WO2007094408A1 - PROTÉINE RÉCEPTRICE DE LA RYANODINE DU VER À SOIE, SON ADNc, VECTEUR RECOMBINANT, PLASMIDE D'EXPRESSION, CELLULE PRÉSENTANT UNE EXPRESSION CONSTANTE, PROCÉDÉ DE CRIBLAGE D'UN COMPOSANT INSECTICIDE ET PROCÉDÉ DE CRIBLAGE D'UNE SUBSTANCE RÉAGISSANT AVEC LE RÉCEPTE - Google Patents

PROTÉINE RÉCEPTRICE DE LA RYANODINE DU VER À SOIE, SON ADNc, VECTEUR RECOMBINANT, PLASMIDE D'EXPRESSION, CELLULE PRÉSENTANT UNE EXPRESSION CONSTANTE, PROCÉDÉ DE CRIBLAGE D'UN COMPOSANT INSECTICIDE ET PROCÉDÉ DE CRIBLAGE D'UNE SUBSTANCE RÉAGISSANT AVEC LE RÉCEPTE Download PDF

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Publication number
WO2007094408A1
WO2007094408A1 PCT/JP2007/052733 JP2007052733W WO2007094408A1 WO 2007094408 A1 WO2007094408 A1 WO 2007094408A1 JP 2007052733 W JP2007052733 W JP 2007052733W WO 2007094408 A1 WO2007094408 A1 WO 2007094408A1
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WIPO (PCT)
Prior art keywords
silkworm
ryanodine receptor
cell
ryr
cdna
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PCT/JP2007/052733
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English (en)
Japanese (ja)
Inventor
Yasuo Mori
Shigeki Kiyonaka
Kenta Kato
Emiko Mori
Yusuke Mizuno
Takao Masaki
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Kyoto University
Nihon Nohyaku Co., Ltd.
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Priority to JP2008500542A priority Critical patent/JPWO2007094408A1/ja
Publication of WO2007094408A1 publication Critical patent/WO2007094408A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G01N2333/43578—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from silkworm
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • Silkworm ryanodine receptor protein its C DNA, recombinant vector, expression plasmid, stationary expression cell, screening method for insecticidal component, screening method for silkworm ryanodine receptor reactive substance
  • the present invention relates to a silkworm ryanodine receptor protein, a full-length cDNA thereof, and a technique using the same.
  • ryanodine receptor a target protein of ryanodine, one of the known pesticides, is present in the endoplasmic reticulum membrane, and in response to ligands, calcium ions (Ca 2 + ) Is released outside the cell, causing muscle contraction in the body.
  • ryanodine is an insecticide that targets RyR as a calcium release channel, and it is mainly used for controlling phytophagous pests. If a screening system for insecticidal components using RyR can be established, it will be a significant contribution to the search for new and useful pesticides.
  • Non-Patent Document 1 Xuehong Xu, “Molecmar loning of cDNA Encoding a Drosophila Ryanodine Receptorand Functional Studies of the Carboxyl— Terminal Calcium Release Channel], Biophysical Journal, Volume 78, ppl270—1281, March 2000
  • Patent Document 1 International Publication WO2004-027042 (Special Table 2006-516884) Disclosure of Invention
  • Patent Document 1 discloses that the RyR gene of Drosophila melanogaster was introduced and expressed in Sf9 cells, which are insect cells, and host cells of RyR gene include bacteria, fungi, and insects. , Plants and animals are stated to be included. ⁇ ⁇ No specific facts are mentioned!
  • Drosophila when searching for insecticidal components targeting lepidopterous insects, Drosophila is distant in terms of biological evolution. Therefore, Drosophila RyR is expressed in insect and mammalian cell lines. Even so, it is difficult to confirm that the insecticidal components that are effective in the test are also effective for foliar insects.
  • the RyR protein of Tobacco budworm a leaf-eating insect, must be isolated as described above! However, its genome has not yet been decoded, and a screening method for insecticidal components can be easily established. Is a word, difficulty,.
  • the RyR protein is relatively large, and the RyR protein is highly cytotoxic to the cell. For many reasons, it is thought that a large amount of ruthenium ions causes cell death. Animal cells are considered difficult to make.
  • the RyR protein of silkworm (silkworm, Bombyx Mori), which is the same lepidopterous insect as many leaf-eating insects, and its cDNA are elucidated and the ryanodine receptor gene of silkworm is steadily expressed.
  • the main challenge is to establish a method for screening insecticidal components against leaf-eating insects, such as Tobacco budworm, by making cells that express it, especially animal cells, and using the properties of silkworm RyR as a calcium release channel. .
  • the present invention is first a silkworm ryanodine receptor (RyR) protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
  • This silkworm RyR protein has 5084 amino acid residues It consists of numbers and was identified for the first time in the present invention.
  • the present invention consists of the amino acid sequence shown in SEQ ID NO: 4 (number of amino acid residues; 5058), or a sequence in which a partial region of the amino acid sequence shown in SEQ ID NO: 2 is deleted, substituted, or added, It is a splicing variant of the silkworm RyR protein described above.
  • the present invention is a full-length cDNA encoding the silkworm RyR protein according to SEQ ID NO: 2, which has the nucleotide sequence shown in SEQ ID NO: 1.
  • This cDNA consists of 15255 bases and was identified for the first time in the present invention.
  • the present invention comprises a nucleotide sequence (number of bases: 15177) described in SEQ ID NO: 3, or a sequence ability in which a partial region of the nucleotide sequence described in SEQ ID NO: 1 is deleted, substituted or added.
  • a cDNA encoding the silkworm ryanodine receptor protein which is a splicing variant of the ryanodine receptor protein described in 1.
  • the cDNA having the nucleotide sequence of SEQ ID NO: 3 is a full-length cDNA encoding the silkworm RyR protein of SEQ ID NO: 4.
  • the recombinant vector of the present invention containing the cDNA can be obtained.
  • Such a recombinant vector is useful for preparing the following expression plasmid used for screening insecticidal components against leaf-eating insects.
  • the expression plasmid of the present invention is an expression plasmid that allows animal cells to express silkworm ryanodine receptor activity, wherein the above-described thread-replaceable vector has the base sequence described in SEQ ID NO: 1 or 3 remaining.
  • the animal cell expressing the silkworm RyR is preferably a mammalian cell, and examples thereof include CHO cell, COS7 cell, Caco-2 cell, HEK293 cell and the like.
  • the present invention also provides a cell into which the above-mentioned cDNA encoding the silkworm RyR protein has been introduced, and which stably expresses the silkworm ryanodine receptor gene. Gin receptor gene constant expression cells.
  • the present invention is a silkworm ryanodine receptor gene steady-state expression cell that stably expresses the silkworm ryanodine receptor gene using the above-described silkworm ryanodine receptor expression plasmid.
  • the biological species of the cells may be bacteria, fungi, insects, plants, and animals.
  • the ryanodine receptor gene constant expression of the silkworm is expressed by animal cells, preferably mammalian cells. Cells can be realized. As such animal cells (mammalian cells), silkworm ryanodine receptor gene constitutively expressing cells can be obtained from human kidney-derived HEK293 cells.
  • the test reagent is administered to silkworm ryanodine receptor gene constant expression cells to search for substances that react with RyR protein.
  • the screening method according to the present invention comprises a step of introducing the silkworm ryanodine receptor expression plasmid into an animal cell into which a Ca 2+ -sensitive fluorescent dye is incorporated, and a test reagent is allowed to act on the animal cell to cause a fluorescence response. Measuring.
  • Ca 2+ -sensitive fluorescent dyes include Fum2, Furo3, Furo4, Indol, Quin2, and Rhod2.
  • the amino acid sequence of the silkworm ryanodine receptor protein and the base sequence of the cDNA encoding it were elucidated. Furthermore, by expressing the silkworm ryanodine receptor protein in animal cells, we were able to establish a screening method for insecticidal components against leaf-eating insects such as lepidopterous insects, so that the insecticidal components could be efficiently searched. Is possible. In particular, in order to conduct a detailed examination including the action of insecticidal components, it can be said that the present invention using the RyR of silkworms, whose full-length genome has been elucidated, is extremely suitable! /. In addition, we were able to create cells that steadily express the silkworm ryanodine receptor gene, especially animal cells. It can be carried out.
  • FIG. 1 is a diagram showing an evolutionary lineage of ryanodine receptor protein.
  • FIG. 2 is a list showing PCR primers used for preparation of silkworm RyR cDNA.
  • FIG. 3 Structure of PCR fragment (cDNA fragment) for obtaining silkworm RyR cDNA
  • FIG. 4 is a block diagram of a vector obtained by subcloning silkworm RyR cDNA.
  • FIG. 5 is a structural diagram of an expression plasmid obtained by subcloning the same vector.
  • FIG. 6 is a graph showing the results of intracellular Ca 2+ concentration change test using caffeine.
  • FIG. 7 is a graph showing the results of intracellular Ca 2+ concentration change test using ryanodine.
  • FIG. 8 is a graph showing the results of intracellular Ca 2+ concentration change test using flubendiamide.
  • FIG. 9 is a diagram showing a comparative test of the amount of increase in fluorescence before and after stimulation with fulvendiamide in a cell line in which the silkworm RyR gene mRNA was detected by Northern blotting.
  • FIG. 10 is a diagram showing a concentration-response curve of flubendiamide to silkworm RyR gene constant expression cells.
  • FIG. 11 is a graph showing the results of a comparative test of fluorescence response by fulvendiamide stimulation between cells expressing the silkworm RyR gene constantly and transiently expressing cells.
  • RNA extraction reagent (trade name; ISOGEN, manufactured by Poppon Gene), followed by polymerase reverse transcription chain reaction (RT-PC R, product) Name: RNA LA PCR Kit, manufactured by Takara Bio Co., Ltd.) was used, and reverse transcription was performed on cDNA.
  • the designed PCR reaction was performed (see SEQ ID NO: 5 to SEQ ID NO: 27). Nested PCR was performed for those in which no specific band was confirmed in the first PCR.
  • the PCR method is carried out using a BD advantage 2 PCR Enzyme System (trade name) (manufactured by BD Biosciences) according to the attached manual.
  • Figure 3 shows the structure of six cDNA fragments (each approximately 3000 bases long) that contain complementary regions. By superimposing these cDNA fragments, the silkworm RyR cDNA sequence was obtained. However, since this sequence did not contain the start codon, the 5 ′ end was determined by the following method.
  • the 5 'RACE method was used as a method for determining the 5' end of silkworm RyR cDNA.
  • 5 'RACE method BD SMART RACE cDNA Amplification Kit (trade name) (manufactured by BD Biosciences) was used.
  • the silkworm was subjected to PCR using silkworm total RNA, and using the BD SMART II A oligo and random 9 mer supplied with the kit as primers.
  • PCR was performed using Nested Universal Primer A attached to the Kit and the gene-specific primer described in SEQ ID NO: 28 as primers.
  • the nucleotide sequence of the obtained PCR product was determined with a DNA sequencer, whereby the sequence of the 5 ′ end of the silkworm RyR cDNA was determined. From the above results, it was possible to determine the entire nucleotide sequence of the cDNA encoding silkworm RyR and the entire amino acid sequence of silkworm RyR.
  • SEQ ID NO: 1 in the sequence listing shows the base sequence of the full-length cDNA of silkworm RyR
  • SEQ ID NO: 2 shows the amino acid sequence of silkworm RyR.
  • SEQ ID NOs: 3 and 4 are splicing mutants of force RyR cDNA and silkworm RyR.
  • the KOZAK sequence was first introduced into the 5 'end of the silkworm RyRcDNA (SEQ ID NO: 1), and its full-length cDNA (Fig. Among these, the reference 1) was subcloned into a vector plasmid (trade name pGEM-T-Easy, Promega) to prepare a recombinant vector (reference 2 in the figure) (see FIG. 4).
  • the recombinant vector 2 was excised with the restriction enzyme Notl, and subcloned again into a vector plasmid (trade name; pC ⁇ Neo vector, Promega), which is an expression vector for mammalian cells.
  • a silkworm RyR expression plasmid (symbol 3 in the figure) was constructed (see FIG. 5).
  • HEK293 cells derived from human kidney are used as mammalian cells in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum), 30unit / ml penicillin, 30mg / ml streptomycin sulfate. After culturing, the expression plasmid obtained in the previous step was transfected. Transfusion was performed using SuperFect Transfection Reagent (trade name) (manufactured by Qiagen) according to its user's manual. Twenty-four hours after the transfer, the cells were detached from the medium with trypsin ZEGTA (glycol ether diamine tetraacetic acid) and re-spread on a glass plate. Thereafter, after 6 to 12 hours, a change in Ca 2+ concentration was measured. [0027] ⁇ Measurement of intracellular Ca 2+ concentration change>
  • the cells attached to the glass plate were incubated for 40 minutes with a DMEM solution containing ImM Fura2ZAM (manufactured by Dojindo Laboratories). Thereafter, the glass plate was placed on the reflux chamber of the microscope stage together with the test reagent, and fluorescence measurement was performed. The fluorescence image of the cells was performed with a video image analysis system (trade name; Aqua cosmos, manufactured by Hamamatsu Photonics).
  • the fluorescence of the fluorescent dye Fura2 is therefore to measure the fluorescence of 510nm after irradiation alternately 340nm and 380nm of the excitation light, and calculates the fluorescence intensity ratio (ratio (340/380)), intracellular Ca 2 + Concentration ratio was evaluated.
  • FIG. 6 shows the test results using caffeine as a test reagent.
  • Caffeine is known to react with RyR at relatively high concentrations.
  • Figure (a) shows the Ca 2+ response (average trace, average value of 20 cells) when cells were caffeinated, and (b) shows the increase in fluorescence before and after caffeine stimulation. A comparison is shown.
  • HEK293 cells not expressing silkworm RyR are used as controls.
  • HEK293 cells expressing silkworm RyR (kaikoRyR in the figure) clearly show a greater Ca 2+ response after caffeine stimulation compared to non-expressed controls. .
  • FIG. 7 shows the test results using ryanodine as a test reagent.
  • Figure (a) shows the Ca 2+ response (average trace, average of 20 cells) when cells were stimulated with ryanodine, and (b) shows a comparison of the increase in fluorescence before and after ryanodine stimulation. Show.
  • HEK293 cells expressing silkworm RyR show a greater Ca 2+ response after ryanodine stimulation compared to the non-expressed control.
  • ryanodine reacts with silkworm RyR at a lower concentration than caffeine. From the above results, it was strongly suggested that silkworm RyR is functionally expressed in HEK293 cells in this example.
  • Flubendiamide is a compound with high control activity against lepidopterous insects, and is reported to specifically activate the ryanodine receptor (ryanodine-sensitive Ca 2+ release channel) present on the insect endoplasmic reticulum membrane.
  • ryanodine receptor ryanodine-sensitive Ca 2+ release channel
  • two rabbit ryanodine receptors were also expressed in HEK293 cells to evaluate the flubenamide Ca 2+ response.
  • Figure 8 shows the test results.
  • Figure (a) shows the Ca 2+ response (average trace, average of 20 cells) when cells were stimulated with flubenamide, and (b) shows the fluorescence before and after flubenamide stimulation.
  • the silkworm RyR expression system is useful as a screening method for insecticidal components targeting the ryanodine receptor.
  • Ry R of species other than silkworms is expressed in the same manner and evaluated at the same time as silkworm RyR, the selectivity of the insecticidal component among the species (toxicity to other animal species) can also be evaluated.
  • the silkworm RyR expression plasmid into which the silkworm RyR cDNA was inserted was introduced into HEK293 cells by the lipofusion method using the Superfect transfection reagent. See section>). Twenty-four hours after the transfer treatment, di ⁇ nestin (G418) (manufactured by SIGMA) was seeded in a DMEM medium containing lmg / mL, and cultured at 37 ° C for 2 weeks to form colonies. Thereafter, only a single colony was recovered and further cultured.
  • RNA was extracted from the cultured cells by a conventional method, and mRNA derived from silkworm RyR was detected by Northern blotting to select those having activity against flubendiamide.
  • Figure 9 shows the northern blotty.
  • FIG. 10 shows a concentration-response curve of flubendiamide to the silkworm RyR gene constant expression cells (line 7) thus obtained.
  • the silkworm RyR gene is clearly expressed and the silkworm RyR protein is activated by flubendiamide.
  • Fig. 11 shows the results of a comparison test of the fluorescence response of silkworm RyR gene constitutively expressing cells (Stable) and silkworm RyR gene transiently expressing cells (Transient) by fulvendiamide stimulation.
  • Fig. (A) shows the concentration-response curves of flubendiamide to cells that express steady-state and transiently expressed cells. The comparison of the fluorescence increase amount before and after benzamide stimulation is shown. From these results, the silkworm RyR gene constant expression cells have EC values of 10 to 30 as in the transient expression cells.
  • nM can be estimated. Furthermore, it can be said that the silkworm RyR gene constant-expressing cells have much higher fluorescence response intensity by fulvendiamide stimulation than the silkworm RyR gene transiently-expressing cells.
  • the base sequence of the silkworm ryanodine receptor (RyR) cDNA and the amino acid sequence of the silkworm RyR protein were elucidated, and the silkworm RyR protein was expressed in animal cells, and thus, the silkworm RyR constant expression Even cells can be created.
  • This As a result it becomes possible to easily apply insecticidal ingredients to leaf-eating insects such as lepidopterous insects, which greatly contributes to the search for new insecticidal ingredients.

Abstract

Pour déterminer une protéine réceptrice de la ryanodine du ver à soie et son ADNc et établir un procédé de criblage d'un nouveau composant insecticide, la séquence d'acides aminés de la protéine RyR du ver à soie, qui n'avait jamais été déterminée auparavant, et la séquence de base d'un ADNc (1) codant pour cette protéine ont été élucidées. Cet ADNc (1) est sous-cloné dans un plasmide afin de préparer un plasmide d'expression (3). En outre, une cellule présentant une expression constante du gène RyR du ver à soie est construite. Il est ainsi établi un procédé de criblage d'un composant insecticide, comprenant le transfert du plasmide d'expression dans des cellules animales ou l'utilisation de la cellule présentant l'expression constante et la mesure par fluorescence de la réponse Ca2+-réactive à un réactif de test dans la cellule.
PCT/JP2007/052733 2006-02-16 2007-02-15 PROTÉINE RÉCEPTRICE DE LA RYANODINE DU VER À SOIE, SON ADNc, VECTEUR RECOMBINANT, PLASMIDE D'EXPRESSION, CELLULE PRÉSENTANT UNE EXPRESSION CONSTANTE, PROCÉDÉ DE CRIBLAGE D'UN COMPOSANT INSECTICIDE ET PROCÉDÉ DE CRIBLAGE D'UNE SUBSTANCE RÉAGISSANT AVEC LE RÉCEPTE WO2007094408A1 (fr)

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JP2008500542A JPWO2007094408A1 (ja) 2006-02-16 2007-02-15 カイコのリアノジンレセプタータンパク質、そのｃＤＮＡ、組換えベクター、発現プラスミド、定常発現細胞、殺虫成分のスクリーニング方法、カイコのリアノジンレセプター反応物質のスクリーニング方法

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Cited By (1)

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CN105456285A (zh) * 2015-12-14 2016-04-06 湖州市农业科学研究院 一种采用蚕或蚕蛹生产生物制剂时减少细菌感染的方法

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WO2004027042A2 (fr) * 2002-09-23 2004-04-01 E. I. Du Pont De Nemours And Company Isolement et utilisation de recepteurs de la ryanodine

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WO2004027042A2 (fr) * 2002-09-23 2004-04-01 E. I. Du Pont De Nemours And Company Isolement et utilisation de recepteurs de la ryanodine

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105456285A (zh) * 2015-12-14 2016-04-06 湖州市农业科学研究院 一种采用蚕或蚕蛹生产生物制剂时减少细菌感染的方法

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