KR101546485B1 - A pair of amino acid sequences for monitoring tau-tau interaction in living cells via bimolecular fluorescence complementation - Google Patents

Abstract

The present invention relates to a method for monitoring the interaction between a tau protein in a living cell, a protein sequence for monitoring the interaction between the tau protein, a base sequence pair encoding the same, a vector pair comprising the same, a cell line transformed with the vector pair, The present invention relates to a method for monitoring Tau-Tau interactions in cells, more particularly, to a method for monitoring the interaction of intracellular tau proteins with a bimolecular fluorescent complementary amino acid sequence pair, A first fluorescent complementary amino acid sequence to which an N-terminal sequence of a Venus amino acid sequence is linked; And a second fluorescent complementary amino acid sequence comprising a C-terminal sequence of the Venus amino acid sequence at the C-terminus of the full-length human tauro amino acid sequence, a pair of base sequences encoding the same, A cell line transformed by the vector pair, and a method for monitoring Tau-Tau interaction in living cells using the cell line.
According to the present invention, it is possible to monitor and quantify the tau oligomerization process by directly visualizing the Tau-Tau interaction in living cells, thus studying the occurrence of the disease that tau is involved in and the oligomerization process It can be used as a useful means for developing a method for preventing and restoring the data.

Description

A pair of amino acid sequences for monitoring tau-tau interaction in living cells via bimolecular fluorescence complementation to monitor the interaction of intracellular tau protein

The present invention relates to a method for monitoring the interaction between a tau protein in a living cell, a protein sequence for monitoring the interaction between the tau protein, a base sequence pair encoding the same, a vector pair comprising the same, a cell line transformed with the vector pair, Lt; RTI ID = 0.0 > Tau-Tau < / RTI >

Abnormal tau aggregation is a major feature in Alzheimer's disease (AD) and many other neurodegenerative diseases (collectively, tauopathies) (Brandt R, Hundelt M, Shahani N (2005) Tau alteration and neuronal degeneration in tauopathies: mechanisms and models, Biochimica et Biophysica Acta 1739: 331-354). In healthy nerves, tau stabilizes microtubules by promoting growth from the axon and polarizing neurons. When pathologically hyperphosphorylated, the tau is separated from the microtubules and produces insoluble aggregates (Gendron TF, Petrucelli L (2009).) The role of tau in neurodegeneration.

Over the years there has been proposed a structural skeleton for tau aggregation, evidence that insoluble filaments are formed from ten soluble monomers and that these filaments bind to a higher dimensional structure called neurofibrillary tangles (NFTs) It has been proposed. However, there is still little known pathophysiological significance of NFTs, causal and molecular mechanisms that cause this process in Taurospora, as there is a reliable method for monitoring tau aggregation under physiological conditions It is because it does not. Most studies on tau aggregation were carried out under non-physiological conditions using purified tau or tau fragments. Furthermore, because tau is extremely high in solubility, tau aggregation needs to be artificially induced by the addition of cofactors such as heparin. Because of this, a cell-based model that can monitor tau assemblies in living cells can be a useful tool in studying tau pathology and developing ways to prevent and reverse the process.

The human battleground tau contains a microtubule-binding domain consisting of four conserved sequence repeats. Positively charged residues in these sequence repeats play an important role in binding to highly negatively charged microtubules (20-30 electrons per αβ-tubulin dimer) (Minoura I, Muto E (2006) Dielectric measurement of individual microtubules using the 5 electroorientation method Biophys. J. 90: 3739-3748 .; Mukrasch MD, Biernat J, von Bergen M, Griesinger C, Mandelkow E, et al. beta} -structure and bind to microtubules and polyanions. J. Biol. Chem. 280: 24978-24986). The binding affinity of tau for microtubules is also actively regulated by phosphorylation, which results in dynamic rearrangement of the microtubule network. Unusually excessive phosphorylation of tau inhibits the balance of this dynamic rearrangement and dramatically reduces the affinity for microtubules (Drewes G, Trinczek B, Illenberger S, Biernat J, Schmitt-Ulms G, et al (1995) Microtubule-associated protein / microtubule affinity-regulating kinase (p110mark) A novel protein kinase that regulates tau-microtubule interactions and dynamic instability by phosphorylation at the Alzheimer-specific site serine 262. J. Biol. (1993) Phosphorylation of Ser262 strongly reduces the binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding. Neuron 11: 153-163; Biernat J; Gustke N; Drewes G; Mandelkow EM; Mandelkow E .

In brains suffering from Alzheimer's disease, abnormally hyperphosphorylated tau and its aggregates are observed as an onset. Thus, excessive phosphorylation is generally considered to be a cause of tau aggregation. However, this relationship has not yet been fully described, as overly phosphorylated tau has a very high solubility. Regardless of whether it is spontaneous hyperphosphorylation or induced hyperphosphorylation, overexpressed tau does not implicitly show a tendency to aggregate in most cell lines (Guo JL, Lee VM (2011) Seeding of normal Tau by pathological Tau conformers drives pathogenesis J. Alzheimer-like tangles. J. Biol. Chem. 286: 15317-15331 .; Jenkins SM, Johnson GV (1999) Modulation of tau phosphorylation within its microtubule-binding domain by cellular thiols J. Neurochem. Chun W, Johnson GV (2007) Activation of glycogen synthase kinase 3beta promotes the intermolecular association of tau. The use of fluorescence resonance energy transfer microscopy. J. Biol. Chem. 282: 23410-23417).

In order to study the linkage between tau phosphorylation and aggregation, there is a need to study the soluble forms of tau aggregates, and recent studies have suggested that soluble tau oligomers induce memory decay and neural degeneration (Lasagna Tau oligomers impair memory and induce synaptic and mitochondrial dysfunction in wild-type mice, Mol. Neurodegener 6: 39 .; Brunden et al. J. Alzheimers Dis. 14: 393-399), soluble oligomers may indeed be toxic to neurons, and that the soluble oligomers may actually be toxic to neurons Is widely accepted.

BiFC (bimolecular fluorescence complementation), on the other hand, is a method for visualizing protein-protein interactions based on the technique of forming fluorescent protein complexes from non-transmissive components attached to proteins of interest (Kerppola TK (2008) Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells Annu. Rev. Biophys. 37: 465-487). Conventionally, a green fluorescent protein (GFP) complementary technique was used to quantify tau aggregation (Chun W, Waldo GS, and Johnson GV (2011) Split GFP complementation assay for quantitative measurement of tau aggregation in situ. Mol. Biol. 670: 109-123 .; Chun W, Waldo GS, Johnson GV (2007) Split GFP complementation assay: a novel approach to quantitatively measure aggregation of tau in situ: effects of GSK3beta activation and caspase 3 cleavage. Neurochem., 103: 2529-2539). In these assays, tau was fused to smaller GFP fragments (GFP 11) and expressed in cells with larger GFP fragments (GFP 1-10). When tau is present as a monomer or small aggregate, a large GFP fragment can access a small GFP fragment fused to the tau, thereby binding GFP with fluorescence activity. However, when tau coagulates, reconstitution of active GFP is inhibited and GFP fluorescence decreases in the cells. Although the segmentation-GFP assay has been spotlighted as a method for quantifying aggregation, there is a limitation in monitoring the tau oligomer because of its limited range and low resolution.

Thus, the present invention provides a reliable system for monitoring tau interactions under physiological conditions of living cells without the addition of an artificial cofactor such as heparin, and it is a reliable system for monitoring the disadvantages of the conventional green fluorescent protein complementary technique, We want to provide a new tau interaction monitoring system that solves the problem.

In order to achieve the above object,

As a bimolecular fluorescent complementary amino acid sequence pair for monitoring the interaction of intracellular tau protein,

A first fluorescent complementary amino acid sequence in which an N-terminal sequence of a Venus amino acid sequence is joined to a C-terminus of a full-length human tau amino acid sequence; And

And a second fluorescent complementary amino acid sequence comprising a C-terminal sequence of the Venus amino acid sequence at the C-terminus of the full-length human tauro amino acid sequence.

According to an embodiment of the present invention, the first fluorescent complementary amino acid sequence may be an amino acid sequence represented by SEQ ID NO: 1, and the second fluorescent complementary amino acid sequence may be an amino acid sequence represented by SEQ ID NO: 2.

Further, in order to achieve the above object,

A first nucleotide sequence encoding the first fluorescent complementary amino acid sequence; And

And a second nucleotide sequence encoding the second fluorescent complementary amino acid sequence, wherein the second nucleotide sequence encodes the second fluorescent complementary amino acid sequence.

According to an embodiment of the present invention, the first nucleotide sequence may be the nucleotide sequence shown in SEQ ID NO: 3, and the second nucleotide sequence may be the nucleotide sequence shown in SEQ ID NO: 4.

Further, in order to achieve the above object,

A first pCMV vector comprising the first nucleotide sequence; And

And a second pCMV vector comprising the second nucleotide sequence.

In addition, the present invention provides a cell line transformed with said vector for transformation.

According to one embodiment of the present invention, the cell line may be an HEK293 cell strain transformed by the vector pair.

Finally, the present invention relates to a method for producing a transformed cell line, comprising the steps of: treating the transformed cell line with a substance inducing hyperphosphorylation of tau protein and culturing the treated cell line; And performing immunoblot analysis on the cell line. The present invention also provides a method for monitoring Tau-Tau interaction in living cells.

According to an embodiment of the present invention, the substance inducing hyperphosphorylation of the tau protein may be a phoscholine or an okada acid.

According to the present invention, it is possible to monitor and quantify the tau oligomerization process by directly visualizing the Tau-Tau interaction in living cells, thus studying the occurrence of the disease that tau is involved in and the oligomerization process It can be used as a useful means for developing a method for preventing and restoring the data.

1A is a schematic view of a pair of protein constructs according to the present invention in which the N-terminal and C-terminal regions of a Venus protein are fused to full-length tau, and 1b is a photograph showing the basal fluorescence intensity of a tau-GFP and a tau- 1c is a photograph showing immunoblot results for measuring the expression and basal phosphorylation level of tau-GFP and tau-BiFC cell lines, and immunoblotting with anti-tau (ser 262) antibody shows the level of total tau expression , Immunoblotting with anti-tau phosphorylation (ser 396) antibody indicates the basal level of phosphorylated tau, and anti-actin means loading control.
FIG. 2A is a photograph showing the cell distribution of tau-GFP, 2b is a cell distribution of tau-BiFC, and the cells were incubated with nocodazole or vinblastine (3 μM) for 30 minutes and photographed.
Fig. 3A is a schematic diagram of a process of matureting BiFC according to tau phosphorylation, 3b is a photograph taken after incubation of tau-BiFC cells for 24 hours with acid (30 nM) and phoscholine (20 μM) Is a graph quantifying the increase of BiFC fluorescence in various time zones, and 3d-f is the result of culturing Tau-BiFC cells together with the respective compounds for 24 hours to prepare a cell lysate solution and performing immunoblotting As shown in the figure, a black arrow indicates an electric field tau, a red arrow indicates a tau dimmer, and a blue arrow indicates a tau fragment.
Fig. 4 is a photograph showing the cell distribution of tau-BiFC fluorescence. Fig. 4a is a tau-GFP control group, and Fig. 4b is a photograph of Tau-BiFC cells incubated with each compound for 24 hours.
FIG. 5 is a diagram for classifying Tau-BiFC cells by FACS. FIG. 5a is a graph of HEK293 cells and HEK293 cells expressing a tau-BiFC sequence as a result of flow cytometry analysis. Cells transfected with geneticin were selected, and then cells showing BiFC-fluorescence (green dots in the P2 region in the graph) were further classified using FACS. As fluorescence images for Tau-BiFC cells, it can be seen that the tau-BiFC cells exhibiting basal BiFC-fluorescence are greatly increased after the classification.

In the present invention, a tau-BiFC cell model was completed in order to visualize the Tau-Tau interaction at the oligomer level. The cell model according to the present invention can solve the narrow application range and the low resolution problem of the conventional split-GFP assay, specifically, the N-terminal and C-terminal non-fluorescent portions of the Venus protein The present invention has been made to solve the problems of the prior art by employing a BiFC method based on a Venus protein fused. As a fluorescent "turn-on" approach, fluorescence is not observed when the tau is present as a monomer in the present invention, but Venus fluorescence is observed when tau is coupled together. Thus, by removing background noise from monomer tau, tau dimerization and oligomerization in live cells can be analyzed spatiotemporally without staining with foreign molecules.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to assist the understanding of the present invention and should not be construed as limiting the scope of the present invention.

Tau - BiFC  Manufacture of sensor

In order to manufacture a tau-BiFC sensor, a Venus-based BiFC system was used in the present invention. Venus protein is a type of yellow fluorescence protein (YFP) that is well suited for the spatio-temporal analysis of tau binding, which is either (i) fast and effective maturation of the Venus protein, (ii) (Iii) the fluorescence intensity of the Venus-based BiFC is 10 times higher than that of the EYFP-based BiFC (Shyu YJ, Liu H, Deng X, Hu CD (2006) Identification of new fluorescent protein fragments for bimolecular fluorescence complementation assay under physiological conditions. Biotechniques 40: 61-66 .; Kodama Y, Hu CD (2010) Biotechniques 49: 793-805).

To produce the Venus tau-BiFC sensor, full-length human tau (441 a.a.) was fused to the N-terminal fragment (1-172 a.a., VN173) and the C-terminal fragment (155-238 a.a, VC155) of Venus. Specifically, the mammalian expression vector pCMV6-hTau40-GFP was purchased from OriGene Technologies Inc. (Rockville, Md.). To replace GFP with BiFC moieties, pBiFC-VN173 and pBiFC-VC155 were purchased from Addgene (Cambridge, Mass.) And then amplified using PCR primers with XhoI / PmeI restriction enzyme sequences. Each forward and back primer sequence is as follows:

(VC155-F) 5'-AATTCGGTCG ACCGAGATCT CTCGAGGTAC-3 '

(VC155-R) 5'-CTAGTTGTGG TTTGTTTAAA CTCATCAATG TATC-3 '

(VN173-F) 5'-ATGACGACAA GCTCGAGGCC GCGAATTCAT CG-3 '

(VN173-R) 5'-CTAGTTGTGG TTTGTTTAAA CTCATCAATG TATC-3 '

pCMV6-hTau40-VN173 and pCMV6-hTau-VC155 according to the present invention were prepared by digesting and splicing pCMV6-hTau40-GFP and PCR amplified insights using XhoI / PmeI.

Then, two DNA sequences of the tau-BiFC prepared as described above were stably expressed in HEK293 cells (Fig. 1A). Specifically, HEK293 cells were purchased from ATCC (Manassas, Va.) And cultured in Dulbecco's modified eagle medium containing 10% fetal bovine serum and 10,000 units / mL of penicillin and 10,000 ug / mL of streptomycin, CO ₂ -containing humidified atmospheric conditions. On the day before transfection, HEK-293 cells were plated on 12-well plates with OPTI-MEM medium (Invitrogen - Gibco, Carlsbad, CA). On the other hand, cells were transfected with pCMV6-hTau40-GFP using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif.) According to the manufacturer's guidelines in order to prepare a tau-GFP cell line as a control. Further, in order to produce a tau-BiFC cell line, cells were transfected together with pCMV6-hTau40-VN173 and pCMV6-hTau40-VC155 by the same procedure. To establish a stable cell line, cells were selected and stored in a culture medium containing 400 ng / mL Genecticin.

Subsequently, cells expressing both of the two BiFC constructs were selected by sorting the cells using Tau-BiFC cells using fluorescence cytometry (FIGS. 5A and 5B). Specifically, fluorescent cells were sorted using FACSAria (BD Bioscience, San Jose, Calif.). Tau-GFP cell lines were also prepared for comparison. To compare the expression levels of recombinant tau, cell lysates were prepared and subjected to SDS-PAGE analysis. Specifically, for fluorescent image analysis, cells were grown in 96-well plates and Tau-BiFC cells were treated with various concentrations of okadaic acid or phocolin and cultured at 37 ° C for 24 hours. Fluorescence images were acquired and analyzed using Operetta (PerkinElmer).

Immunoblot analysis of cell protein extracts using Tao antibodies revealed two bands for tau-BiFC cells near 85 kD and one band for tau-GFP cells at ~ 100 kD (Fig. 1C). Specifically, the cells cultured as described above were dissolved in a cell lysis reagent CelLytic M (Sigma, St. Louis, MO) containing protease and phosphatase inhibitory cocktail. Protein concentrations were determined using a Bradford (Thermo scientific, Waltham, MA) assay and protein samples were loaded on a 10% SDS PAGE gel and transferred to a PVDF membrane. Tau antibodies were purchased from abcam (Cambridge, MA): pSer199 + Ser202 (abcam, ab4864), pSer396 (abcam, ab64193) and Ser262 (abcam, ab32057).

Notable is that HEK293 did not cause endogenous tau expression, and this endogenous tau may reduce the efficiency of tau-BiFC maturation. In addition, immunoblot analysis using the phosphorylated-tau antibody (phospho-Ser396) revealed a basal level of tau phosphorylation in both cell lines (Fig. 1c). As expected, the fluorescence intensity of the tau-BiFC cells was significantly lower than that of the tau-GFP cells despite a similar level of expression (Fig. 1b). This fact implies that the majority of tau molecules expressed in HEK293 are present as monomers under basal conditions. Therefore, the tau-conjugated BiFC moieties can not be close enough to induce BiFC complementation.

Tau - BiFC  And microtubule assembly

Next, the cell distribution pattern of tau-BiFC fluorescence was examined. In the case of tau-GFP cells, GFP-fluorescence was abundantly observed throughout the cytoplasm without any specific association with microtubules (Fig. 2a). In contrast, BiFC-fluorescence, although very weak, showed a clear association with microtubules (Figure 2b). These results indicate that highly over-expressed tau binds closely enough to microtubules to induce BiFC maturation on microtubules; BiFC maturation occurs only when two parts are in close proximity (less than 10 nm) (Shyu YJ, Liu H, Deng X, Hu CD (2006) Identification of new fluorescent protein fragments for biphasic fluorescence complementation analysis under physiological conditions. Biotechniques 40 : 61-66 .; Kodama Y, Hu CD (2010) An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio. Biotechniques 49: 793-805).

Furthermore, strong fluorescence was observed in the cytoplasm of the tau-GFP cells, but not in the tau-BiFC cells. This means that excess tau expressed in the cytoplasm is present in the monomeric state. To further investigate the interaction between tau and microtubules, cells were treated with small molecules destabilizing microtubules. Microtubule-associated BiFC fluorescence disappeared when treated with nocodazole, which degrades microtubules. When treated with vinblastine to precipitate microtubules, BiFC fluorescence was enhanced to a very high degree in load-shaped precipitates. The results clearly demonstrate that the fusion of BiFC moieties does not interfere with the interaction between tau and microtubules.

Tau  Due to hyperphosphorylation Tau - BiFC of Maturity

To investigate the possibility of using the tau-BiFC as an indicator of the tau assembly, the tau-BiFC cells were treated with forskolin and okadaic acid, which induced hyperphosphorylation of tau . The battle tau has 79 putative serine and threonine residues. Phosphorylation of these residues is precisely controlled by protein kinases and phosphatases to maintain the microtubule mechanics required for nerve flexibility. Among these regulatory enzymes, the predominant tau phosphatase in the human brain is the protein phosphatase 2A (PP2A), which regulates the activity of some protein kinases that phosphorylate tau. Okada acid is known to induce Alzheimer-like tau phosphorylation in rat brain with low PP2A (Arias C, Sharma N, Davies P, Shafit-Zagardo B (1993) 2 and tau phosphorylation prior to neurodegeneration in cultured cortical neurons. J. Neurochem. 61: 673-682 .; Zhang Z, Simpkins JW (2010) Okadaic acid induces tau phosphorylation in SH-SY5Y cells in an estrogen-preventable manner. 1345: 176-181). Activation of protein kinase A (PKA) by phokolin is known to induce hyperphosphorylation and memory decline of tau (Liu SJ, Zhang JY, Li HL, Fang ZY, Wang Q, et al. a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain. 279: 50078-50088 .; Tian Q, Zhang JX, Zhang Y, Wu F, Tang Q, et al. 2009) Biphasic effects of forskolin on tau phosphorylation and spatial memory in rats J. Alzheimers Dis. 17: 631-642). As expected, when the tau-BiFC cells were incubated with okadaic acid and phoscholin for 24 hours, the BiFC fluorescence increased by 2.2 and 1.9 times, respectively (Fig. 3b, c). The increase in BiFC fluorescence directly means that the level of Tau-Tau interaction in the cells has increased.

As a result of immunoblot analysis, the level of phosphorylation was increased in Ser199 and Ser202 of full-length tau and several tau fragments by treatment with Okada acid (30 nM) (Fig. 3f), which means that the okada acid induces hyperphosphorylation of tau. In addition, some tau fragments showed a strong phosphorylation at Ser396 (indicated by the blue arrow in Fig. 3e) and not at full length tau (Fig. 3e), indicating that the truncated fragments of tau It means that it becomes vulnerable temperament. More importantly, bands of phosphorylated tau dimers at Ser199, Ser202, and Ser396 were observed at around 150 kD (indicated by the red arrows in Figure 3e, f). These results imply that okadaic acid treatment initiates tauopathy characterized by abnormal tau hyperphosphorylation, fragmentation, and dimerization. Furthermore, the fact that an actual tau-BiFC aggregation phenomenon is observed in cells treated with okada strongly supports the occurrence of tau pathology induced by acidosis (Fig. 4).

In contrast, treatment with phosholin (20 [mu] M) induced tau phosphorylation at Ser396, whereas no Tau fragmentation or dimerization was observed on the immunoblot (Fig. 3e). Although phoscholin induced tau phosphorylation and assembly, these effects may not be sufficient to promote the development of tauopathy as seen in the okadaic acid treatment. As an activator of adenylyl cyclase, the phosholin stimulates the cyclic AMP (cAMP) dependent signaling pathway, which is involved in a variety of cellular processes including transcription, metabolism, cell cycle progression and apoptosis. In particular, in nerve cells, it is well known that phoshocin-induced cAMP activation promotes neuronal differentiation and neurite growth (Richter-Landsberg C, Jastorff B (1986). The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells. J. Cell Biol. 102: 821-829 .; Cheng HC, Shih HM, Chern Y (2002) Essential role of cAMP-response element-binding protein activation by A2A adenosine receptors in rescuing the Hansen T, Rehfeld JF, Nielsen FC (2003) KCl potentiates forskolin-induced PC12 cell neurite outgrowth via protein kinase A and extracellular signal-regulated kinase signaling pathways. Neurosci. Lett. 347: 57-61). Even in Tau BiFC cells, neurite-like structures were significantly increased on HEK293 cells by phosholin treatment (Fig. 4A). Phoscholine and Okada acid are small molecules that are commonly used to induce tau phosphorylation, but their contribution to tau aggregation has not yet been elucidated. Thus, in the present invention, the effect of small molecules on the tau assembly in living cells can be directly monitored and quantified by visualizing the Tau-Tau interaction.

Patients with Alzheimer's Disease Since the discovery of abnormal fiber content in the nerves of the brain, nerve fiber knots have been considered pathological structures of the tau. However, recent evidence suggests that smaller, soluble tau oligomers are toxic aggregates, closely related to neural degeneration and memory decay. Large chunks may be a survival strategy to protect neurons by isolating toxic oligomers. Thus, preventing tau oligomerization can be an effective strategy for treating tau-related neurodegenerative diseases. A reliable system for monitoring the tau oligomerization process is needed to identify the cause and molecular mechanism of tau aggregation and to reverse this process.

The Tau-BiFC system according to the present invention provides a means for monitoring and quantifying the tau oligomerization process by allowing the Tau-Tau interaction to be directly visualized in living cells. Thus, the Tau-BiFC sensor according to the present invention will be a useful tool in studying the occurrence of diseases involving tau and developing a method for preventing and reversing the oligomerization process.

<110> Korea Institute of Scienc and Technology <120> A pair of amino acid sequences for monitoring tau-tau interaction          in living cells via bimolecular fluorescence complementation <130> HPC4394 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 628 <212> PRT <213> Artificial Sequence <220> <223> Tau-VN173 <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 Thr Arg Thr Arg Pro Leu Glu         435 440 445 Ser Arg Arg Ser Ser Ale Thr Met Val Ser Lys Gly Glu Glu Leu Phe     450 455 460 Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 465 470 475 480 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly                 485 490 495 Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro             500 505 510 Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Gln Cys Phe Ala         515 520 525 Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met     530 535 540 Pro Glu Gly Tyr Val Glu Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly 545 550 555 560 Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val                 565 570 575 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile             580 585 590 Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile         595 600 605 Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg     610 615 620 His Asn Ile Glu 625 <210> 2 <211> 531 <212> PRT <213> Artificial Sequence <220> <223> Tau-VC155 <400> 2 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 Thr Arg Thr Arg Pro Leu Glu         435 440 445 Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile     450 455 460 Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 465 470 475 480 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr                 485 490 495 Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val             500 505 510 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu         515 520 525 Leu Tyr Lys     530 <210> 3 <211> 1887 <212> DNA <213> Artificial Sequence <220> <223> Tau-VN173 <400> 3 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 accagactg 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 aggtgcagat aattaataag 840 aagctggatc ttagcaacgt ccagtccaag tgtggctcaa aggataatat caaacacgtc 900 ccgggaggcg gcagtgtgca aatagtctac aaaccagttg acctgagcaa ggtgacctcc 960 aagtgtggct cattaggcaa catccatcat aaaccaggag gtggccaggt ggaagtaaaa 1020 tctgagaagc ttgacttcaa ggacagagtc cagtcgaaga ttgggtccct ggacaatatc 1080 acccacgtcc ctggcggagg aaataaaaag attgaaaccc acaagctgac cttccgcgag 1140 aacgccaaag ccaagacaga ccacggggcg gagatcgtgt acaagtcgcc agtggtgtct 1200 ggggacacgt ctccacggca tctcagcaat gtctcctcca ccggcagcat cgacatggta 1260 gactcgcccc agctcgccac gctagctgac gaggtgtctg cctccctggc caagcagggt 1320 ttgacgcgta cgcggccgct cgagtctaga agatccatcg ccaccatggt gagcaagggc 1380 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 1440 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 1500 aagctgatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 1560 ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc 1620 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 1680 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 1740 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 1800 tacaacagcc acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac 1860 ttcaagatcc gccacaacat cgagtag 1887 <210> 4 <211> 1596 <212> DNA <213> Artificial Sequence <220> <223> Tau-VC155 <400> 4 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 accagactg 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 aggtgcagat aattaataag 840 aagctggatc ttagcaacgt ccagtccaag tgtggctcaa aggataatat caaacacgtc 900 ccgggaggcg gcagtgtgca aatagtctac aaaccagttg acctgagcaa ggtgacctcc 960 aagtgtggct cattaggcaa catccatcat aaaccaggag gtggccaggt ggaagtaaaa 1020 tctgagaagc ttgacttcaa ggacagagtc cagtcgaaga ttgggtccct ggacaatatc 1080 acccacgtcc ctggcggagg aaataaaaag attgaaaccc acaagctgac cttccgcgag 1140 aacgccaaag ccaagacaga ccacggggcg gagatcgtgt acaagtcgcc agtggtgtct 1200 ggggacacgt ctccacggca tctcagcaat gtctcctcca ccggcagcat cgacatggta 1260 gactcgcccc agctcgccac gctagctgac gaggtgtctg cctccctggc caagcagggt 1320 ttgacgcgta cgcggccgct cgagaagcag aagaacggca tcaaggccaa cttcaagatc 1380 cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc 1440 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccaaactg 1500 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 1560 gggatcactc tcggcatgga cgagctgtac aagtaa 1596

Claims (9)

A first fluorescent complementary amino acid sequence in which an N-terminal sequence of a Venus amino acid sequence is joined to a C-terminus of a full-length human tau amino acid sequence; And
A second fluorescent complementary amino acid sequence in which the C-terminal sequence of the Venus amino acid sequence is bound to the C-terminus of the full-length human tauro amino acid sequence,
Wherein the first fluorescent complementary amino acid sequence is represented by SEQ ID NO: 1,
Wherein the second fluorescent complementary amino acid sequence is represented by SEQ. ID. NO. 2, a pair of bifunctional fluorescent complementary amino acid sequences for monitoring the interaction of intracellular tau protein. delete A nucleotide sequence pair encoding an amino acid sequence pair according to claim 1,
A first nucleotide sequence encoding the first fluorescent complementary amino acid sequence; And
And a second nucleotide sequence encoding said second fluorescent complementary amino acid sequence,
The first nucleotide sequence is represented by SEQ ID NO: 3,
Wherein the second nucleotide sequence is represented by SEQ ID NO: 4, wherein the pair of nucleotide sequences encoding the bimolecular fluorescent complementary amino acid sequence pair is encoded. delete A first pCMV vector comprising a first base sequence according to claim 3; And
A second pCMV vector comprising the second nucleotide sequence according to claim 3; A cell line transformed with the vector for transformation according to claim 5. 7. The transformed cell line according to claim 6, wherein the cell line is an HEK293 cell line transformed by the vector pair. Treating the cell line according to claim 6 with a substance inducing hyperphosphorylation of tau protein and culturing the treated cell line; And
Performing immunoblot analysis on the cell line
Lt; RTI ID = 0.0 &gt; Tau-Tau &lt; / RTI &gt; 9. The method of claim 8, wherein the substance inducing hyperphosphorylation of the tau protein is a phosholin or an okada acid.

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