JP2008220302A - System for screening therapeutic drug of neurodegeneration disease caused by agglomeration of protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a system for screening a therapeutic drug of neurodegeneration disease caused by the agglomeration of a protein. <P>SOLUTION: The system for screening the therapeutic drug of the neurodegeneration disease uses a gene encoding a variant γPKC caused by the protein agglomeration in spinocerebellar degeneration, a gene encoding GFP, and a gene encoding a Gene Switch protein regulating the expression of a protein by a medicament referred to mifepristone, or a cell containing a vector including a Tet-controlling system regulating the expression of the protein by tetracycline. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a vector for screening a drug for treating a neurodegenerative disease caused by protein accumulation resulting from accumulation of abnormally aggregated proteins in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, cells incorporating the same, and them Relates to a screening system comprising

One of the characteristics common to the onset of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease is hereditary onset, which is overwhelmingly hereditary compared to other organ diseases. A second feature common to neurodegenerative diseases is often accompanied by selective cell death. Another point that should be pointed out in relation to neurodegenerative diseases is that neuronal cell death in neurodegenerative diseases is considered to be apoptosis (Non-patent Document 1). Neurodegenerative diseases are thought to result from aggregation of proteins such as polyglutamine, Huntington, Pick, Alzheimer, Parkinson, amyotrophic lateral sclerosis (ALS), spinocerebellar degeneration, and prion disease. For example, polyglutamine for Huntington's disease, Machado-Joseph disease, etc., β-amyloid (Aβ) for Alzheimer's disease, Tau protein, presenilin, α-synuclein for Parkinson's disease, superoxide mutase 1 (SOD1) for ALS, In spinocerebellar degeneration, it is believed that neurodegenerative diseases develop when proteins aggregate and accumulate inside and outside cells including the nucleus due to mutations such as gamma protein kinase (γPKC) (Non-patent Document 2). Some of these diseases are known, such as the position of the gene to be mutated. For example, many C1B mutations in the regulatory domain of the γPKC gene have been discovered in spinocerebellar degeneration (FIG. 1). However, it is difficult to find abnormalities in point mutations that cause mutations by normal nucleic acid hybridization. In addition, a method using a transgenic mouse (Patent Document 1) has been disclosed as a screening for therapeutic agents for neurodegenerative diseases, but it is difficult to screen many compounds in an assay system using a transgenic mouse.
The latest internal medicine system, volume 68, neurodegenerative diseases, edited by Hiroo Imura, Goro Ogata, Fumiaki Takahisa, Seiichiro Tarui, publisher: Nakayama Shoten (1997), 3-7p Cell Engineering, Vol. 20, No. 11, Takeshi Iwatsubo, 1474-1477 (2001) JP 2003-267874 A

  As described above, there are a wide variety of neurodegenerative diseases caused by protein aggregation, and it is desired to establish a screening method capable of accurately and simply using these therapeutic drug candidates.

As a result of diligent research in view of the above circumstances, the present inventors have found that a gene encoding a mutant protein that forms an aggregate, a gene encoding a green fluorescent protein that enables tracking of protein aggregation, a mutant protein and a green fluorescent protein depending on the drug A vector containing a gene whose expression can be regulated by a drug was introduced into mammalian cells to establish a system for screening a drug for the treatment of neurodegenerative diseases caused by protein aggregation. For example, a gene encoding a mutant protein γPKC resulting from protein aggregation in spinocerebellar degeneration, a gene encoding green fluorescent protein (GFP) for tracking protein aggregation, and a drug can regulate expression of the protein A system for aggregating proteins was established using a vector containing the GeneSwitch gene. However, when protein aggregates are formed, the cells undergo apoptosis, and in cerebellar Purkinje cells, the spread of dendrites becomes small and morphologically different, making appropriate evaluation impossible. For example, when using a CHO cell, a gene encoding a gene switch protein that can regulate protein expression with mifepristone, or when using a cerebellar Purkinje cell, a tetracycline transactivator gene that can be controlled by tetracycline is used. In the case of cerebellar Purkinje cells, suppression of dendritic morphology abnormalities makes it possible to evaluate drugs to treat protein aggregation, and further treatment with such agents After confirming that the formation was improved, the present invention was reached.

  Vectors comprising genes encoding mutant γPKC, GFP and GeneSwitch proteins are provided.

  The present invention provides a vector containing a gene whose γPKC-GFP expression can be controlled by mutant γPKC, GFP and tetracycline.

  Provided are screening kits and methods for drug candidates for neurodegenerative disease by protein accumulation using the above vectors.

  Hereinafter, the present invention will be described in detail.

  In the present specification, in accordance with the provisions of IUPAC and IUB “Guidelines for the creation of a description including a base sequence or amino acid sequence” (edited by the Japan Patent Office) and common symbols in the field, the base sequence, nucleic acid sequence, gene sequence, The amino acid sequence and the like are indicated by various abbreviations.

  In the present specification, for example, “H101Y” representing a mutation indicates that histidine at position 101 from the amino terminus has been mutated to tyrosine. For example, “S119F” indicates that serine at position 119 from the amino acid terminal is mutated to phenylalanine.

  Mammalian vectors containing genes encoding mutant proteins that form aggregates, genes encoding green fluorescent proteins that allow aggregation of proteins to be tracked, and genes that can regulate the expression of mutant proteins and green fluorescent proteins by drugs. And established a system for screening drugs for the treatment of neurodegenerative diseases caused by protein aggregation. Examples of mutant proteins include, but are not limited to, polyglutamine, β-amyloid (Aβ), Tau protein, presenilin, α-synuclein, superoxide mutase 1 (SOD1), and gamma protein kinase (γPKC).

  Examples of genes that can regulate protein expression by a drug include GeneSwitch gene and tetracycline transactivator gene. It is not limited to these. For example, more specifically, a gene encoding a mutant protein γPKC-GFP resulting from protein aggregation in spinocerebellar degeneration, a gene encoding green fluorescent protein (GFP) to track protein aggregation, and a drug A vector containing a gene capable of regulating expression was introduced into cells to establish a system for suppressing protein aggregation. Examples of cells to be introduced include Chinese hamster ovary cells (CHO), human nerve-derived cells, and cerebellar Purkinje cells, which may be established or primary cultures, but are not limited thereto.

When protein aggregates are formed, cells undergo apoptosis, and in cerebellar Purkinje cells, the spread of dendrites becomes small and morphologically different, making appropriate evaluation impossible. For example, when CHO cells are used, expression can be regulated by using a gene encoding a gene switch protein that can regulate protein expression with mipripritone. When human nerve-derived cells (SH-SY 5Y) are used, it is possible to express mutant γPKC-GFP with an adenoviral vector using a Tet control system that can regulate expression with tetracycline. In this case, it can be expressed with high efficiency and is suitable for screening a drug against cell death. When cerebellar Purkinje cells are used, it is possible to express mutant γPKC-GFP with an adenoviral vector using a Tet control system that can be controlled by tetracycline. Since it is a screening system using Purkinje cells, which are the main lesion of SCA14, it has high clinical value.

(Expression of protein aggregates in cells)
It is known that a mutation in γPKC is a causative gene of hereditary spinocerebellar ataxia type 14 (SCA14). Many of these mutations have been found in the C1B regulatory domain of the γPKC expression gene (FIG. 1).

  When this gene and a gene encoding GFP for tracking aggregation are introduced into a cell, for example, CHO, to express a protein, the cytoplasm is better when the mutant γPKC is used compared to the wild-type γPKC and the mutant γPKC. Aggregation in the inside was remarkable (FIG. 2). The degree of protein aggregation in the cytoplasm can be performed using image analysis software such as Image Pro Plus and MetaMorph.

  Further, if the fluidity of a protein is measured using FRAP analysis, quantitative analysis can be performed as an index of protein aggregation (FIG. 12).

  However, when protein aggregation occurs in the cytoplasm, cell death is induced (FIG. 3), which is inappropriate for a drug screening system. Therefore, a vector in which the GeneSwitch gene was introduced as an expression regulatory region on the 5 'side of the mutant γPKC-GFP gene was prepared so that the mutant γPKC-GFP was expressed only when a specific drug was present (FIGS. 4 and 5). That is, γPKC-GFP cDNA was subcloned into pGene / V5-His, γPKC-GFP subcloned into pGene / V5-His was lipofected into CHO cells into which pSwitch was incorporated, cultured in a medium containing Hydromycin / Zeocin, and the above vector The CHO strain in which the expression of γPKC-GFP was observed was established by cloning the cells in which γ was incorporated. The expression of γPKC-GFP was confirmed on the third day after the treatment using Mifepristone 10 nM as the inducing drug. Moreover, it was confirmed that γPKC-GFP was not expressed in the absence of the induced drug (FIG. 6). The expression of mutant γPKC-GFP was similarly examined for drug induction. The expression level was lower than that of the wild type (FIG. 7).

  The effect of trehalose having an effect of improving protein aggregation was confirmed by the GeneSwitch system (FIGS. 8 and 9 Table 6).

    When human nerve-derived cells (SH-SY 5Y) are used, it is possible to express mutant γPKC-GFP with an adenoviral vector using a Tet control system that can regulate expression with tetracycline. In this case, it can be expressed with high efficiency and is suitable for screening a drug against cell death. An outline of the Tet control system is shown in FIG. Using this system, it is possible to express mutant γPKC-GFP in human nerve-derived cells (SH-SY 5Y) (FIG. 11), and in this system also the effect of trehalose having an effect of improving protein aggregation was confirmed. .

    It is also possible to express mutant γPKC-GFP in cerebellar Purkinje cells with an adenoviral vector using a Tet control system that can be controlled by tetracycline (FIG. 11). Since it is a screening system using Purkinje cells, which are the main lesion of SCA14, it has high clinical value. When cerebellar Purkinje cells were used, mutant γPKC-GFP formed aggregates (FIG. 13), and when FRAP analysis was performed, a decrease in fluidity was observed (FIGS. 12 and 14). The usefulness of FRAP analysis was confirmed as a quantitative analysis method.

(Experimental material)
F-12 used for cell culture, Dulbecco's modified Eagle medium (DMEM) and trehalose are Sigma, fetal bovine serum (FBS) is Biologic Industries, penicillin, anti-P. Peroxidase-conjugate AffiniPure Goat anti-mouse IgG was purchased from Jackson Immuno Research Laboratories.

(Cell culture)
GeneSwitch CHO cells, HEK293 cells, and SHSY5Y cells were cultured in a mixed medium of F-12, DMEM, and F-12 / DMEM, respectively, at 37 ° C. and 5% CO ₂ . To these media, 10% FBS and penicillin (100 units / ml) and streptomycin (100 μg / ml) were added.

(Example 1: Preparation of plasmid DNA encoding protein of GFP-fused γPKC)
Quick Change Multi Site-Directed Mutagenesis Kit with (Stratagene, Inc.), the following two types in the procedure of single nucleotide ^(S119P: replacing ^{Ser 119} to Pro, ^G128D: substituting ^{Gly 128} to Asp, Figure 1-1) the It was introduced into human γPKC cDNA.

(Synthesis of mutated γPKC cDNA)
A target gene mutation was introduced, and an oligonucleotide having a phosphorylated 5 ′ end was synthesized as a primer for DNA polymerase (requested from Sigma).
According to the protocol of QuickChange Multi Site-Directed Mutagenesis Kit, a DNA synthesis reaction was performed using a thermal cycler (Mastercycler personal, Eppendorf) under the following conditions.

  The composition of the reaction solution is shown in Table 1.

    The reaction conditions are shown in Table 2.

  After completion of the reaction, 1 μl of DpnI was added to the reaction solution and reacted at 37 ° C. for 1.5 to 2 hours to cleave the template DNA. This leaves a single stranded plasmid containing the mutated γPKC cDNA.

(Transduction into E. coli)
XL-10 Gold (E. coli capable of amplifying a single-stranded plasmid as a double strand) was thawed on ice, 2 μl of mercaptoethanol was added to 45 μl of XL-10 Gold, and 10 minutes on ice. Incubated. Next, 1.5 μl of DpnI-treated reaction solution was added to XL-10 Gold and incubated on ice for 30 minutes. Further, using a block inverter, a heat shock was given at 42 ° C. for 30 seconds, incubated on ice for 2 minutes, and 500 μl of NZY medium warmed to 42 ° C. was added, and a bioshaker (Tytec Corp., BR-40LF at 37 ° C.) was added. ) For 1 hour. Thereafter, the microorganisms are precipitated by centrifugation at 3000 rpm for 5 minutes, 400 μl of the supernatant is removed, the bacteria are suspended in the remaining liquid, and ampicillin-containing (50 mg / l) L-Broth (LB) agar medium (10 g Bacto-tryptone). , 5 g Bact-yeat extract, 10 g NaCl, 1.5% (w / v) Bacto-ager / L) and cultured at 37 ° C. overnight.

(Isolation of plasmid containing mutant γPKC cDNA)
Colonies were inoculated from LB plates in 2 ml of ampicillin-containing (50 mg / l) liquid LB medium and shaken at 37 ° C. with a bioshaker. Inoculate ampicillin-containing (50 mg / l) agar LB medium at the time of inoculation, and use it as a master plate. After 12 to 16 hours, plasmid was extracted from the culture solution using a plasmid purifier (Kurabo, PI-50). Extracted. An appropriate amount of the extracted plasmid was reacted with ApaI at 37 ° C. for 1 hour and treated with a restriction enzyme. An appropriate amount of a loading buffer was added to the reaction product, and electrophoresis was performed using a 1% agarose gel, and whether or not the plasmid contained γPKC cDNA was confirmed by the length of the restriction enzyme fragment.

(Agarose gel)
The required amount of agarose was added to 0.5 × TBE (43.2 g Tris-base,
22 g boric acid, 16 ml 0.5 M EDTA, pH 8.0 / total 8 L), autoclaved, cooled to about 65 ° C., and 10 mg / ml ethidium bromide solution was added at 100 μl / L to prepare a gel The mixture was poured into a vessel and solidified, and the gene sequence at the site where the mutation was introduced was confirmed by cycle sequencing, and a plasmid containing the γPKC cDNA into which the mutation was introduced was selected.

(Cycle sequence method)
Using BigDye Terminators v1.1 Cycle Sequencing Kit (Applied Biosystems), thermal cycle
The sequence reaction was carried out by the Table 3 shows the composition of the sequence reaction solution.

    The reaction conditions are shown in Table 4.

    DNA was purified using Sigma Spin Post-Reaction Purification Columns (Sigma), and the base sequence was confirmed using ABI Prism 310 (Applied Biosystems).

(Subcloning of mutant γPKC-GFP cDNA into expression vector pcDNA3)
Plasmids encoding γPKC (S119P) and γPKC (G128D) whose nucleotide sequences were confirmed by sequencing were reacted at 37 ° C. for 1 hour using EcoRI and BglII, respectively, subjected to restriction enzyme treatment, and migrated to agarose gel did. The target band was cut out and the DNA fragment was eluted in 30 μl of AC water using QIAquick Gel Extraction Kit (Qiagen). Already prepared wild-type γPKC-GFP in pcDNA3 was reacted with Bgl II and Not I at 37 ° C. for 1 hour, treated with restriction enzyme, and the DNA fragment was recovered in the same manner as in 1, and pcDNA3 was recovered as restriction enzyme. The restriction enzyme was inactivated by treating with EcoRI and Not I at 37 ° C. for 1 hour and incubating at 80 ° C. for 10 minutes. In order to insert the two DNA fragments obtained above into EcoRI / Not I site of pcDNA3, Ligation kit ver. 2 (Takara) was used for ligation by incubating at 16 ° C. for 2 hours. Table 5 shows the composition of the Ligation reaction solution.

(Transduction into E. coli)
Thaw the competent cell DH5α on ice and place it in the tube after ligation.
Competent cells were added at 100 μl / tube, mixed and incubated on ice for 30 minutes. Further, heat shock was added at 42 ° C. for 45 seconds in a block incubator, and the mixture was incubated on ice for 3 minutes, and then SOC medium (20.0 g Bact-tryptone, 5.0 g Bact-yeat extract, 2.0 ml 5 M NaCl, 1.25 ml 2 M) 2M Mg ²⁺ solution (1M MgSO ₄ · 7 H ₂ O + 1M MgCl ₂ · 6) autoclaved in a separate reagent bottle in 970 ml of KCl / total
900 μl / tube of 10 ml of H ₂ O) and 20 ml of filter-sterilized 1M glucose solution was added, and the mixture was cultured with shaking on a bioshaker at 37 ° C. for 30 to 60 minutes. Centrifugation was carried out at 3000 rpm for 5 minutes, 880 μl of the supernatant was discarded, and the bacteria were suspended in the remaining liquid, inoculated into ampicillin-containing (50 mg / l) agar LB medium and cultured at 37 ° C. overnight. A plasmid containing cDNA encoding the mutant γPKC-GFP was isolated and confirmed by the same method as in III.

(Plasma mass purification)
E. coli transduced with plasmids containing the target cDNA (γPKC (S119P) -GFP, γPKC (G128D) -GFP) was inoculated from a master plate into 250 ml of ampicillin-containing (50 mg / l) liquid LB medium, And cultured for 16 hours or more. Purification of the plasmid was performed using Genopure Plasmid Maxi Kit (Roche). An appropriate amount of the extracted plasmid was taken and subjected to restriction enzyme treatment and cycle sequencing to identify a plasmid containing γPKC (S119P) -GFP and γPKC (G128D) -GFP cDNA.

(Establishment of a CHO cell line having a mutant γPKC-GFP gene)
Gene Switch system (Invitrogen), which induces gene expression using mipripristone, was used to establish a mutant γPKC-GFP-expressing CHO cell line. An outline of this system is shown in FIG.

  As shown in FIG. 4, GeneSwitch System is a system that allows a target gene to be expressed only in the presence of an inducing drug mifepristone, and is a system that can express a cytotoxic protein or the like at any time. Using this, GeneSwitch CHO, which stably holds the genes of γPKC (S119P) -GFP and γPKC (G128D) -GFP, which are considered to be cytotoxic, was prepared by the method described below.

(Subcloning of mutant γPKC-GFP cDNA into pGene / V5-His A)
γPKC (S119P) -GFP in pcDNA3 and γPKC (G128D) -GFP in pcDNA3 were subjected to restriction enzyme treatment using Hind III / Xba I, respectively, and DNA fragments were recovered. pGene / V5-His A (FIG. 5) was cleaved with Hind III / Spe I and ligated with the DNA fragment obtained in 1. The ligation product was transduced into DH5α, inoculated into ampicillin-containing (50 mg / l) agar LB medium, and cultured overnight at 37 ° C. PGene / V5-His A containing mutant γPKC-GFP was purified and isolated by the same method as in III above. A large amount of pGene / V5-His A containing mutant γPKC-GFP was purified by the same method as in V above.

(Production of GeneSwitch CHO having a stably mutated γPKC-GFP gene)
GeneSwitch CHO cells (CHO cells in which pSwitch (FIG. 5) has already been stably incorporated, purchased from Invitrogen) were seeded in 2 × 10 ⁵ cells / dish on 3 6ch dishes. Wild type (WT) γPKC-GFP, γPK
Three plasmids of pGene / V5-His obtained by subcloning C (S119P) -GFP and γPKC (G128D) -GFP, respectively, were prepared using FuGene6 Transfection Reagent (Roche Diagnostics) at 6 μg / dishes for 6 ch. The cells were cultured in F-12 medium containing Hygromycin (100 μg / ml).

(Gene transfer into cultured cells by lipofection method)
Cells were prepared in a 6 cm dish, and 10 μl of FuGene6 Transfection Reagent was placed in a 1.5 ml Eppendorf tube, and 500 μl of serum-free medium was added thereto. 5 μg of the plasmid solution was added to the above solution, vortexed, centrifuged, incubated at room temperature for 10 minutes, the lipofection solution was added to the 6 cm dish, and cultured in a 37 ° C. incubator.
On the next day, the medium was replaced with F-12 medium containing Zeocin (240 μg / ml) / Hygromycin (100 μg / ml), and the cells were collected when they became confluent at 6 cm dish and suspended in 10 ml of culture medium. In order to obtain colonies derived from a single cell, 1 μl (1/10000), 2 μl (1/5000), and 10 μl (1/1000) are poured into three 10 cm dishes in an F-12 medium containing Zeocin / Hygromycin. And cultured. After confirming the formation of colonies, cells were collected from each colony with a micropipette under a microscope, and transplanted to 24 plates in which 1 ml of medium (with Zeocin / Hygromycin) had been put in advance and cultured. When the cells grew, the cells in each well were removed with trypsin, a part was transferred to another 24 well plate or 6 cm dish, and cultured in the presence of the induction drug mifpristone (10 nM). Several days later, the cells in which expression was induced in 6 were observed with a fluorescence microscope or Western blotting analysis to select clones in which γPKC-GFP expression was observed, and the cells of the selected clones were placed in a 3.5 cm dish glass bottom dish. Inoculated and cultured for 3 days in the presence or absence of 10 nM mifpriston. Three days later, the cells were observed with a confocal laser microscope, and a clone in which γPKC-GFP was expressed in all cells only when Mifepristone was present was defined as a CHO cell line stably having the γPKC-GFP gene.

(Examination of effect of trehalose on aggregate formation of mutant γPKC-GFP in GeneSwitch CHO cells)
The GeneSwitch CHO cell line established above is suspended in the medium at 3 × 10 ⁴ cells / 1 ml and seeded on a 3.5 cm glass bottom dish. After cell adhesion, mifpristone with a final concentration of 10 nM was treated and cultured for 6 days. Thereby, expression of γPKC-GFP was induced, and aggregate formation of mutant γPKC-GFP was observed. Thereafter, trehalose treatment was carried out at a final concentration of 0.1 mM, 0.5 mM, and 1 mM simultaneously with Mifepristone, followed by culturing for 6 days. The effect of trehalose on the formation of mutant γPKC-GFP aggregates was examined by observing cells using a confocal laser microscope. Table 6 shows the ratio of aggregated cells / expressing cells and the result of chi-square test.

・ P <0.01
In both S119P and G128D, aggregation was significantly suppressed with Trehalose 0.1 mM. In S119P, an effect was also observed at Trehalose 0.5 mM.

(Example 2: Production of recombinant adenovirus vector)
Gene transfer of γPKC-GFP by an adenovirus vector was performed using a tetracycline (Tet) control system that can induce expression (FIG. 10). By using this system, expression can be suppressed by the addition of tetracycline, so that transient gene expression can be induced [33]. An adenovirus vector is prepared using AdEasy Adenirus Vector System (Stratagene), and adenovirus vector (d) expressing WT γPKC-GFP, γPKC (S119P) -GFP, γPKC (G128D) -GFP downstream of the TetOp promoter. TetOp-γPKC-GFP) was prepared. It is a tetracycle transactivator downstream of the CMV promoter.
By co-infection with an adenoviral vector that expresses (tTA), γPKC-GFP was expressed in cultured cells (FIG. 11).

The TetOp minimal promoter can express a downstream target gene by binding to a tetracycline transactivator (tTA). At this time, if tetracycline exists, tTA is TetOp.
It becomes impossible to bind to the minimal promoter, preventing the expression of the target gene. Therefore, gene expression can be controlled by tetracycline.

(Incorporation of γPKC-GFP into pShuttle vector)
The TetOp promoter-pShuttle vector contains poly
Since there is no A signal, WT γPKC-GFP, γPKC (S119P) -GFP, and γPKC (G128D) -GFP are incorporated into the expression vector pcDNA3 as a template, and a fragment containing polyA signal and γPKC-GFP is subjected to PCR reaction. Amplified.

  Table 6 shows the composition of the PCR reaction solution.

  Table 7 shows the PCR reaction conditions.

  Primer sequences are as follows.

PKC-EcoRI-forward primer GAATTCGCC
ATGGCTGGTCTG
cDNA3-polyA-reverse primer ATCCCCAG
CATGCCCTGCCTATT
An appropriate amount of loading buffer is added to 25 μl of PCR product, electrophoresed using 1% agarose gel, and 3000 b. Using QIAquick Gel Extraction Kit. p. Were recovered from the gel. At that time, the DNA fragment was eluted in 100 μl AC water.

(Ethanol precipitation)
Mix DNA eluted in 100 μl of AC water with 10 μl of 2.5 M sodium acetate and 250 μl of 100% ethanol, centrifuge at 15,000 rpm for 10 minutes, aspirate the supernatant with a pipette, add 100 μl of 70% ethanol The mixture was further centrifuged for 1 minute, and the supernatant was aspirated with a pipette. Both ends of the γPKC-GFP-polyA fragment were phosphorylated using DNA Kination kit (TOYOBO), dissolved in 75 μl of Denaturation buffer, incubated at 90 ° C. for 2 minutes, further incubated on ice for 2 minutes, rapidly cooled. Then, the solutions shown in Table 8 were added, mixed, and incubated at 37 ° C. for 1 hour.

The DNA fragment was recovered using QIAquick PCR Purification Kit (Qiagen) and eluted in 30 μl of AC water. Since the PCR fragment obtained above has a blunt end, the vector for incorporating it must also have a blunt end. For this purpose, TetOp promoter-pShuttle was cleaved with the restriction enzyme Hind III, and then the restriction enzyme fragment was blunted using a DNA blunting kit (Takara). TetOp promoter-pShuttle Hind III cleavage product was purified with QIAquick PCR Purification kit, ethanol precipitation as shown above was performed, dissolved in 8 μl AC water, 1 μl 10 × blunting buffer was added, and incubated at 70 ° C. for 5 minutes, Add 1 μl of T4 DNA polymerase and incubate at 37 ° C. for 5 minutes.
After inactivating the enzyme by ortexing, it was immediately purified with the QIAquick PCR Purification kit and eluted in 44 μl of AC water. In order to suppress self ligation at the time of ligation, dephosphorylation was performed using cal intestine alkali phosphatase (CIAP, GE Healthcare Bioscience). To the blunting product eluted with 44 μl, 1 μl of CIAP and 5 μl of One-Phor-All PLUS buffer were added and incubated at 37 ° C. for 30 minutes, and further 1 μl of CIAP was added and incubated at 55 ° C. for 1 hour. Run on a 1% agarose gel, about 7000b. p. Were cut from the gel. The DNA fragment was recovered using QIAquick Gel Extraction Kit and eluted into 30 μl of AC water. The γPKC-GFP-polyA fragment and TetOp promoter-pShuttle were ligated in the same procedure as above. After completion of ligation, the cells were transformed into DH5α competent cells, inoculated into kanamycin-containing (30 mg / l) agar LB medium, and cultured at 37 ° C. overnight. Thereafter, 2 ml of kanamycin-containing (30 mg / l) liquid LB medium was inoculated with colonies from the LB plate and shaken at 37 ° C. with a bioshaker. At the time of inoculation, a kanamycin-containing (50 mg / l) agar LB medium was also inoculated to form a master plate. TetOp promoter-pShuttle (TetOp-γPKC-GFP in pShuttle) containing γPKC-GFP cDNA was purified and isolated by the same method as described above. The gene sequence of γPKC-GFP cDNA amplified by PCR was confirmed by the cycle sequencing method as described above. A large amount of TetOp-γPKC-GFP in pShuttle was purified by the same method as described above.

(Preparation of recombinant adenovirus plasmid in E. coli)
A shuttle vector (TetOp-γPKC-GFP in pShuttle) incorporating WTγPKC-GFP, γPKC (S119P) -GFP, and γPKC (G128D) -GFP was cleaved with PmeI and linearized. After electrophoresis on a 1% (w / v) agarose gel for about 40 minutes, the band was cut out slightly above the gel. The linearized plasmid DNA was purified using QIAquick Gel Extraction Kit and eluted into 30 μl of AC water. Adenovirus backbone vector (pAdEasy-1) was suspended in 1 μl of AC water to 1 μg / μl, and 15 μg of plasmid DNA purified in 1 was added and mixed.

  Furthermore, 20 μl of E. coli BJ5183 (competent cell adjusted for electroporation) thawed on ice was added and transferred to a 2.0 mm cuvette. Electroporation was performed using Gene Pulser (Bio-Rad) under conditions of 2500 V, 200Ω, and 25 μFD, and co-translation of the shuttle vector and the adenovirus backbone vector was performed. Thereby, when homologous recombination occurs in E. coli, an adenovirus plasmid containing TetOp-γPKC-GFP (TetOp-γPKC-GFP in pAdEasy 1) is produced.

  After electroporation, 500 μl of LB medium was added to the cuvette, transferred to a 1.5 ml Eppendorf tube, and shaken at 37 ° C. for about 1 hour. Centrifuge for 5 minutes at 3000 rpm.

  The supernatant (450 μl) was discarded, and the remainder was inoculated into an agar LB medium containing kanamycin (60 mg / l) and cultured at 37 ° C. for 16 hours or more. The next day several colonies were inoculated into 2 ml of LB medium containing kanamycin, shaken for 12 to 16 hours, and the plasmid was purified by the miniprep method.

The size of the plasmid in the supercoiled state (35 kb) and Pac by electrophoresis
The cleavage pattern by I (20 kb or more and 3.5 kb (or 4 kb)) was examined, and referring to them, homologous recombination of shuttle vector and adenovirus backbone vector occurred, and TetOp-γPKC-GFP in pAdEasy 1 was completed. confirmed.

  10 μl of clones in which homologous recombination occurred were transformed into 100 μl of DH5α competent cells, inoculated into kanamycin-containing (30 mg / l) agar LB medium, and cultured at 37 ° C. for 12 to 16 hours. The next day, colonies were picked up and shaken in 2 ml of LB medium containing kanamycin for 12 to 16 hours, and the plasmid was purified by the miniprep method.

  The extracted plasmid was treated with PacI and confirmed to be TetOp-γPKC-GFP in pAdEasy 1. A large amount of TetOp-γPKC-GFP in pAdEasy 1 was purified by the same method as above.

  The sequence of γPKC (WT, S119P, G128D) in TetOp-γPKC-GFP in pAdEasy 1 was confirmed by cycle sequencing (FIG. 10).

(Fluorescence observation of γPKC-GFP in mouse primary cerebellum cultured Purkinje cells)
(Observation of living cells)
DIV28 observes γPKC-GFP fluorescence of primary cultured cells of mouse cerebellum with a fluorescence microscope, and counts the number of cells where γPKC-GFP aggregates are found in Purkinje cells expressing wild type and mutant γPKC-GFP. did. A confocal laser microscope (LSM510META, Carl Zeiss) was used to capture GFP fluorescence images of Purkinje cells. GFP fluorescence was excited with a 488 nm argon laser and 505-535 nm band
Detection was performed using a pass filter. Since Purkinje cells are thick, 10 to 30 Z-axis continuous images were taken into one Purkinje cell, and an overlay image of the Z-axis continuous images was created using LSM510META software. Using the distribution of GFP fluorescence as an index, the surface area of γPKC-GFP-expressing Purkinje cells was measured using image analysis software Image-Pro Plus 5.1J (Media Cybernetics).

(Stimulation-induced translocation observation of γPKC-GFP in living cells) A medium of primary cultured cells of mouse cerebellum was added to DIV28 as HEPES buffer (NaCl
165 mM, KCl 5 mM, CaCl ₂ 1 mM, MgCl ₂ 1 mM
, HEPES 5 mM, Glucose 10 mM pH 7.4) 950 μL.

    50 μL of stimulating drug prepared to 20 times the final concentration was added to stimulate γPKC-GFP. Observation of stimulation-induced γPKC-GFP translocation was performed by capturing continuous images of GFP fluorescence before and after stimulation using a confocal laser microscope. It was examined whether there was a difference in translocation between wild type and mutant γPKC-GFP.

  Stimulation with 50 mM KCl + 50 μM glutamate was performed, and continuous images were captured at 1-second intervals, and images for 10 minutes were acquired.

    Stimulation with 1 μM TPA (12-O-tetradecanoylphospho1-l3-acetate) was given, and continuous images were captured at 30-second intervals, and 30-minute images were acquired.

(FRAP analysis of γPKC-GFP in living cells)
FRAP (Fluorescence recovery after photobleaching) is a process of irradiating a part of a living cell expressing a fluorescent protein with strong excitation light, and fading off the fluorescence of the part (photobleaching), and then fading with a fluorescent protein that has moved from the surroundings. This is an experimental method for analyzing how quickly the fluorescence of the portion recovers (FIG. 12). Thereby, the fluidity | liquidity in the cell of the fluorescent protein expressed can be examined (Meyvis et al., 1999).

  The medium of the mouse cerebellar primary culture cells was replaced with 1 mL of HEPES buffer in DIV28, and GFP fluorescence images of cells without aggregates in Purkinje cells expressing wild type and mutant γPKC-GFP were captured with a confocal laser microscope. A part of the cell body of the cell was continuously irradiated with strong excitation light, and the GFP fluorescence of the part was faded. Subsequent changes in GFP fluorescence were observed by taking successive images at regular intervals.

  The time-dependent change of the fluorescence intensity of the irradiated part was analyzed by LSM510 META software, and it was examined whether there was a difference in intracellular fluidity between the wild type and the mutant γPKC-GFP. The above procedure was repeated three times using cells in the same dish, and the average was taken as the result of one experiment. And this experiment was performed 3 times and the fluorescence recovery of the wild type and the mutant type was compared.

FIG. 1 shows the gene structure of γPKC and the gene mutation found in SCA14. FIG. 2 is an observation result of mutant γPKC-GFP expressed in CHO cells. Wild type γPKC-GFP is uniformly distributed in the cytoplasm. In the case of mutant γPKC (S119P) -GFP and mutant γPKC (Q127R) -GFP, aggregates are observed in the cytoplasm. In the lower panel, cell death occurs due to overexpression of mutant γPKC forming an aggregate. FIG. 3 is a diagram showing that cell death is caused by expressing mutant γPKC-GFP. When S119P mutant γPKC-GFP is expressed, the proportion of dead cells is higher than when wild type γPKC-GFP is expressed. FIG. 4 shows an overview of the GeneSwitch system. When Mifepristone is not present, plasmid 1 and plasmid 2 do not bind and GeneSwitch protein and γPKC-GFP are hardly expressed, whereas when Mifepristone is present, plasmid 1 and plasmid 2 dimerize through it, and GeneSwitch Increased expression of protein and γPKC-GFP. FIG. 5 shows the structure of plasmid 1 incorporating the cDNA of GeneSwitch protein and the structure of plasmid 2 incorporating the cDNA of mutant γPKC-GFP. FIG. 6 shows that CHO cells containing the wild type γPKC-GFP gene differ in the expression of wildtype γPKC-GFP in the presence and absence of Mifepristone. FIG. 7 shows the expression of wild-type γPKC-GFP and mutant γPKC-GFP by the GeneSwitch system. Wild type γPKC-GFP does not show aggregation, but mutant γPKC-GFP forms aggregates. FIG. 8 shows a decrease in aggregate formation when mutant γPKC-GFP (S119P) is treated with trehalose. FIG. 9 shows a decrease in aggregate formation when mutant γPKC-GFP (G128D) is treated with trehalose. FIG. 10 shows an expression-inducible tetracycline 'Tet) control system used for gene transfer of γPKC-GFP by an adenovirus vector. FIG. 11 shows the expression of γPKC-GFP and the formation of aggregates of mutant γPKC-GFP in SH-SY5Y cells by an adenovirus vector. FIG. 12 shows a schematic diagram (A) of FRAP analysis and an example (B) of a change in fluorescence intensity of a fading portion. A (a): Before photobleaching. (B): Immediately after Photobleaching, the fluorescence intensity of the irradiated part is significantly reduced. (C): Fluorescence recovery by moving the surrounding fluorescent protein to the irradiated part with time. (D): After a while, the fluorescence of the irradiated part recovers to the same as the surroundings.

B Since the highly fluid fluorescent protein continues to move rapidly to the irradiated part even during excitation light irradiation, the decrease in fluorescence intensity immediately after photobleaching is weak, and the subsequent fluorescence recovery is fast. On the other hand, a fluorescent protein with low fluidity can move only slowly in the cell, so that the fluorescence intensity immediately after photobleaching is significantly reduced and the subsequent fluorescence recovery is slow.
FIG. 13 shows the percentage (%) of cells in which aggregates are observed in γPKC-GFP-expressing Purkinje cells. Mutant γPKC-GFP tends to form aggregates in Purkinje cells. FIG. 14 shows the results of FRAP analysis (change in fluorescence intensity over time, recovery time) of γPKC-GFP-expressing Purkinje cells.

  SEQ ID NO: 1 is the nucleic acid sequence of the mutant γPKC-GFP (S119P) gene.

  SEQ ID NO: 2 is the nucleic acid sequence of the green jellyfish green fluorescent protein gene.

  SEQ ID NO: 3 is the nucleic acid sequence of pGeneV5His.

SEQ ID NO: 4 is the nucleic acid sequence of pSwitch.
SEQ ID NO: 5 is the nucleic acid sequence of tetracycline transactivator (tTA).
SEQ ID NO: 6 is the nucleic acid sequence of tetracycline-operated promoter (TetOp promoter).

Claims (12)

A vector for expressing a protein associated with neurodegenerative diseases caused by protein accumulation,
i) a nucleic acid sequence of a gene encoding one protein selected from the group consisting of γPKC, Aβ, Tau protein, polyglutamine, preserinin, α-synuclein and SOD1 and variants thereof;
ii) a nucleic acid sequence of a gene encoding GFP and 111) a nucleic acid sequence selected from the group consisting of a GeneSwitch protein and a Tet control system that allows for the regulation of protein expression by an agent. A vector comprising the nucleic acid sequence shown in SEQ ID NO: 1, the nucleic acid sequence shown in SEQ ID NO: 2, and the nucleic acid sequence shown in SEQ ID NO: 3 and the nucleic acid sequence shown in SEQ ID NO: 4. A vector comprising the nucleic acid sequence shown in SEQ ID NO: 1, the nucleic acid sequence shown in SEQ ID NO: 2, and the nucleic acid sequence shown in SEQ ID NO: 5 and the nucleic acid sequence shown in SEQ ID NO: 6. A cell comprising the vector according to claim 2. A cell comprising the vector according to claim 3. The vector according to claim 1 for screening a drug-degenerative disease therapeutic drug candidate or a neurodegenerative disease therapeutic drug candidate. 6. The cell according to claim 4 or claim 5 for screening a protein degenerative disease therapeutic drug candidate or a neurodegenerative disease therapeutic drug candidate. The neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, polyglutamine disease, Pick's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, and prion disease. Vector. The neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, polyglutamine disease, Pick's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, and prion disease. Or the cell of Claim 7. The cell according to claim 4, 5 or 7, for screening a drug candidate for treating a neurodegenerative disease, which utilizes regulation of protein expression by a drug. A screening kit for a neurodegenerative disease therapeutic drug comprising the vector according to claim 8 or the cell according to claim 9. A screening method for neurodegenerative diseases using the vector according to claim 8 or the cell according to claim 9.