓ　ｇａｍｂｉａｅ：以下、「ハマダラカ」又は「Ａｇ」という場合もある。）
ネッタイシマカ（Ａｅｄｅｓ　ａｅｇｙｐｔｉ：以下、「シマカ」又は「Ａａ」という場合もある。）
キイロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ：以下、「ショウジョウバエ」又は「Ｄｍ」という場合もある。）
セイヨウミツバチ（Ａｐｉｓ　ｍｅｌｌｉｆｅｒａ：以下、「ミツバチ」又は「Ａｍ」という場合もある。）
カイコガ（Ｂｏｍｂｙｘ　ｍｏｒｉ：以下、「カイコガ」又は「Ｂｍ」という場合もある。） The insects described in this example are as follows.
Anopheles gambiae (hereinafter also referred to as “Anopheles gambiae” or “Ag”)
Aedes aegypti (hereinafter sometimes referred to as “Shimaka” or “Aa”)
Drosophila melanogaster (hereinafter sometimes referred to as “Drosophila” or “Dm”)
Apis melifera (hereinafter, also referred to as “bee” or “Am”)
Bombyx mori (hereinafter, also referred to as “silkworm” or “Bm”)
＜共受容体の発現プラスミドの作製＞
ハマダラカ共受容体（以下、ＡｇＯＲＣＯという場合もある。）の発現プラスミド
　ガンビエハマダラカ（Ａｎｏｐｈｅｌｅｓ　ｇａｍｂｉａｅ）Ｋｉｓｕｍ系統又はＧ３系統の頭部を液体窒素で凍結させた後、ＴＲＩｚｏｌ（インビトロジェン社製）を加えて液体窒素存在下で乳鉢と乳棒を用いて破砕した。得られた破砕物から、ＴＲＩｚｏｌ試薬に添付の説明書に従いＲＮＡを抽出した。抽出されたＲＮＡを、ＲＮＡ精製キット（ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ；ＱＩＡＧＥＮ社製）を用い、当該キットに添付された説明書に従って精製した。得られたＴｏｔａｌ　ＲＮＡを鋳型にし、Ｓｕｐｅｒ　Ｓｃｒｉｐｔ　ＩＩＩ逆転写酵素（インビトロジェン社製）を添加し、５５℃で５０分間、次いで７５℃で１５分間保温することにより逆転写反応を行い、ｃＤＮＡを得た。得られたｃＤＮＡ1μＬを鋳型に用い、１０μＭのフォワードプライマーＡｇＯｒｃｏ－５’（5’-caccatgcaagtccagccgaccaagtacgtcggcct；配列番号２）１μＬ、１０μＭのリバースプライマーＡｇＯｒｃｏ－３’（5’-ttacttcagctgcaccagcaccatgaagt；配列番号３）１μＬ及びＫＯＤ　ＰｌｕｓＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、５分間、（２）９８℃、１０秒間、（３）６０℃、３０秒間、（４）６８℃、１．５分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物をアガロースゲル電気泳動した後、当該ゲル上で検出された約１．４ｋｂのＤＮＡを回収した。回収されたＤＮＡをｐＥＮＴＲ／Ｄ－ＴＯＰＯベクター（インビトロジェン社製）へ導入し、ｐＥＮＴＲ－ＡｇＯｒｃｏと名付けたプラスミドを得た。４μＬのｐＥＮＴＲ－ＡｇＯｒｃｏ、１μＬのｐｃＤＮＡ６．２Ｖ５－ＤＥＳＴ及び２μＬのＬＲクロナーゼＩＩ（インビトロジェン社製）を混合して室温で１時間保持した。得られた混合物を大腸菌へ導入し培養することにより、ｐｃＤＮＡ６．２－ＡｇＯｒｃｏと名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ６．２－ＡｇＯｒｃｏの塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号４で示される塩基配列を含有することが判った。配列番号４で示される塩基配列は配列番号５で示されるアミノ酸配列をコードする。 <Preparation of co-receptor expression plasmid>
An expression plasmid of Anopheles co-receptor (hereinafter also referred to as AgORCO) After freezing the head of the Anopheles gambiae Kisum line or G3 line with liquid nitrogen, TRIzol (manufactured by Invitrogen) was added. The mixture was crushed using a mortar and pestle in the presence of liquid nitrogen. From the obtained crushed material, RNA was extracted according to the instructions attached to the TRIzol reagent. The extracted RNA was purified using an RNA purification kit (RNeasy Mini Kit; manufactured by QIAGEN) according to the instructions attached to the kit. Using the obtained total RNA as a template, Super Script III reverse transcriptase (manufactured by Invitrogen) was added, and the mixture was incubated at 55 ° C. for 50 minutes and then at 75 ° C. for 15 minutes to obtain a cDNA. . Using 1 μL of the obtained cDNA as a template, 10 μM forward primer AgOrco-5 ′ (5′-caccatgcaagtccagccgaccaagtacgtcggcct; SEQ ID NO: 2) 1 μL, 10 μM reverse primer AgOrco-3 ′ (5′-ttacttcagctgcaccagcaccatgaagt; SEQ ID NO: 3) 1 μL PCR was performed using 1 μL of Plus DNA polymerase (Toyobo). The PCR reaction was carried out at (1) 94 ° C for 5 minutes, (2) 98 ° C for 10 seconds, (3) 60 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles. The obtained PCR product was subjected to agarose gel electrophoresis, and then about 1.4 kb of DNA detected on the gel was recovered. The recovered DNA was introduced into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-AgOrco. 4 μL of pENTR-AgOrco, 1 μL of pcDNA6.2V5-DEST and 2 μL of LR clonase II (Invitrogen) were mixed and kept at room temperature for 1 hour. The resulting mixture was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA6.2-AgOrco. Analysis of the nucleotide sequence of the expression plasmid pcDNA6.2-AgOrco using a DNA sequencer revealed that it contained the nucleotide sequence represented by SEQ ID NO: 4. The base sequence represented by SEQ ID NO: 4 encodes the amino acid sequence represented by SEQ ID NO: 5.
ショウジョウバエ共受容体（以下、ＤｍＯＲＣＯという場合もある。）の発現プラスミド
　ショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ）成虫ＲＮＡ（ＴＡＫＡＲＡ社製）を鋳型に用いて、Ｓｕｐｅｒ　Ｓｃｒｉｐｔ　ＩＩＩ逆転写酵素（インビトロジェン社製）を添加し、５５℃で５０分間、次いで７５℃で１５分間保温することにより逆転写反応を行い、ｃＤＮＡを得た。得られたｃＤＮＡ１μＬを鋳型に用い、１０μＭのフォワードプライマーＤｍＯｒｃｏ－５’（5’-caccatgacaacctcgatgcagccgagcaagt；配列番号６）１μＬ、１０μＭのリバースプライマーＤｍＯｒｃｏ－３’（5’-ttacttgagctgcaccagcaccataaagt；配列番号７）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏ　ＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、２分間、（２）９８℃、１０秒間、（３）６８℃、１．５分間で行い、（２）～（３）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物をアガロースゲル電気泳動した後、当該ゲル上で検出された約１．５ｋｂのＤＮＡを回収した。回収されたＤＮＡをｐＥＮＴＲ／Ｄ－ＴＯＰＯベクター（インビトロジェン社製）へ導入し、ｐＥＮＴＲ－ＤｍＯｒｃｏと名付けたプラスミドを得た。４μＬのｐＥＮＴＲ－ＤｍＯｒｃｏ、１μＬのｐｃＤＮＡ６．２Ｖ５－ＤＥＳＴ及び２μＬのＬＲクロナーゼＩＩ（インビトロジェン社製）を混合して室温で１時間保持した。得られた混合物を大腸菌へ導入し培養することにより、ｐｃＤＮＡ６．２－ＤｍＯｒｃｏと名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ６．２－ＤｍＯｒｃｏの塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号８で示される塩基配列を含有することが判った。配列番号８で示される塩基配列は配列番号９で示されるアミノ酸配列をコードする。 Expression plasmid of Drosophila co-receptor (hereinafter also referred to as DmORCO) Using Drosophila melanogaster adult RNA (manufactured by TAKARA) as a template, Super Script III reverse transcriptase (manufactured by Invitrogen) was added, A reverse transcription reaction was performed by incubating at 55 ° C. for 50 minutes and then at 75 ° C. for 15 minutes to obtain cDNA. Using 1 μL of the obtained cDNA as a template, 10 μM forward primer DmOrco-5 ′ (5′-caccatgacaacctcgatgcagccgagcaagt; SEQ ID NO: 6) 1 μL, 10 μM reverse primer DmOrco-3 ′ (5′-ttacttgagctgcaccagcaccatcataaagt; SEQ ID NO: 7) 1 μL PCR was performed using 1 μL of Plus neo DNA polymerase (Toyobo). PCR reaction was performed at (1) 94 ° C. for 2 minutes, (2) 98 ° C. for 10 seconds, (3) 68 ° C. for 1.5 minutes, and steps (2) to (3) were repeated 35 cycles. . The obtained PCR product was subjected to agarose gel electrophoresis, and then about 1.5 kb of DNA detected on the gel was recovered. The recovered DNA was introduced into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-DmOrco. 4 μL of pENTR-DmOrco, 1 μL of pcDNA6.2V5-DEST and 2 μL of LR clonase II (Invitrogen) were mixed and kept at room temperature for 1 hour. The resulting mixture was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA6.2-DmOrco. When the nucleotide sequence of the expression plasmid pcDNA6.2-DmOrco was analyzed by a DNA sequencer, it was found to contain the nucleotide sequence represented by SEQ ID NO: 8. The base sequence represented by SEQ ID NO: 8 encodes the amino acid sequence represented by SEQ ID NO: 9.
シマカ共受容体（以下、ＡａＯＲＣＯという場合もある。）の発現プラスミド
　ネッタイシマカ（Ａｅｄｅｓ　ａｅｇｙｐｔｉ）の頭部を液体窒素で凍結させた後、ＴＲＩｚｏｌ（インビトロジェン社製）を加えて液体窒素存在下で乳鉢と乳棒を用いて破砕した。得られた破砕物から、ＴＲＩｚｏｌ試薬に添付の説明書に従いＲＮＡを抽出した。抽出されたＲＮＡを、ＲＮＡ精製キット（ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ；ＱＩＡＧＥＮ社製）を用い、当該キットに添付された説明書に従って精製した。得られたＴｏｔａｌ　ＲＮＡを鋳型にし、Ｓｕｐｅｒ　Ｓｃｒｉｐｔ　ＩＩＩ逆転写酵素（インビトロジェン社製）を添加し、５５℃で５０分間、次いで７５℃で１５分間保温することにより逆転写反応を行い、ｃＤＮＡを得た。得られたｃＤＮＡ１μＬを鋳型に用い、１０μＭのフォワードプライマーＡａＯｒｃｏ－５’（5’-tggaattctgcagatcaccatgaacgtccaaccgacaaagtacc；配列番号１０）１μＬ、１０μＭのリバースプライマーＡａＯｒｃｏ－３’（5’-gccactgtgctggatttatttcaactgcaccaacacc；配列番号１１）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、２分間、（２）９８℃、１０秒間、（３）６３℃、３０秒間、（４）６８℃、１．５分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物を、ＥｃｏＲＶで消化されたｐｃＤＮＡ３．１（インビトロジェン社製）にＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（ＴＡＫＡＲＡ社製）を使用して連結した。その後、これを大腸菌へ導入して培養することにより、ｐｃＤＮＡ３．１－ＡａＯｒｃｏと名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ３．１－ＡａＯｒｃｏの塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号１２で示される塩基配列を含有することが判った。配列番号１２で示される塩基配列は配列番号１３で示されるアミノ酸配列をコードする。 The head of an expression plasmid of Aedes aegypti was frozen in liquid nitrogen, and then TRIzol (manufactured by Invitrogen) was added to the mortar in the presence of liquid nitrogen. Crush using a pestle. From the obtained crushed material, RNA was extracted according to the instructions attached to the TRIzol reagent. The extracted RNA was purified using an RNA purification kit (RNeasy Mini Kit; manufactured by QIAGEN) according to the instructions attached to the kit. Using the obtained total RNA as a template, Super Script III reverse transcriptase (manufactured by Invitrogen) was added, and the mixture was incubated at 55 ° C. for 50 minutes and then at 75 ° C. for 15 minutes to obtain a cDNA. . Using 1 μL of the obtained cDNA as a template, 10 μM forward primer AaOrco-5 ′ (5′-tggaattctgcagatcaccatgaacgtccaaccgacaaagtacc; SEQ ID NO: 10) 1 μL, 10 μM reverse primer AaOrco-3 ′ (5′-gccactgtgctggatttatttcaactgKaccaacaccL) PCR was performed using 1 μL of Plus neo DNA polymerase (Toyobo). The PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles. The obtained PCR product was ligated to pcDNA3.1 (manufactured by Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Then, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-AaOrco. Analysis of the base sequence of expression plasmid pcDNA3.1-AaOrco using a DNA sequencer revealed that it contained the base sequence represented by SEQ ID NO: 12. The base sequence represented by SEQ ID NO: 12 encodes the amino acid sequence represented by SEQ ID NO: 13.
ミツバチ共受容体（以下、ＡｍＯＲＣＯという場合もある。）の発現プラスミド
　いずれか一方のＤＮＡ鎖に配列番号１４で示される塩基配列を有する二本鎖ＤＮＡを合成した。得られたＤＮＡ１００ｎｇを鋳型に用い、１０μＭのフォワードプライマーＡｍＯｒｃｏ－５’（5’-tggaattctgcagatcaccatgaagttcaagcaacaagggctaa；配列番号１５）１μＬ、１０μＭのリバースプライマーＡｍＯｒｃｏ－３’（5’-gccactgtgctggattcacttcagttgcaccaacaccatgaa；配列番号１６）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、２分間、（２）９８℃、１０秒間、（３）６３℃、３０秒間、（４）６８℃、１．５分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物を、ＥｃｏＲＶで消化されたｐｃＤＮＡ３．１（インビトロジェン社製）にＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（ＴＡＫＡＲＡ社製）を使用して連結した。その後、これを大腸菌へ導入して培養することにより、ｐｃＤＮＡ３．１－ＡｍＯｒｃｏと名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ３．１－ＡｍＯｒｃｏの塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号１４で示される塩基配列を含有することが判った。配列番号１４で示される塩基配列は配列番号１で示されるアミノ酸配列をコードする。 Expression plasmid of honeybee co-receptor (hereinafter sometimes referred to as AmORCO) A double-stranded DNA having the base sequence shown in SEQ ID NO: 14 was synthesized in one of the DNA strands. 100 μg of the obtained DNA was used as a template, 10 μM forward primer AmOrco-5 ′ (5′-tggaattctgcagatcaccatgaagttcaagcaacaagggctaa; SEQ ID NO: 15) PCR was performed using 1 μL of Plus neo DNA polymerase (Toyobo). The PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles. The obtained PCR product was ligated to pcDNA3.1 (manufactured by Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Subsequently, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-AmOrco. Analysis of the base sequence of expression plasmid pcDNA3.1-AmOrco using a DNA sequencer revealed that it contained the base sequence represented by SEQ ID NO: 14. The base sequence represented by SEQ ID NO: 14 encodes the amino acid sequence represented by SEQ ID NO: 1.
カイコガ共受容体（以下、ＢｍＯＲＣＯという場合もある。）の発現プラスミド
　いずれか一方のＤＮＡ鎖に配列番号１７で示される塩基配列を有する二本鎖ＤＮＡを合成した。得られたＤＮＡ１００ｎｇを鋳型に用い、１０μＭのフォワードプライマーＢｍＯｒｃｏ－５’（5’-tggaattctgcagatcaccatgatgaccaaggtcaagacgcagggcct；配列番号１８）１μＬ、１０μＭのリバースプライマーＢｍＯｒｃｏ－３’（5’-gccactgtgctggatctacttcagttggatcaacaccatga；配列番号１９）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、２分間、（２）９８℃、１０秒間、（３）６３℃、３０秒間、（４）６８℃、１．５分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物を、ＥｃｏＲＶで消化されたｐｃＤＮＡ３．１（インビトロジェン社製）に、Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（ＴＡＫＡＲＡ社製）を使用して連結した。その後、これを大腸菌へ導入して培養することにより、ｐｃＤＮＡ３．１－ＢｍＯｒｃｏと名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ３．１－ＢｍＯｒｃｏの塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号１７で示される塩基配列を含有することが判った。配列番号１７で示される塩基配列は配列番号２０で示されるアミノ酸配列をコードする。 Expression plasmid of silkworm co-receptor (hereinafter sometimes referred to as BmORCO) Double-stranded DNA having a base sequence represented by SEQ ID NO: 17 was synthesized on either DNA strand. Using 100 ng of the obtained DNA as a template, 10 μM forward primer BmOrco-5 ′ (5′-tggaattctgcagatcaccatgatgaccaaggtcaagacgcagggcct; SEQ ID NO: 18) 1 μL, 10 μM reverse primer BmOrco-3 ′ (5′-gccactgtgctggatctactataccatgatggatctactataccatgatggatctacttaccatgatggatctacttaccat PCR was performed using 1 μL of Plus neo DNA polymerase (Toyobo). The PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles. The obtained PCR product was ligated to pcDNA3.1 (Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Thereafter, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-BmOrco. Analysis of the nucleotide sequence of the expression plasmid pcDNA3.1-BmOrco using a DNA sequencer revealed that it contained the nucleotide sequence represented by SEQ ID NO: 17. The base sequence represented by SEQ ID NO: 17 encodes the amino acid sequence represented by SEQ ID NO: 20.
＜嗅覚受容体の発現プラスミドの作製＞
シマカ嗅覚受容体ＯＲ８（以下、ＡａＯＲ８という場合もある。）の発現プラスミド
　上記方法により調製されたシマカ頭部由来のｃＤＮＡ１μＬを鋳型に用い、１０μＭのフォワードプライマーＡａＯＲ８－５’（5’-tggaattctgcagatcaccatgaacgacctggtgaagtttgagt；配列番号２１）１μＬ、１０μＭのリバースプライマーＡａＯＲ８－３’（5’-gccactgtgctggattcacttctgacttggttcatagatggt；配列番号２２）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、２分間、（２）９８℃、１０秒間、（３）６８℃、１分間で行い、（２）～（４）の工程は３０サイクル繰り返し行った。得られたＰＣＲ産物を、ＥｃｏＲＶで消化されたｐｃＤＮＡ３．１（インビトロジェン社製）にＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（ＴＡＫＡＲＡ社製）を使用して連結した。その後、これを大腸菌へ導入して培養することにより、ｐｃＤＮＡ３．１－ＡａＯＲ８と名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ３．１－ＡａＯＲ８の塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号２３で示される塩基配列を含有することが判った。配列番号２３で示される塩基配列は配列番号２４で示されるアミノ酸配列をコードする。 <Preparation of olfactory receptor expression plasmid>
An expression plasmid for the mosquito olfactory receptor OR8 (hereinafter sometimes referred to as AaOR8) 10 μM of the forward primer AaOR8-5 ′ (5′-tggaattctgcagatcaccatgaacgacctggtgaagtttgagt; sequence) No. 21) PCR was performed using 1 μL of 1 μL and 10 μM of reverse primer AaOR8-3 ′ (5′-gccactgtgctggattcacttctgacttggttcatagatggt; SEQ ID NO: 22) and 1 μL of KOD Plus neoDNA polymerase (manufactured by Toyobo). The PCR reaction was performed (1) 94 ° C. for 2 minutes, (2) 98 ° C. for 10 seconds, (3) 68 ° C. for 1 minute, and steps (2) to (4) were repeated 30 cycles. The obtained PCR product was ligated to pcDNA3.1 (manufactured by Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Thereafter, this was introduced into Escherichia coli and cultured to obtain an expression plasmid named pcDNA3.1-AaOR8. Analysis of the nucleotide sequence of expression plasmid pcDNA3.1-AaOR8 using a DNA sequencer revealed that it contained the nucleotide sequence represented by SEQ ID NO: 23. The base sequence represented by SEQ ID NO: 23 encodes the amino acid sequence represented by SEQ ID NO: 24.
シマカ嗅覚受容体ＯＲ１０（以下、ＡａＯＲ１０という場合もある。）の発現プラスミド
　上記方法により調製されたシマカ頭部由来のｃＤＮＡ１μＬを鋳型に用い、１０μＭのフォワードプライマーＡａＯＲ１０－５’（5’-tggaattctgcagatcaccatggcaagcattcttgattgcccg；配列番号２５）１μＬ、１０μＭのリバースプライマーＡａＯＲ１０－３’（5’-gccactgtgctggatttaattataaacccgacgcagcag；配列番号２６）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、２分間、（２）９８℃、１０秒間、（３）６３℃、３０秒間、（４）６８℃、１．５分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物を、ＥｃｏＲＶで消化されたｐｃＤＮＡ３．１（インビトロジェン社製）にＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（ＴＡＫＡＲＡ社製）を使用して連結した。その後、これを大腸菌へ導入して培養することにより、ｐｃＤＮＡ３．１－ＡａＯＲ１０と名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ３．１－ＡａＯＲ１０の塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号２７で示される塩基配列を含有することが判った。配列番号２７で示される塩基配列は配列番号２８で示されるアミノ酸配列をコードする。 An expression plasmid for the mosquito olfactory receptor OR10 (hereinafter sometimes referred to as AaOR10) 10 μM of the forward primer AaOR10-5 ′ (5′-tggaattctgcagatcaccatggcaagcattcttgattgcccg; sequence) No. 25) PCR was performed using 1 μL of 1 μL, 10 μM of reverse primer AaOR10-3 ′ (5′-gccactgtgctggatttaattataaacccgacgcagcag; SEQ ID NO: 26) and 1 μL of KOD Plus neoDNA polymerase (manufactured by Toyobo). The PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles. The obtained PCR product was ligated to pcDNA3.1 (manufactured by Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Thereafter, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-AaOR10. Analysis of the base sequence of expression plasmid pcDNA3.1-AaOR10 using a DNA sequencer revealed that it contained the base sequence represented by SEQ ID NO: 27. The base sequence represented by SEQ ID NO: 27 encodes the amino acid sequence represented by SEQ ID NO: 28.
ハマダラカ嗅覚受容体ＯＲ１０（以下、ＡｇＯＲ１０という場合もある。）の発現プラスミド
　上記方法により調製されたハマダラカ頭部由来のｃＤＮＡ１μＬを鋳型に用い、１０μＭのフォワードプライマーＡｇＯＲ１０－５’（5’-caccatggaggtcctcaactgtccgctact；配列番号２９）１μＬ、１０μＭのリバースプライマーＡｇＯＲ１０－３’（5’-ttaattgtaaactcttctcagaagcgtaa；配列番号３０）１μＬ及びＫＯＤ　ＰｌｕｓＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、５分間、（２）９８℃、１０秒間、（３）６０℃、３０秒間、（４）６８℃、１．５分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物をアガロースゲル電気泳動した後、当該ゲル上で検出された約１．１ｋｂのＤＮＡを回収した。回収されたＤＮＡをｐＥＮＴＲ／Ｄ－ＴＯＰＯベクター（インビトロジェン社製）へ導入し、ｐＥＮＴＲ－ＡｇＯＲ１０と名付けたプラスミドを得た。４μＬのｐＥＮＴＲ－ＡｇＯＲ１０、１μＬのｐｃＤＮＡ６．２Ｖ５－ＤＥＳＴ及び２μＬのＬＲクロナーゼＩＩ（インビトロジェン社製）を混合して室温で１時間保持した。得られた混合物を大腸菌へ導入し培養することにより、ｐｃＤＮＡ６．２－ＡｇＯＲ１０と名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ６．２－ＡｇＯＲ１０の塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号３１で示される塩基配列を含有することが判った。配列番号３１で示される塩基配列は配列番号３２で示されるアミノ酸配列をコードする。 Anopheles olfactory receptor OR10 (hereinafter, sometimes referred to as AgOR10) expression plasmid 10 μM of forward primer AgOR10-5 ′ (5′-caccatggaggtcctcaactgtccgctact; sequence) No. 29) PCR was performed using 1 μL of 1 μL, 10 μM reverse primer AgOR10-3 ′ (5′-ttaattgtaaactcttctcagaagcgtaa; SEQ ID NO: 30) and 1 μL of KOD Plus DNA polymerase (manufactured by Toyobo). The PCR reaction was carried out at (1) 94 ° C for 5 minutes, (2) 98 ° C for 10 seconds, (3) 60 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles. The obtained PCR product was subjected to agarose gel electrophoresis, and then about 1.1 kb of DNA detected on the gel was recovered. The recovered DNA was introduced into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-AgOR10. 4 μL of pENTR-AgOR10, 1 μL of pcDNA6.2V5-DEST and 2 μL of LR clonase II (Invitrogen) were mixed and kept at room temperature for 1 hour. The resulting mixture was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA6.2-AgOR10. When the nucleotide sequence of the expression plasmid pcDNA6.2-AgOR10 was analyzed by a DNA sequencer, it was found to contain the nucleotide sequence represented by SEQ ID NO: 31. The base sequence represented by SEQ ID NO: 31 encodes the amino acid sequence represented by SEQ ID NO: 32.
ハマダラカ嗅覚受容体ＯＲ２８（以下、ＡｇＯＲ２８という場合もある。）の発現プラスミド
　上記方法により調製されたハマダラカ頭部由来のｃＤＮＡ１μＬを鋳型に用い、
１０μＭのフォワードプライマーＡｇＯＲ２８－５’（5’-caccatggcccgtttggtactgcacgaggt；配列番号３３）１μＬ、１０μＭのリバースプライマーＡｇＯＲ２８－３’（5’-ttattgctgattgatggtttgcagtaagg；配列番号３４）１μＬ及びＫＯＤ　ＰｌｕｓＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、５分間、（２）９８℃、１０秒間、（３）６０℃、３０秒間、（４）６８℃、１．５分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物をアガロースゲル電気泳動した後、当該ゲル上で検出された約１．３ｋｂのＤＮＡを回収した。回収されたＤＮＡをｐＥＮＴＲ／Ｄ－ＴＯＰＯベクター（インビトロジェン社製）へ導入し、ｐＥＮＴＲ－ＡｇＯＲ２８と名付けたプラスミドを得た。４μＬのｐＥＮＴＲ－ＡｇＯＲ１０、１μＬのｐｃＤＮＡ６．２Ｖ５－ＤＥＳＴ及び２μＬのＬＲクロナーゼＩＩ（インビトロジェン社製）を混合して室温で１時間保持した。得られた混合物を大腸菌へ導入し培養することにより、ｐｃＤＮＡ６．２－ＡｇＯＲ２８と名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ６．２－ＡｇＯＲ２８の塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号３５で示される塩基配列を含有することが判った。配列番号３５で示される塩基配列は配列番号３６で示されるアミノ酸配列をコードする。 Anopheles olfactory receptor OR28 (hereinafter, sometimes referred to as AgOR28) expression plasmid 1 μL of an anopheles head-derived cDNA prepared by the above method was used as a template.
10 μM forward primer AgOR28-5 ′ (5′-caccatggcccgtttggtactgcacgaggt; SEQ ID NO: 33) 1 μL, 10 μM reverse primer AgOR28-3 ′ (5′-ttattgctgattgatggtttgcagtaagg; SEQ ID NO: 34) 1 μL and KOD Plus DNA polymerase (1 made by Toyobo) PCR was performed. The PCR reaction was carried out at (1) 94 ° C for 5 minutes, (2) 98 ° C for 10 seconds, (3) 60 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles. The obtained PCR product was subjected to agarose gel electrophoresis, and then about 1.3 kb of DNA detected on the gel was recovered. The recovered DNA was introduced into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-AgOR28. 4 μL of pENTR-AgOR10, 1 μL of pcDNA6.2V5-DEST and 2 μL of LR clonase II (Invitrogen) were mixed and kept at room temperature for 1 hour. The resulting mixture was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA6.2-AgOR28. Analysis of the base sequence of expression plasmid pcDNA6.2-AgOR28 using a DNA sequencer revealed that it contained the base sequence represented by SEQ ID NO: 35. The base sequence represented by SEQ ID NO: 35 encodes the amino acid sequence represented by SEQ ID NO: 36.
カイコガ嗅覚受容体ＯＲ５６（以下、ＢｍＯＲ５６という場合もある。）の発現プラスミド
　いずれか一方のＤＮＡ鎖に配列番号３７で示される塩基配列を有する二本鎖ＤＮＡを合成した。配列番号３７で示される塩基配列を有する二本鎖ＤＮＡ１００ｎｇを鋳型に用いて、１０μＭのフォワードプライマーＢｍＯＲ５６－５’（5’-tggaattctgcagatcaccatgaagctcctggagaagctag；配列番号３８）１μＬ、１０μＭのリバースプライマーＢｍＯＲ５６－３’（5’-gccactgtgctggattcatgttttattcatttgcgactgac；配列番号３９）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、２分間、（２）９８℃、１０秒間、（３）６３℃、３０秒間、（４）６８℃、１．５分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物を、ＥｃｏＲＶで消化されたｐｃＤＮＡ３．１（インビトロジェン社製）に、Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（ＴＡＫＡＲＡ社製）を使用して連結した。その後、これを大腸菌へ導入して培養することにより、ｐｃＤＮＡ３．１－ＢｍＯＲ５６と名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ３．１－ＢｍＯＲ５６の塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号３７で示される塩基配列を含有することが判った。配列番号３７で示される塩基配列は配列番号４０で示されるアミノ酸配列をコードする。 Expression plasmid of the silkworm olfactory receptor OR56 (hereinafter sometimes referred to as BmOR56) A double-stranded DNA having the base sequence represented by SEQ ID NO: 37 was synthesized in one of the DNA strands. 10 μM of forward primer BmOR56-5 ′ (5′-tggaattctgcagatcaccatgaagctcctggagaagctag; SEQ ID NO: 38) 1 μL, 10 μM of reverse primer BmOR56-3 ′ (5 PCR was performed using 1 μL of '-gccactgtgctggattcatgttttattcatttgcgactgac; SEQ ID NO: 39) and 1 μL of KOD Plus neo DNA polymerase (Toyobo). The PCR reaction was carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 63 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4 ) Was repeated 35 cycles. The obtained PCR product was ligated to pcDNA3.1 (Invitrogen) digested with EcoRV using In-Fusion HD Cloning Kit (manufactured by TAKARA). Thereafter, this was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA3.1-BmOR56. Analysis of the base sequence of expression plasmid pcDNA3.1-BmOR56 using a DNA sequencer revealed that it contained the base sequence represented by SEQ ID NO: 37. The base sequence represented by SEQ ID NO: 37 encodes the amino acid sequence represented by SEQ ID NO: 40.
ショウジョウバエ嗅覚受容体ＯＲ４７ａ（以下、ＤｍＯＲ４７ａという場合もある。）の発現プラスミド
　上記方法により調製されたショウジョウバエ由来のｃＤＮＡ１μＬを鋳型に用い、１０μＭのフォワードプライマーＤｍＯＲ４７ａ－５’（5’-caccatggacagttttctgcaagtacagaaga；配列番号４１）１μＬ、１０μＭのリバースプライマーＤｍＯＲ４７ａ－３’（5’-ttaggagaatgatctcagcattgtgatgta；配列番号４２）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏ　ＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９４℃、２分間、（２）９８℃、１０秒間、（３）６８℃、１．５分間で行い、（２）～（３）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物をアガロースゲル電気泳動した後、当該ゲル上で検出された約１．５ｋｂのＤＮＡを回収した。回収されたＤＮＡをｐＥＮＴＲ／Ｄ－ＴＯＰＯベクター（インビトロジェン社製）へクローニングし、ｐＥＮＴＲ－ＤｍＯＲ４７ａと名付けたプラスミドを得た。４μＬのｐＥＮＴＲ－ＤｍＯＲ４７ａ、１μＬのｐｃＤＮＡ６．２Ｖ５－ＤＥＳＴ及び２μＬのＬＲクロナーゼＩＩ（インビトロジェン社製）を混合して室温で１時間保持した。得られた混合物を大腸菌へ導入し培養することにより、ｐｃＤＮＡ６．２－ＤｍＯＲ４７ａと名付けた発現プラスミドを得た。発現プラスミドｐｃＤＮＡ６．２－ＤｍＯＲ４７ａの塩基配列をＤＮＡシーケンサーにより解析したところ、配列番号４３で示される塩基配列を含有することが判った。配列番号４３で示される塩基配列は配列番号４４で示されるアミノ酸配列をコードする。 Expression plasmid of Drosophila olfactory receptor OR47a (hereinafter sometimes referred to as DmOR47a) Using 1 μL of Drosophila-derived cDNA prepared by the above method as a template, 10 μM forward primer DmOR47a-5 ′ (5′-caccatggacagttttctgcaagtacagaaga; SEQ ID NO: 41 ) PCR was performed using 1 μL of 1 μL and 10 μM reverse primer DmOR47a-3 ′ (5′-ttaggagaatgatctcagcattgtgatgta; SEQ ID NO: 42) and 1 μL of KOD Plus neo DNA polymerase (Toyobo). PCR reaction was performed at (1) 94 ° C. for 2 minutes, (2) 98 ° C. for 10 seconds, (3) 68 ° C. for 1.5 minutes, and steps (2) to (3) were repeated 35 cycles. . The obtained PCR product was subjected to agarose gel electrophoresis, and then about 1.5 kb of DNA detected on the gel was recovered. The recovered DNA was cloned into a pENTR / D-TOPO vector (Invitrogen) to obtain a plasmid named pENTR-DmOR47a. 4 μL of pENTR-DmOR47a, 1 μL of pcDNA6.2V5-DEST and 2 μL of LR clonase II (Invitrogen) were mixed and kept at room temperature for 1 hour. The resulting mixture was introduced into E. coli and cultured to obtain an expression plasmid named pcDNA6.2-DmOR47a. The nucleotide sequence of the expression plasmid pcDNA6.2-DmOR47a was analyzed by a DNA sequencer and found to contain the nucleotide sequence represented by SEQ ID NO: 43. The base sequence represented by SEQ ID NO: 43 encodes the amino acid sequence represented by SEQ ID NO: 44.
＜イクオリン（Ａｅｑｕｏｒｉｎ）発現プラスミドの作製＞
　Ａｅｑｕｏｒｉｎをコードする配列番号４５で示される塩基配列がｐＭＤ１９ベクターにクローニングされたプラスミド（ｐＭＤ１９－ＡＥＱ）５０ｎｇを鋳型に用い、１０μＭのフォワードプライマーＡＥＱ－５’（5’-caccatgacaagcaaacaatactc；配列番号４６）１μＬ、１０μＭのリバースプライマーＡＥＱ－３’（5’-ttaggggacagctccaccgtag；配列番号４７）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９５℃、３分間、（２）９５℃、３０秒間、（３）６０℃、３０秒間、（４）６８℃、１分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＰＣＲ産物をアガロースゲル電気泳動した後、当該ゲル上で検出された約６００ｂｐのＤＮＡを回収した。回収されたＤＮＡをｐＥＮＴＲ／Ｄ－ＴＯＰＯベクター（インビトロジェン社製）へ導入し、ｐＥＮＴＲ－ＡＥＱと名付けたプラスミドを得た。配列番号４５で示される塩基配列は配列番号４８で示されるアミノ酸配列をコードする。４μＬのｐＥＮＴＲ－ＡＥＱ、１μＬのｐｃＤＮＡ６．２Ｖ５－ＤＥＳＴ及び２μＬのＬＲクロナーゼＩＩ（インビトロジェン社製）を混合し室温で１時間保持した。得られた混合物を大腸菌へ導入し増幅することにより、ｐｃＤＮＡ６．２－ＡＥＱと名付けたプラスミドを得た。プラスミドｐｃＤＮＡ６．２－ＡＥＱ１００ｎｇを鋳型に用い、１０μＭのフォワードプライマーＩｎ－Ｆｕｓｉｏｎ　ＡＥＱ－５’（5’-gtggcggccgctcgaggccaccatgacaagcaaacaatactc；配列番号４９）１μＬ、１０μＭのリバースプライマーＩｎ－Ｆｕｓｉｏｎ　ＡＥＱ－３’（5’-gccctctagactcgagttaggggacagctccaccgtag；配列番号５０）１μＬ及びＫＯＤ　ｎｅｏ　Ｐｌｕｓ　ＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９５℃、３分間、（２）９５℃、３０秒間、（３）６０℃、３０秒間、（４）６８℃、１分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＤＮＡをアガロースゲル電気泳動した後、当該ゲル上で検出された約６００ｂｐのＤＮＡを回収してこれをインサートＤＮＡ１とした。ｐｃＤＮＡ３．１（インビトロジェン社製）をＸｈｏＩで消化したものをベクターとして用い、これに上記インサートＤＮＡ１をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（ＴＡＫＡＲＡ社製）を用いて連結した。その後、これを大腸菌へ導入し培養することによりｐｃＤＮＡ３．１－ＡＥＱと名付けたプラスミドを得た。ｐｃＤＮＡ６．２－ＡＥＱ５０ｎｇを鋳型に用い、１０μＭのフォワードプライマーＩｎ－Ｆｕｓｉｏｎ　ＡＥＱ－５’（5’-gtggcggccgctcgaggccaccatgacaagcaaacaatactc；配列番号５１）１μＬ、１０μＭのリバースプライマーＩｎ－Ｆｕｓｉｏｎ　ＡＥＱ－３’（5’-gccctctagactcgagttaggggacagctccaccgtag；配列番号５２）１μＬ及びＫＯＤ　Ｐｌｕｓ　ｎｅｏ　ＤＮＡポリメラーゼ（東洋紡製）１μＬを用いてＰＣＲを行った。ＰＣＲ反応は、（１）９５℃、３分間、（２）９５℃、３０秒間、（３）６０℃、３０秒間、（４）６８℃、１分間で行い、（２）～（４）の工程は３５サイクル繰り返し行った。得られたＤＮＡをアガロースゲル電気泳動した後、当該ゲル上で検出された約６００ｂｐのＤＮＡを回収してこれをインサートＤＮＡ２とした。ｐｃＤＮＡ３．１（ｈｙｇｒｏ）（インビトロジェン社製）をＸｈｏＩで消化したものをベクターとして用い、これに上記インサートＤＮＡ２をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（ＴＡＫＡＲＡ社製）を用いて連結した後、大腸菌へ導入し培養することによりｐｃＤＮＡ３．１（ｈｙｇｒｏ）－ＡＥＱと名付けた発現プラスミドを得た。 <Preparation of Aequorin Expression Plasmid>
Using 50 ng of a plasmid (pMD19-AEQ) in which the nucleotide sequence represented by SEQ ID NO: 45 encoding Aequorin was cloned in the pMD19 vector as a template, 10 μM forward primer AEQ-5 ′ (5′-caccatgacaagcaaacaatactc; SEQ ID NO: 46) 1 μL PCR was performed using 1 μL of 10 μM reverse primer AEQ-3 ′ (5′-ttaggggacagctccaccgtag; SEQ ID NO: 47) and 1 μL of KOD Plus neo DNA polymerase (Toyobo). The PCR reaction was performed at (1) 95 ° C. for 3 minutes, (2) 95 ° C. for 30 seconds, (3) 60 ° C. for 30 seconds, (4) 68 ° C. for 1 minute, and (2) to (4) The process was repeated 35 cycles. The obtained PCR product was subjected to agarose gel electrophoresis, and then about 600 bp of DNA detected on the gel was recovered. The recovered DNA was introduced into a pENTR / D-TOPO vector (manufactured by Invitrogen) to obtain a plasmid named pENTR-AEQ. The base sequence represented by SEQ ID NO: 45 encodes the amino acid sequence represented by SEQ ID NO: 48. 4 μL of pENTR-AEQ, 1 μL of pcDNA6.2V5-DEST and 2 μL of LR clonase II (Invitrogen) were mixed and kept at room temperature for 1 hour. The resulting mixture was introduced into E. coli and amplified to obtain a plasmid named pcDNA6.2-AEQ. 10 μM forward primer In-Fusion AEQ-5 ′ (5′-gtggcggccgctcgaggccaccatgacaagcaaacaatactc; SEQ ID NO: 49) 1 μL, 10 μM reverse primer In-Fusion AEQ-3 ′ (5′-gccctaccgtaggatagga) ; SEQ ID NO: 50) PCR was carried out using 1 μL and 1 μL of KOD neo Plus DNA polymerase (manufactured by Toyobo). The PCR reaction was performed at (1) 95 ° C. for 3 minutes, (2) 95 ° C. for 30 seconds, (3) 60 ° C. for 30 seconds, (4) 68 ° C. for 1 minute, and (2) to (4) The process was repeated 35 cycles. After the obtained DNA was subjected to agarose gel electrophoresis, about 600 bp of DNA detected on the gel was recovered and used as insert DNA1. A cDNA obtained by digesting pcDNA3.1 (manufactured by Invitrogen) with XhoI was used as a vector, and the insert DNA1 was ligated thereto using In-Fusion HD Cloning Kit (manufactured by TAKARA). Thereafter, this was introduced into E. coli and cultured to obtain a plasmid named pcDNA3.1-AEQ. pcDNA6.2-AEQ 50 ng as a template, 10 μM forward primer In-Fusion AEQ-5 ′ (5′-gtggcggccgctcgaggccaccatgacaagcaaacaatactc; SEQ ID NO: 51) 1 μL, 10 μM reverse primer In-Fusion AEQ-3 ′ (5′-gccctcggtagtaggaga SEQ ID NO: 52) PCR was performed using 1 μL and 1 μL of KOD Plus neo DNA polymerase (manufactured by Toyobo). The PCR reaction was performed at (1) 95 ° C. for 3 minutes, (2) 95 ° C. for 30 seconds, (3) 60 ° C. for 30 seconds, (4) 68 ° C. for 1 minute, and (2) to (4) The process was repeated 35 cycles. After the obtained DNA was subjected to agarose gel electrophoresis, about 600 bp DNA detected on the gel was recovered and used as insert DNA2. A pcDNA3.1 (hygro) (manufactured by Invitrogen) digested with XhoI was used as a vector, and the insert DNA2 was ligated thereto using In-Fusion HD Cloning Kit (manufactured by TAKARA), and then introduced into Escherichia coli. By culturing, an expression plasmid named pcDNA3.1 (hygro) -AEQ was obtained.
＜発現プラスミドの細胞への導入＞
　ＨＥＫ２９３ＦＴ細胞（Ｉｎｖｉｔｒｏｇｅｎ社から購入）を１０ｃｍシャーレに３×１０６ｃｅｌｌｓ／シャーレで播種し、１０％ＦＢＳを含むＤＭＥＭ培地（ナカライテスク社製）中で、３７℃、５％ＣＯ２条件下にて約２４時間培養した。作製したいずれかの共受容体発現プラスミド３μｇと、作製したいずれかの嗅覚受容体発現プラスミド１．５μｇと、Ａｅｑｕｏｒｉｎ発現プラスミド８μｇとを、１２．５μＬのＰｌｕｓ試薬及び３１．２５μＬのリポフェクトアミンＬＴＸ（インビトロジェン社製）と混合し、３０分間保持した。その後、この混合液を上記細胞にトランスフェクションした。トランスフェクション開始４時間後に９６ｗｅｌｌプレートに９×１０４ｃｅｌｌｓ／ｗｅｌｌで播種し、１０％ＦＢＳを含むＤＭＥＭ培地（ナカライテスク社製）中で、３７℃、５％ＣＯ２条件下にて約２４時間培養した。これにより、共受容体発現プラスミド、嗅覚受容体発現プラスミド及びＡｅｑｕｏｒｉｎ発現プラスミドが導入されたトランジェントな形質転換細胞を得た。 <Introduction of expression plasmid into cells>
HEK293FT cells (purchased from Invitrogen) were seeded in a 10 cm petri dish at 3 × 10 6 cells / petri dish, and in DMEM medium (manufactured by Nacalai Tesque) containing 10% FBS under conditions of 37 ° C. and 5% CO 2 . Cultured for about 24 hours. 3 μg of any of the prepared co-receptor expression plasmids, 1.5 μg of any of the prepared olfactory receptor expression plasmids, and 8 μg of the Aequorin expression plasmid, 12.5 μL of Plus reagent and 31.25 μL of lipofectamine LTX (Invitrogen) and mixed for 30 minutes. Thereafter, this mixed solution was transfected into the cells. 4 hours after the start of transfection, the cells were seeded on a 96-well plate at 9 × 10 4 cells / well, and were cultured in DMEM medium (manufactured by Nacalai Tesque) containing 10% FBS for about 24 hours at 37 ° C. under 5% CO 2 Cultured. Thereby, a transient transformed cell into which the co-receptor expression plasmid, the olfactory receptor expression plasmid, and the Aequorin expression plasmid were introduced was obtained.
＜活性測定＞
　イクオリンはカルシウムイオンと結合すると活性化されてセレンテラジン等の基質を酸化して発光する。したがって、イクオリンを発現する細胞の細胞内カルシウムイオン濃度の上昇は直接、発光量の増大として現れることになるため、被検物質の添加により嗅覚受容体複合体がイオンチャネルとして機能しているかどうかを発光量の変化から測定することができる。
　上記形質転換細胞の培養液を除去してＡｓｓａｙ　ｂｕｆｆｅｒ（０．５μＭセレンテラジンｈ（プロメガ社製）及び０．３％のＢＳＡを含むＨａｎｋｓ－ＨＥＰＥＳ（２０　ｍＭ　ｐＨ　７．４））に置換し、更に４時間、室温で静置した。次いで、Ｆｌｅｘｓｔａｔｉｏｎ３（Ｍｏｌｅｃｕｌａｒ　ｄｅｖｉｃｅｓ社製）を用いて、嗅覚受容体に対応する被検物質を細胞の培養液中に添加するとともに細胞の発光量を測定した。対照物質としてジメチルスルホキシドを添加した細胞での発光量を１とし、被検物質を添加した細胞の発光量を相対値として算出した。 <Activity measurement>
Aequorin is activated when bound to calcium ions, and oxidizes a substrate such as coelenterazine to emit light. Therefore, since the increase in intracellular calcium ion concentration in cells expressing aequorin directly appears as an increase in luminescence, whether or not the olfactory receptor complex functions as an ion channel by adding a test substance It can be measured from the change in the amount of luminescence.
The culture solution of the transformed cells was removed and replaced with Assay buffer (Hanks-HEPES (20 mM pH 7.4) containing 0.5 μM coelenterazine h (Promega) and 0.3% BSA), and The mixture was allowed to stand at room temperature for 4 hours. Next, using Flexstation 3 (Molecular devices), a test substance corresponding to the olfactory receptor was added to the cell culture medium, and the amount of luminescence of the cells was measured. The amount of luminescence in cells to which dimethyl sulfoxide was added as a control substance was taken as 1, and the amount of luminescence in cells to which a test substance was added was calculated as a relative value.
（実施例１）
　嗅覚受容体ＡａＯＲ８の発現プラスミド、
共受容体ＡａＯＲＣＯ又はＡｍＯＲＣＯのいずれかの発現プラスミド、及び
Ａｅｑｕｏｒｉｎ発現プラスミド
を有する細胞を用いて、上述のように発光量を測定した。被検物質としては１－オクテン－３－オールを用い、１０－４Ｍ、１０－５Ｍ、１０－６Ｍ、１０－７Ｍ、１０－８Ｍ、又は１０－９Ｍの濃度で培養液に添加した。その結果を図１に示す。シマカのＡａＯＲ８とＡａＯＲＣＯとの複合体を発現する細胞よりも、シマカＡａＯＲ８とミツバチＡｍＯＲＣＯとの複合体を発現する細胞の方が、１－オクテン－３－オールに対して、強く応答することが示された。 (Example 1)
Expression plasmid of olfactory receptor AaOR8,
The amount of luminescence was measured as described above using cells having either the co-receptor AaORCO or AmORCO expression plasmid and the Aequorin expression plasmid. 1-octen-3-ol was used as the test substance, and the culture solution was at a concentration of 10 −4 M, 10 −5 M, 10 −6 M, 10 −7 M, 10 −8 M, or 10 −9 M. Added to. The result is shown in FIG. It is shown that cells expressing the complex of the mosquito AaOR8 and the bee AmORCO respond more strongly to 1-octen-3-ol than cells expressing the complex of the mosquito AaOR8 and AaORCO. It was done.
（実施例２）
　嗅覚受容体ＡａＯＲ１０の発現プラスミド、
共受容体ＡａＯＲＣＯ又はＡｍＯＲＣＯのいずれかの発現プラスミド、及び
Ａｅｑｕｏｒｉｎ発現プラスミド
を有する細胞を用いて、上述のように発光量を測定した。被検物質としてはベンズアルデヒドを用い、１０－３Ｍ、１０－４Ｍ、１０－５Ｍ、１０－６Ｍ、又は１０－７Ｍの濃度で培養液に添加した。その結果を図２に示す。シマカのＡａＯＲ１０とＡａＯＲＣＯとの複合体を発現する細胞よりも、シマカＡａＯＲ１０とミツバチＡｍＯＲＣＯとの複合体を発現する細胞の方が、ベンズアルデヒドに対して、強く応答することが示された。 (Example 2)
Expression plasmid of olfactory receptor AaOR10,
The amount of luminescence was measured as described above using cells having either the co-receptor AaORCO or AmORCO expression plasmid and the Aequorin expression plasmid. Benzaldehyde was used as a test substance and added to the culture solution at a concentration of 10 −3 M, 10 −4 M, 10 −5 M, 10 −6 M, or 10 −7 M. The result is shown in FIG. It has been shown that cells expressing the complex of the mosquito AaOR10 and the bee AmORCO respond more strongly to benzaldehyde than cells expressing the complex of the mosquito AaOR10 and AaORCO.
（実施例３）
　嗅覚受容体ＡｇＯＲ１０の発現プラスミド、
共受容体ＡｇＯＲＣＯ又はＡｍＯＲＣＯのいずれかの発現プラスミド、及び
Ａｅｑｕｏｒｉｎ発現プラスミド
を有する細胞を用いて、上述のように発光量を測定した。被検物質としてはベンズアルデヒドを用い、１０－３Ｍ、１０－４Ｍ、１０－５Ｍ、１０－６Ｍ、又は１０－７Ｍの濃度で培養液に添加した。その結果を図３に示す。ハマダラカのＡｇＯＲ１０とＡｇＯＲＣＯとの複合体を発現する細胞よりも、ハマダラカＡｇＯＲ１０とミツバチＡｍＯＲＣＯとの複合体を発現する細胞の方が、ベンズアルデヒドに対して、強く応答することが示された。 (Example 3)
Expression plasmid of olfactory receptor AgOR10,
The amount of luminescence was measured as described above using cells containing either the co-receptor AgORCO or AmORCO expression plasmid and the Aequorin expression plasmid. Benzaldehyde was used as a test substance and added to the culture solution at a concentration of 10 −3 M, 10 −4 M, 10 −5 M, 10 −6 M, or 10 −7 M. The result is shown in FIG. It was shown that cells expressing the complex of Anopheles AgOR10 and honey bee AmORCO respond more strongly to benzaldehyde than cells expressing the complex of Anopheles AgOR10 and AgORCO.
（実施例４）
　嗅覚受容体ＡｇＯＲ２８の発現プラスミド、
共受容体ＡｇＯＲＣＯ又はＡｍＯＲＣＯのいずれかの発現プラスミド、及び
Ａｅｑｕｏｒｉｎ発現プラスミド
を有する細胞を用いて、上述のように発光量を測定した。被検物質としては２，４，５－トリメチルチアゾールを用い、１０－３Ｍ、１０－４Ｍ、１０－５Ｍ、１０－６Ｍ、１０－７Ｍ、又は１０－８Ｍの濃度で培養液に添加した。その結果を図４に示す。ハマダラカのＡｇＯＲ２８とＡｇＯＲＣＯとの複合体を発現する細胞よりも、ハマダラカＡｇＯＲ２８とミツバチＡｍＯＲＣＯとの複合体を発現する細胞の方が、２，４，５－トリメチルチアゾールに対して、強く応答することが示された。 Example 4
Expression plasmid of olfactory receptor AgOR28,
The amount of luminescence was measured as described above using cells containing either the co-receptor AgORCO or AmORCO expression plasmid and the Aequorin expression plasmid. 2,4,5-Trimethylthiazole is used as a test substance and cultured at a concentration of 10 −3 M, 10 −4 M, 10 −5 M, 10 −6 M, 10 −7 M, or 10 −8 M. Added to the liquid. The result is shown in FIG. Cells expressing the complex of Anopheles AgOR28 and honey bee AmORCO may respond more strongly to 2,4,5-trimethylthiazole than cells expressing the complex of Anopheles AgOR28 and AgORCO Indicated.
（実施例５）
　嗅覚受容体ＢｍＯＲ５６の発現プラスミド、
共受容体ＢｍＯＲＣＯ又はＡｍＯＲＣＯのいずれかの発現プラスミド、及び
Ａｅｑｕｏｒｉｎ発現プラスミド
を有する細胞を用いて、上述のように発光量を測定した。被検物質としてはｃｉｓ－ジャスモンを用い、１０－６Ｍ、１０－７Ｍ、１０－８Ｍ、１０－９Ｍ又は１０－１０Ｍの濃度で培養液に添加した。その結果を図５に示す。カイコガのＢｍＯＲ５６とＢｍＯＲＣＯとの複合体を発現する細胞よりも、カイコガＢｍＯＲ５６とミツバチＡｍＯＲＣＯとの複合体を発現する細胞の方が、ｃｉｓ－ジャスモンに対して、強く応答することが示された。 (Example 5)
Expression plasmid for olfactory receptor BmOR56,
The amount of luminescence was measured as described above using cells containing either the co-receptor BmORCO or AmORCO expression plasmid and the Aequorin expression plasmid. As a test substance, cis-jasmon was used and added to the culture solution at a concentration of 10 −6 M, 10 −7 M, 10 −8 M, 10 −9 M or 10 −10 M. The result is shown in FIG. It was shown that cells expressing the complex of silkworm BmOR56 and honey bee AmORCO responded more strongly to cis-jasmon than cells expressing the complex of silkworm BmOR56 and BmORCO.
（実施例６）
　嗅覚受容体ＤｍＯＲ４７ａの発現プラスミド、
共受容体ＤｍＯＲＣＯ又はＡｍＯＲＣＯのいずれかの発現プラスミド、及び
Ａｅｑｕｏｒｉｎ発現プラスミド
を有する細胞を用いて、上述のように発光量を測定した。被検物質としてはペンチルアセテートを用い、１０－４Ｍ、５×１０－４Ｍ、１０－５Ｍ、５×１０－５Ｍ、１０－６Ｍ、５×１０－６Ｍ、１０－７Ｍ、又は５×１０－７Ｍの濃度で培養液に添加した。その結果を図６に示す。ショウジョウバエのＤｍＯＲ４７ａとＤｍＯＲＣＯとの複合体を発現する細胞よりも、ショウジョウバエＤｍＯＲ４７ａとミツバチＡｍＯＲＣＯとの複合体を発現する細胞の方が、ペンチルアセテートに対して、強く応答することが示された。 (Example 6)
Expression plasmid of olfactory receptor DmOR47a,
The amount of luminescence was measured as described above using cells containing either the co-receptor DmORCO or AmORCO expression plasmid and the Aequorin expression plasmid. As a test substance, pentyl acetate is used. 10 −4 M, 5 × 10 −4 M, 10 −5 M, 5 × 10 −5 M, 10 −6 M, 5 × 10 −6 M, 10 −7 M Or at a concentration of 5 × 10 −7 M. The result is shown in FIG. It was shown that cells expressing the Drosophila DmOR47a and honey bee AmORCO complex responded more strongly to pentyl acetate than cells expressing the Drosophila DmOR47a and DmORCO complex.

Claims (6)

  1. 　ミツバチ科に属さない昆虫の嗅覚受容体と、ミツバチ科昆虫の嗅覚受容体共受容体とから形成される、嗅覚受容体複合体。 An olfactory receptor complex formed from an olfactory receptor of an insect that does not belong to the honeybee family and an olfactory receptor co-receptor of an honeybee insect.
  2. 　前記嗅覚受容体が、カ科昆虫、カイコガ科昆虫、及びショウジョウバエ科昆虫からなる群から選択される昆虫の嗅覚受容体である、請求項１に記載の嗅覚受容体複合体。 The olfactory receptor complex according to claim 1, wherein the olfactory receptor is an olfactory receptor of an insect selected from the group consisting of mosquito insects, Bombycidae insects, and Drosophila insects.
  3. 　前記嗅覚受容体が、１－オクテン－３－オール、ベンズアルデヒド、２，４，５－トリメチルチアゾール、ｃｉｓ－ジャスモン、及びペンチルアセテートからなる群から選択される化合物をリガンドとする嗅覚受容体である、請求項１又は２に記載の嗅覚受容体複合体。 The olfactory receptor is an olfactory receptor having a compound selected from the group consisting of 1-octen-3-ol, benzaldehyde, 2,4,5-trimethylthiazole, cis-jasmon, and pentyl acetate as a ligand. The olfactory receptor complex according to claim 1 or 2.
  4. 　前記嗅覚受容体が、ネッタイシマカ（Ａｅｄｅｓ　ａｅｇｙｐｔｉ）のＯＲ８若しくはＯＲ１０、ガンビエハマダラカ（Ａｎｏｐｈｅｌｅｓ　ｇａｍｂｉａｅ）のＯＲ１０若しくはＯＲ２８、カイコガ（Ｂｏｍｂｙｘ　ｍｏｒｉ）のＯＲ５６、又はキイロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ）のＯＲ４７ａ、或いは、これらのうちいずれかの嗅覚受容体全体のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を有する嗅覚受容体であり、
    　前記嗅覚受容体共受容体が、セイヨウミツバチ（Ａｐｉｓ　ｍｅｌｌｉｆｅｒａ）又はその近縁種昆虫の嗅覚受容体共受容体である、請求項１～３のいずれか一項に記載の嗅覚受容体複合体。     The olfactory receptor is OR8 or OR10 of Aedes aegypti, OR10 or OR28 of Anopheles gambiae, OR56 of Bombyx mori, or Drosole of Drosophila, An olfactory receptor having an amino acid sequence having 90% or more identity with the entire amino acid sequence of any olfactory receptor,
    The olfactory receptor complex according to any one of claims 1 to 3, wherein the olfactory receptor co-receptor is an olfactory receptor co-receptor of Apis mellifera or its related insects.
  5. 　前記嗅覚受容体が、ネッタイシマカ（Ａｅｄｅｓ　ａｅｇｙｐｔｉ）のＯＲ８若しくはＯＲ１０、ガンビエハマダラカ（Ａｎｏｐｈｅｌｅｓ　ｇａｍｂｉａｅ）のＯＲ１０若しくはＯＲ２８、カイコガ（Ｂｏｍｂｙｘ　ｍｏｒｉ）のＯＲ５６、又はキイロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ）のＯＲ４７ａ、或いは、これらのうちいずれかの嗅覚受容体全体のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を有する嗅覚受容体であり、
    　前記嗅覚受容体共受容体が、配列番号１に記載のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を有し、かつ、昆虫嗅覚受容体と共役して陽イオンを細胞内へ流入させる能力を有するタンパク質である、請求項１～３のいずれか一項に記載の嗅覚受容体複合体。     The olfactory receptor is OR8 or OR10 of Aedes aegypti, OR10 or OR28 of Anopheles gambiae, OR56 of Bombyx mori, or Drosole of Drosophila, An olfactory receptor having an amino acid sequence having 90% or more identity with the entire amino acid sequence of any olfactory receptor,
    The olfactory receptor co-receptor has an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and is coupled to an insect olfactory receptor to allow a cation to flow into a cell. The olfactory receptor complex according to any one of claims 1 to 3, which is a protein having ability.
  6. 　請求項１～５のいずれか一項に記載の嗅覚受容体複合体を発現する細胞。 A cell that expresses the olfactory receptor complex according to any one of claims 1 to 5.
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