KR20130091288A - A system for detecting tau aggregation - Google Patents

Abstract

PURPOSE: A tau protein agglomeration detection system is provided to be effectively used in screening treatments for dementia including Alzheimer, as well as a tau protein agglomeration inhibitor by effectively detecting agglomeration of tau protein. CONSTITUTION: Tau protein and a fluorescence protein fraction are linked through a linker peptide in a structure. The tau protein comprises an amino acid sequence of SEQ ID NO:1 or a fraction thereof. The linker peptide comprises an amino acid sequence of SEQ ID NO:3, 4, or 5 or a fraction thereof. A composition for screening a tau protein agglomeration inhibitor includes a plurality of the structures. A composition for detecting tau protein agglomeration includes a plurality of the structures.

Description

Tau protein aggregation detection system {A system for detecting Tau aggregation}

The present invention relates to a tau protein aggregation phenomenon detection system, and more particularly, to a fusion protein structure for detecting tau protein aggregation phenomenon, a composition for detecting the aggregation phenomenon containing the same, and a tau protein aggregation inhibitor screening composition using the same. .

Dementia is a clinical syndrome that refers to a condition in which high-order cerebral dysfunction persists, including acquired memory lesions, language impairments, and changes in personality and sensitivity.

Dementia can be classified into primary dementia, including Alzheimer's dementia, which has no known cause and cannot be improved, and vascular dementia caused by stroke due to cerebrovascular or cardiovascular disorders. While cholinesterase inhibitors such as aricept, reminil, and excellon are used, vascular dementia uses vasodilators or thrombolytic agents, and various treatments are used depending on the type of dementia.

One of the main etiological features of Alzheimer's disease is neurofibrillary tangles (NFTs), which consist of paired helical filaments (PHFs), the result of hyperphosphorylation of tau proteins. Thus, hyperphosphorylation of tau proteins is understood to be an important step in the formation of NFTs.

Tau exist as alternating isomers and contain three or four copies of the repeating sequence corresponding to the microtubule binding domain. Tau in PHF is proteolytically processed into the core domain, consisting of a phase shifted version of the repeat domain; Only three repeats are involved in the stable tau-tau interaction. Aggregates of hyperphosphorylated tau protein lead to destabilization of the microtubules resulting in inhibition of several signaling through axons.

In Alzheimer's dementia, as a prior patent related to tau protein, Patent Document 1 is a patent for a beta-tau aggregation assay, in which bea amyloid aggregated with a tau protein-derived polypeptide is reacted in the presence of a candidate substance, and in the presence of these candidate substances The present invention relates to a technique for measuring the amount of tau-beta amyloid complex formation, and Patent Document 2 relates to a transgenic animal model expressing an tau protein derived from Alzheimer's disease, and an experimental animal that directly presents the tau protein role in Alzheimer's dementia. It's about the model.

On the other hand, there are currently no treatments for primary dementia such as Alzheimer's dementia, and the drugs on the market have only a slight symptomatic effect, and there are no drugs to treat the cause.

Patent Document 1: US 20020164657 Patent Document 2: US20060112437

In order to solve the problems of the prior art, the present inventors recognize that these aggregation inhibitors are targets of dementia treatment agents and screen them based on the aggregation of tau protein as the main pathogen of Alzheimer's dementia. And a system comprising a structure in which fluorescent protein fragments are bound through a linker peptide, thereby completing the present invention.

It is an object of the present invention to provide a tau protein aggregation detection system.

Another object of the present invention is to provide a structure in which tau protein and fluorescent protein fragments are bound through a linker peptide.

In addition, another object of the present invention to provide a composition for screening tau protein aggregation inhibitor comprising a plurality of the above structure.

In addition, another object of the present invention to provide a composition for detecting the aggregation of tau protein comprising a plurality of the above structure.

In addition, another object of the present invention to provide a base sequence encoding the structure.

In addition, another object of the present invention to provide a recombinant vector comprising a base sequence encoding the structure.

In addition, another object of the present invention to provide a cell transformed with the recombinant vector.

In addition, another object of the present invention (a) preparing a culture of the detection composition or the transformed cells; (b) treating the tau protein aggregation inhibition candidate to the composition or culture of step (a); And (c) after the treatment of the candidate substance of step (b), to provide a method for screening tau protein aggregation inhibitor comprising the step of detecting whether the tau protein aggregation.

In order to achieve the above object of the present invention, the present invention provides a tau protein aggregation phenomenon detection system (tau-BIFC). The system includes a plurality of structures in which tau proteins and fluorescent protein fragments are linked by linker peptides. For example, as shown in FIG. 1, the fluorescent protein fragments in each structure are composed of venus 1 and venus 2 fragments, and FIG. 5. As shown in FIG. 2, when the tau protein (or fragment thereof) aggregates, a fluorescence is expressed through binding of fragments of respective fluorescent proteins linked to the tau protein by a linker peptide, thereby detecting the aggregation phenomenon. Such a system may be treated by treating a tau protein aggregation inhibitor candidate substance or an Alzheimer's dementia disease treatment candidate substance with the present system, and confirming the tau protein aggregation or the like by fluorescence expression intensity through these treatments, and comparing it with an untreated group, or Only by quantification can screening for tau protein aggregation inhibitors or Alzheimer's disease treatment.

In this regard, the present invention provides a structure in which tau protein and fluorescent protein fragments are bound via a linker peptide.

In one embodiment of the invention, in the above structure, the tau protein is a structure characterized in that consisting of the amino acid sequence of SEQ ID NO: 1 or fragments thereof.

In one embodiment of the invention, the tau protein may be characterized by consisting of the nucleotide sequence of SEQ ID NO: 2 or fragments thereof.

In one embodiment of the present invention, the fluorescent protein fragment may be a structure characterized in that the venus, Cerulean, Citrine, mKate are fluorescent proteins of different colors, or a part thereof.

In one embodiment of the present invention, the linker peptide may be any structure, for example, may be a structure characterized in that consisting of the amino acid sequence of SEQ ID NO: 3, 4 or 5 or fragments thereof.

The present invention also provides a composition for screening a tau protein aggregation inhibitor comprising a plurality of the above structures.

In one embodiment of the present invention, the structure may be characterized in that the sequence is different from each other, the sequence of the fluorescent protein fragment in the structure may be different from each other.

In one embodiment of the invention, the composition may be characterized in that it further comprises a substance that enhances the aggregation phenomenon of the tau protein, such a substance may be a beta amyloid, okadaic acid or HDAC inhibitor.

In addition, the present invention provides a composition for detecting the aggregation of tau protein comprising a plurality of the above structure.

In addition, the present invention provides a base sequence encoding the structure.

The present invention also provides a recombinant vector comprising a nucleotide sequence encoding the structure.

The present invention also provides a cell transformed with the recombinant vector.

In addition, the present invention comprises the steps of (a) preparing a culture of the detection composition or the transformed cells; (b) treating the tau protein aggregation inhibition candidate to the composition or culture of step (a); And (c) after the treatment of the candidate substance of step (b), it provides a method for screening tau protein aggregation inhibitor comprising the step of detecting whether the tau protein aggregation.

In one embodiment of the present invention, step (b) of the method may be a step of treating the tau protein aggregation inhibitor candidate material to the cell lysate (cell lysate) crushed cells.

In one embodiment of the present invention, step (c) of the method is not particularly limited as long as it is a known method of protein aggregation whether known in the prior art, in particular, the method using a fluorescent or luciferase to determine whether the tau protein aggregation I can measure it.

The present invention also provides a composition for screening Alzheimer's dementia treatment agent comprising a plurality of the above structures.

In one embodiment of the present invention, the tau protein in the construct is a tau protein variant consisting of the amino acid sequence of SEQ ID NO: 9.

The detection system according to the present invention will allow cell biology and biochemical analysis (immunostaining, immunoblotting) using fluorescent proteins to better understand the pathological phenomena of the existing tau protein, and effectively determine whether the tau protein is aggregated. It can be detected and used effectively to screen tau protein aggregation inhibitors, and further, therapeutic agents for dementia diseases, including Alzheimer's.

Figure 1 shows one configuration of the tau protein aggregation phenomenon detection system according to the present invention.
Figure 2 graphically shows the results showing the increase in fluorescence expression by tau protein aggregation and the effect of enhancing the aggregation by amyloid.
Figure 3 is a result graph showing the tau aggregation inhibitory effect by daunorubicin.
4 is a fluorescence micrograph of cells in which tau protein is aggregated.
5 is a diagram showing the structure of the aggregated structure set of a plurality of different sequences according to the present invention.
Figure 6 shows the result of measuring the binding between normal, abnormal tau protein by FACS.
7 is a result of Western blot and phosphorylation of normal and abnormal tau protein expressed in cells.
8 is a result of fluorescence measurement of the binding between normal and abnormal tau protein.
9 is a diagram illustrating a process of constructing a structure in which a tau protein and a fluorescent protein fragment are linked with a linker according to the present invention.

In order to achieve the object of the present invention as described above, the present invention relates to a structure in which tau protein and fluorescent protein fragments are bound through a linker peptide.

In the present invention, the tau protein of the structure may be a protein consisting of the amino acid sequence of SEQ ID NO: 1 or fragments thereof. The nucleotide sequence encoding such a tau protein may comprise the nucleotide sequence of SEQ ID NO: 2 or a fragment thereof.

In the present invention, the fluorescent protein fragments may be venus, Cerulean, Citrine, mKate, fluorescent proteins of different colors, or a part thereof.

In one embodiment of the invention, the fluorescent protein may be a fragment of a venus protein, for example, may have a nucleotide sequence of SEQ ID NO: 6 or 7.

In one embodiment of the invention, the linker peptide may be any structure that does not interfere with the binding of the tau protein, or consequently, the binding of the fluorescent protein is also not hindered, for example, SEQ ID NO: 3 ( GGGGSGGGGS), SEQ ID NO: 4 (RPACKIPNDLKQKVMNH), or SEQ ID NO: 5 (AAANSSIDLISVPVDSR) can be a construct characterized by consisting of a fragment thereof.

In addition, the present invention relates to a composition for screening a tau protein aggregation inhibitor comprising a plurality of the above structure, in one embodiment of the present invention, the fluorescent protein in the structure may be characterized in that the sequence is different from each other. For example, as shown in FIG. 5, a plurality of structures are combined to form a set such that the venus protein, particularly a fluorescent protein, has 1-158 amino acids (MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT) on one structure.

LVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL

VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQ, SEQ ID NO: 10)

, The other construct contains 159-240 amino acids (MKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD)

HMVLLEFVTAAGITLGMDELYK, SEQ ID NO: 11)

This is a method of binding to fluorescence and detecting tau protein aggregation. This can also be utilized for screening aggregation inhibitors.

In one embodiment of the present invention, the composition for screening the aggregation inhibitor may be characterized in that it further comprises a substance that enhances the aggregation phenomenon of the tau protein, such materials include beta amyloid, okadaic acid, alpha-synuclein (a-synuclein) protein or HDAC inhibitor.

In addition, another object of the present invention to provide a composition for detecting the aggregation of tau protein comprising a plurality of the above structure.

In addition, another object of the present invention to provide a base sequence encoding the structure.

In addition, another object of the present invention to provide a recombinant vector comprising a base sequence encoding the structure.

In addition, another object of the present invention to provide a cell transformed with the recombinant vector.

In addition, another object of the present invention (a) preparing a culture of the detection composition or the transformed cells; (b) treating the tau protein aggregation inhibition candidate to the composition or culture of step (a); And (c) after the treatment of the candidate substance of step (b), to provide a method for screening tau protein aggregation inhibitor comprising the step of detecting whether the tau protein aggregation.

In one embodiment of the present invention, step (b) of the method may be a step of treating the tau protein aggregation inhibitor candidate material to the cell lysate (cell lysate) crushed cells.

In one embodiment of the present invention, step (c) of the method is not particularly limited as long as it is a known protein aggregation method known in the prior art, in particular, tau protein aggregation is measured using fluorescence or luciferase can do.

In particular, in the present invention, when the fluorescence measurement method is used as the detection method, it is possible to detect the aggregate in a much shorter time than by directly breaking the cells and confirming the formation of aggregates by biochemical methods (immunoblotting). There are advantages in terms of cost and cost.

In addition, another object of the present invention to provide a composition for screening Alzheimer's dementia therapeutic agent comprising a plurality of the above structure.

 Tau protein variants found in Alzheimer's dementia patients include K257T, I260V, G272V in exon 9, N279K, K280, L284L, P301L, P301S, S305N in exon 10, V337M in exon 12, and G389R in exon 13. Among them, in one embodiment of the present invention, P301L, that is, the 301th amino acid of the amino acid sequence of the normal tau protein has a representative variant found in many patients with Alzheimer's disease in which proline is substituted with leucine, and it is confirmed in terms of aggregation and cohesive strength. Thus, it was confirmed that the constructs of the present invention can be used for screening aggregation inhibitors, and further, for screening therapeutic agents for Alzheimer's disease. Such P301L variant consists of the amino acid sequence of SEQ ID NO: 9, and may be composed of some fragments when these variants are used in the detection method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Fabrication of tau protein aggregation phenomenon according to the present invention

Venus1 (the amino acid sequence of SEQ ID NO: 10 consisting of 1- 198 amino acids, the base sequence of SEQ ID NO: 6) and 10 amino acids (SEQ ID NO: 3), the first fragment of VENUS, a YFP protein, and Tau wt (2N4R, sequence) No. 8) PCR sequence was used to amplify the construction of the sequences linked in this order and cloned into mammalian expression vector pcDNA3.1 (Invitrogen, V790-20) (5'BamHI, 3'EcoRI). The construction of the sequence in which the linker and tau wt (2N4R) of the same sequence as the second fragment Venus2 (amino acid of SEQ ID NO: 11 consisting of 159-240 amino acids and nucleotide sequence of SEQ ID NO: 7) was sequentially connected was also prepared.

Specifically, to briefly show the manufacturing process of the present protein structure in Figure 9,

Obtaining venus1-zipper, venus2-zipper structures prepared according to the method described in Ingrid Remy & Stephen W Michnick, A highly sensitive protein-protein interaction assay based on Gaussia luciferase, Nature Methods 3, 977-979, (2006), As shown in FIG. 9, the linker region was connected after vinus1 / 2, and a zipper domain was attached thereto. The zipper domain has a property of adhering well to each other, so when these two genes are injected into a cell, the protein is expressed in the cell to form a bond between the zippers, and thus the venus protein is completed to fluoresce. We tried to link the tau gene, but it was not easy to cloning using the restriction enzyme. First, we wanted to make a PCR product of venus1 / 2-linker and tau wt / P301L using pcr. In the next step, the resulting venus1 / 2-linker-tau wt / P301L (final PCR product of FIG. 9) was prepared by using hybrid PCR between the thus prepared products, and the PCR product was cloned into the pcDNA3.1 vector using a restriction enzyme. .

First, only the venus1-linker portion of the venus1-linker (GGT GGC GGT GGC TCT GGA GGT GGT GGG TCC: SEQ ID NO: 18) -zipper plasmid was amplified by pcr (as in the case of venus2). Four primers were used in this step (primer 1 ~ 4). tau also used pcr to generate linker-tau (primer5 ~ 6, linker sequence was attached to primer5 to link because linker was not originally in tau gene).

After preparing three PCR products (product 1 ~ 3), the pcr fragments were mixed in two combinations to make a hybrid. (product 1 + product 3, product 2 + product 3). In FIG. 9, only 1 + 3 is expressed, but 2 + 3 is the same). If you mix this and perform pcr with primer 1 and primer6, the two pcr fragments become single strand at high temperature and the linker part is the same, so when the temperature goes down, double strand is made at the linker part, and the rest is DNA polymerization. Hybrid PCR fragments were made and PCR amplification by primers 1 and 6 was possible. The final fragment made was cloned into pcDNA3.1.

The primers used were: (underlined linker, underlined 2, 4 and 5 complementary to each other, 2-5, 4-5.)

Primer 1 (SEQ ID NO: 12) AGCTAGGATCCACCATGGTGAGCAAGGGCGAG

Primer 2 (SEQ ID NO: 13) GGACCCACCACCTCCAGAGCCACCGCCACC CTGCTTGTCGGCGGTGATA

Primer 3 (SEQ ID NO: 14) AGCTAGGATCCACCATGAAGAACGGCATCAAGGC

Primer 4 (SEQ ID NO: 15) GGACCCACCACCTCCAGAGCCACCGCCACC CTTGTACAGCTCGTCCATGC

Primer 5 (SEQ ID NO: 16) GGTGGCGGTGGCTCTGGAGGTGGTGGGTCC ATGGCTGAGCCCCGCCAG

Primer 6 (SEQ ID NO: 17) AGCTAGAATTCTCACAAACCCTGCTTGGCCA

Experiments to increase fluorescence expression by tau protein aggregation and enhance effect of aggregation by amyloid

Hela cells (10 ⁴ cells / well) seeded in a 96well plate were transfected with venus1-tau wt and venus2-tau wt prepared in Example 1 to express the two constructs in the cells (amyloid-b 0, 0.1, 1 μM treatment conditions). The mock transfected only venus1-tau wt, and fluorescence was not observed because venus complex was not formed. Amyloid-beta (bachem, H1368) was treated 24 hours after transfection (final concentration 0, 0.1, 1μM) and washed twice with PBS buffer 24 hours after treatment. After the cells were fixed with 4% paraformaldehyde, the fluorescence of the cells was measured in the fixed solution. (Miltilabel reader Victor X4, Perkin-Elmer)

Amyloid-beta oligomers were formed by incubating at 4 ° C for 24 hours. As a result, two times more fluorescence was observed in the condition in which both constructs were expressed together (non-amyloid-beta treatment) compared to the mock where no fluorescence was observed, indicating that tau aggregation occurred in the cells. This agglomeration phenomenon is shown to increase even more after treatment with amyloid-beta (particularly 1μM) (Fig. 2).

Experiment to confirm the inhibitory effect of tau protein aggregation by daunorubicin

Hela cells (10 ⁴ cells / well) seeded in a 96well plate were transfected with venus1-tau wt and venus2-tau wt prepared in Example 1 to express the two constructs in the cells (amyloid-beta 0, 0.1, 1 μM treatment conditions). The mock transfected only venus1-tau wt, and fluorescence was not observed because venus complex was not formed. After 24 hours of transfection, Daunorubicin was dissolved in DMEM and treated with the final concentration (0, 1, 10 μM) shown in FIG. 2. 24 hours after treatment, the cells were washed twice with PBS buffer. After the cells were fixed with 4% paraformaldehyde, the fluorescence of the cells was measured in the fixed solution.

As a result, two times more fluorescence was observed in the condition in which both constructs were expressed together (non-amyloid-beta treatment) compared to the mock where no fluorescence was observed, indicating that tau aggregation occurred in the cells. This coagulation phenomenon was observed to decrease with the treatment of daunorubicin known as tau coagulation inhibitor. Therefore, together with the results in Example 2, this result indicates that the tau-BIFC system can sensitize the tau aggregation phenomenon (Fig. 3).

Fluorescence Microscopy Observation of Tau Protein Aggregated Cells

Hela cells (10 ⁵ cells / well) seeded in a 12 well plate were transfected with venus1-tau wt and venus2-tau wt prepared in Example 1 to express the two constructs in the cells. The mock transfected only venus1-tau wt, and fluorescence was not observed because venus complex was not formed. After 24 hours after transfection, Aβ oligomer (1mM) was formed by incubating at 4 ° C for 24 hours and fibrill (1mM) at 37 ° C for 24 hours, and further incubated for 24 hours. Cells grown on the coverslip were observed with fluorescence microscopy after 4% paraformaldehyde fixation. As a result, it was observed that the amount of fluorescence cells increased when Amyloid-beta was treated. It was also observed that tau aggregation increased with the oligomer state of amyloid-beta (monomer <oligomer <fibril), resulting in increased fluorescence.

Taken together, it was confirmed that the system according to the present invention has an excellent sensitivity indicating that the tau aggregation state changes under easy fluorescence under conditions that enhance or inhibit tau aggregation.

Quantification of Binding Between Normal and Abnormal Tau Proteins

5-1. The detection system according to the invention was used to quantify the binding between normal tau protein and abnormal tau protein. Obtained from SH-SY5Y cells (ATCC (CRL-2266TM)), neuroblastoma cellline seeded in ⁵ well plates (5x10 ⁵ cells / well), passaged at 3-4 days intervals in DMEM (10% FBS, 1% antibiotics) medium 6) Gene expressions of the seven combinations described in FIG. Measured and quantified. As a result, each of the four genes of v1-tau wt / P301L or v2-tau wt / P301L was unable to observe fluorescence at the time of independent expression, but v1-tau wt / v2-tau wt or v1-tau P301L / In the combination expression of v2-tau P301L, venus complex formation by tau protein binding and fluorescence could be observed. In particular, it was observed that the amount of fluorescence caused by binding between abnormal tau (P301L) proteins was higher than that between normal tau proteins. (Fig. 6)

5-2. In order to confirm whether the fluorescence observed in 5-1 is due to aggregate formation, the protein expressed in cells under the same expression condition was observed by western blot. SH-SY5Y cells, seeded in ⁵ well plates (5x10 ⁵ cells / well), were transfected with the respective combination conditions (v1-tau wt / v2-tau wt, v1-tau P301L / v2-tau P301L). After 48 hours, each protein-expressing cell was disrupted by hypotonic buffer and centrifuged to separate the supernatant (S) and pellet (P) containing the remaining cell tissues and protein aggregates, respectively, by SDS-PAGE. The phosphorylation of protein (YFP-venus) and tau protein (Thr231) was investigated by western blot. As a result, most of the normal tau was found in the supernatant part (S), and most of the abnormal tau (P301L) was observed in the pellets, so the abnormal tau (P301L) protein tended to exist in the aggregate state in the cell. It was found that (Fig. A in Fig. 7). Figures B and C quantify and compare these results and show that both the amount of aggregated tau protein and the amount of phosphorylated tau are increased in abnormal tau (P301L).

5-3. Fluorescence of the expressed fluorescent protein complexes (v1-tau wt / v2-tau wt, v1-tau P301L / v2-tau P301L) was observed under a microscope. Transfection was performed in two combinations (v1-tau wt / v2-tau wt, v1-tau P301L / v2-tau P301L) to SH-SY5Y cells, which are seeded in ⁵ well plates ( ⁵ × 10 ⁵ cells / well), neuroblastoma cells. After 48 hours, cells grown on Coverslip were fixed with 4% paraformaldehyde and treated with thiazine Red and DAPI. As a result, the fluorescence of the tau protein complex formed in the cell was observed and the staining pattern of thiazine Red (red), which reacts specifically with aggregate protein and emits specific fluorescence, was compared. P301L) showed the formation of aggregates in the protein. This result shows that the tau complex observed by the detection system of the present invention forms an aggregate in the case of abnormal tau (P301L) protein, which is consistent with the results obtained in the previous 5-1 and 5-2. Taken together, the system of the present invention which detects the aggregation of tau protein shows that the aggregation of tau protein can be observed by various methods such as microscope, flurometer and FACS using fluorescence, which is suitable for mass drug discovery. Means that.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Nanomol <120> A system for detecting Tau aggregation <130> 2012-01 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 441 <212> PRT <213> Tau protein <400> 1 Met Ala Glu Pro Arg Glu Glu Phe Glu Val Met Glu Asp His Ala Gly   1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His              20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu          35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser      50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val  65 70 75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Gln Pro His Thr Glu                  85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro             100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val         115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly     130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro                 165 170 175 Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly             180 185 190 Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser         195 200 205 Arg Ser Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys     210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Met Pro Asp Leu Lys Asn Val                 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly             260 265 270 Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln         275 280 285 Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly     290 295 300 Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln                 325 330 335 Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser             340 345 350 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn         355 360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala     370 375 380 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser                 405 410 415 Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val             420 425 430 Ser Ala Ser Leu Ala Lys Gln Gly Leu         435 440 <210> 2 <211> 1233 <212> DNA <213> Tau protein <400> 2 atggctgagc cccgccagga gttcgaagtg atggaagatc acgctgggac gtacgggttg 60 ggggacagga aagatcaggg gggctacacc atgcaccaag accaagaggg tgacacggac 120 gctggcctga aagaatctcc cctgcagacc cccactgagg acggatctga ggaaccgggc 180 tctgaaacct ctgatgctaa gagcactcca acagcggaag atgtgacagc acccttagtg 240 gatgagggag ctcccggcaa gcaggctgcc gcgcagcccc acacggagat cccagaagga 300 accacagctg aagaagcagg cattggagac acccccagcc tggaagacga agctgctggt 360 cacgtgaccc aagctcgcat ggtcagtaaa agcaaagacg ggactggaag cgatgacaaa 420 aaagccaagg gggctgatgg taaaacgaag atcgccacac cgcggggagc agcccctcca 480 ggccagaagg gccaggccaa cgccaccagg attccagcaa aaaccccgcc cgctccaaag 540 acaccaccca gctctggtga acctccaaaa tcaggggatc gcagcggcta cagcagcccc 600 ggctccccag gcactcccgg cagccgctcc cgcaccccgt cccttccaac cccacccacc 660 cgggagccca agaaggtggc agtggtccgt actccaccca agtcgccgtc ttccgccaag 720 agccgcctgc agacagcccc cgtgcccatg ccagacctga agaatgtcaa gtccaagatc 780 ggctccactg agaacctgaa gcaccagccg ggaggcggga aggtgcaaat agtctacaaa 840 ccagttgacc tgagcaaggt gacctccaag tgtggctcat taggcaacat ccatcataaa 900 ccaggaggtg gccaggtgga agtaaaatct gagaagcttg acttcaagga cagagtccag 960 tcgaagattg ggtccctgga caatatcacc cacgtccctg gcggaggaaa taaaaagatt 1020 gaaacccaca agctgacctt ccgcgagaac gccaaagcca agacagacca cggggcggag 1080 atcgtgtaca agtcgccagt ggtgtctggg gacacgtctc cacggcatct cagcaatgtc 1140 tcctccaccg gcagcatcga catggtagac tcgccccagc tcgccacgct agctgacgag 1200 gtgtctgcct ccctggccaa gcagggtttg tga 1233 <210> 3 <211> 10 <212> PRT <213> linker peptide <400> 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 <210> 4 <211> 17 <212> PRT <213> linker peptide <400> 4 Arg Pro Ala Cys Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn   1 5 10 15 His     <210> 5 <211> 17 <212> PRT <213> linker peptide <400> 5 Ala Ala Ala Asn Ser Ser Ile Asp Leu Ile Ser Val Pro Val Asp Ser   1 5 10 15 Arg     <210> 6 <211> 474 <212> DNA <213> Venus1 fragment <400> 6 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcag 474 <210> 7 <211> 246 <212> DNA <213> Venus2 fragment <400> 7 atgaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagga cggcggcgtg 60 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 120 gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 180 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 240 tacaag 246 <210> 8 <211> 1233 <212> DNA <213> Tau wt 2N4R <400> 8 atggctgagc cccgccagga gttcgaagtg atggaagatc acgctgggac gtacgggttg 60 ggggacagga aagatcaggg gggctacacc atgcaccaag accaagaggg tgacacggac 120 gctggcctga aagaatctcc cctgcagacc cccactgagg acggatctga ggaaccgggc 180 tctgaaacct ctgatgctaa gagcactcca acagcggaag atgtgacagc acccttagtg 240 gatgagggag ctcccggcaa gcaggctgcc gcgcagcccc acacggagat cccagaagga 300 accacagctg aagaagcagg cattggagac acccccagcc tggaagacga agctgctggt 360 cacgtgaccc aagctcgcat ggtcagtaaa agcaaagacg ggactggaag cgatgacaaa 420 aaagccaagg gggctgatgg taaaacgaag atcgccacac cgcggggagc agcccctcca 480 ggccagaagg gccaggccaa cgccaccagg attccagcaa aaaccccgcc cgctccaaag 540 acaccaccca gctctggtga acctccaaaa tcaggggatc gcagcggcta cagcagcccc 600 ggctccccag gcactcccgg cagccgctcc cgcaccccgt cccttccaac cccacccacc 660 cgggagccca agaaggtggc agtggtccgt actccaccca agtcgccgtc ttccgccaag 720 agccgcctgc agacagcccc cgtgcccatg ccagacctga agaatgtcaa gtccaagatc 780 ggctccactg agaacctgaa gcaccagccg ggaggcggga aggtgcaaat agtctacaaa 840 ccagttgacc tgagcaaggt gacctccaag tgtggctcat taggcaacat ccatcataaa 900 ccaggaggtg gccaggtgga agtaaaatct gagaagcttg acttcaagga cagagtccag 960 tcgaagattg ggtccctgga caatatcacc cacgtccctg gcggaggaaa taaaaagatt 1020 gaaacccaca agctgacctt ccgcgagaac gccaaagcca agacagacca cggggcggag 1080 atcgtgtaca agtcgccagt ggtgtctggg gacacgtctc cacggcatct cagcaatgtc 1140 tcctccaccg gcagcatcga catggtagac tcgccccagc tcgccacgct agctgacgag 1200 gtgtctgcct ccctggccaa gcagggtttg tga 1233 <210> 9 <211> 441 <212> PRT <213> Tau P301L 2N4R <400> 9 Met Ala Glu Pro Arg Glu Glu Phe Glu Val Met Glu Asp His Ala Gly   1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His              20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu          35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser      50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val  65 70 75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Gln Pro His Thr Glu                  85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro             100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val         115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly     130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro                 165 170 175 Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly             180 185 190 Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser         195 200 205 Arg Ser Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys     210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Met Pro Asp Leu Lys Asn Val                 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly             260 265 270 Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln         275 280 285 Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Leu Gly Gly Gly     290 295 300 Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln                 325 330 335 Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser             340 345 350 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn         355 360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala     370 375 380 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser                 405 410 415 Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val             420 425 430 Ser Ala Ser Leu Ala Lys Gln Gly Leu         435 440 <210> 10 <211> 158 <212> PRT <213> Venus 1 fragment <400> 10 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu   1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly              20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile          35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr      50 55 60 Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys  65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu                  85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu             100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly         115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr     130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln 145 150 155 <210> 11 <211> 82 <212> PRT <213> Venus 2 fragment <400> 11 Met Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu   1 5 10 15 Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile              20 25 30 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln          35 40 45 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu      50 55 60 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu  65 70 75 80 Tyr lys         <210> 12 <211> 32 <212> DNA <213> primer 1 <400> 12 agctaggatc caccatggtg agcaagggcg ag 32 <210> 13 <211> 49 <212> DNA <213> primer 2 <400> 13 ggacccacca cctccagagc caccgccacc ctgcttgtcg gcggtgata 49 <210> 14 <211> 34 <212> DNA <213> primer 3 <400> 14 agctaggatc caccatgaag aacggcatca aggc 34 <210> 15 <211> 50 <212> DNA <213> primer 4 <400> 15 ggacccacca cctccagagc caccgccacc cttgtacagc tcgtccatgc 50 <210> 16 <211> 48 <212> DNA <213> primer 5 <400> 16 ggtggcggtg gctctggagg tggtgggtcc atggctgagc cccgccag 48 <210> 17 <211> 31 <212> DNA <213> primer 6 <400> 17 agctagaatt ctcacaaacc ctgcttggcc a 31 <210> 18 <211> 30 <212> DNA <213> linker peptide <400> 18 ggtggcggtg gctctggagg tggtgggtcc 30

Claims (16)

A structure in which tau protein and fluorescent protein fragments are bound via a linker peptide. The structure of claim 1, wherein the tau protein consists of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. The structure of claim 1, wherein the linker peptide consists of an amino acid sequence of SEQ ID NO: 3, 4, or 5 or a fragment thereof. Composition for screening tau protein aggregation inhibitor comprising a plurality of structures of claim 1. The composition of claim 4, wherein the plurality of the structures are structures having different sequences of fluorescent proteins in the structures. The composition of claim 4, wherein the composition further comprises a substance for enhancing the aggregation phenomenon of the tau protein. 7. The composition of claim 6, wherein the substance that enhances the aggregation phenomenon of the tau protein is beta amyloid, okadaic acid, or HDAC inhibitor. Composition for detecting tau protein aggregation, comprising a plurality of structures of claim 1. The composition of claim 8, wherein the plurality of the structures are structures having different sequences of fluorescent proteins in the structures. A base sequence encoding the structure of claim 1. Recombinant vector comprising a nucleotide sequence encoding the structure of claim 1. A cell transformed with the recombinant vector of claim 11. Tau protein aggregation inhibitor screening method comprising the following steps:
(a) preparing a composition of claim 4 or a culture of the transformed cells of claim 12;
(b) treating the tau protein aggregation inhibition candidate to the composition or culture of step (a); And
(c) detecting the tau protein aggregation after the candidate material treatment of step (b). The method of claim 13, wherein detecting whether the tau protein is aggregated comprises measuring fluorescence. A composition for screening Alzheimer's dementia therapeutic agent comprising a plurality of structures of claim 1. 16. The composition of claim 15, wherein the tau protein in the construct is a tau protein variant consisting of the amino acid sequence of SEQ ID NO.

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