CN109824787B

CN109824787B - Chimeras promoting dephosphorylation of tau proteins and genes encoding same and uses thereof

Info

Publication number: CN109824787B
Application number: CN201910160443.XA
Authority: CN
Inventors: 王建枝; 郑杰
Original assignee: Huazhong University of Science and Technology
Current assignee: Azkeli Hangzhou Medical Technology Co ltd
Priority date: 2019-03-04
Filing date: 2019-03-04
Publication date: 2023-06-16
Anticipated expiration: 2039-03-04
Also published as: CN109824787A

Abstract

The invention relates to the technical field of genetic engineering, in particular to a chimeric body for promoting tau protein dephosphorylation, a coding gene thereof and application thereof. The chimera comprises a tau protein binding sequence, a linking sequence, a phosphatase recruiting sequence, and a transmembrane peptide sequence, the tau protein binding sequence and the phosphatase recruiting sequence being linked by the linking sequence. According to the invention, the gene for encoding the chimera for promoting tau protein dephosphorylation is cloned into a plasmid, the obtained recombinant plasmid is used for treating SD rat primary neurons or HEK293 cells transfected with htau-GFP, and the phosphorylation level of tau protein AT Ser396 and AT8 epitopes can be effectively reduced. Thus, it has been shown that the chimeras of the present invention that promote tau protein dephosphorylation may play a role in the prevention and treatment of a range of tau diseases including Alzheimer's disease.

Description

Chimeras promoting dephosphorylation of tau proteins and genes encoding same and uses thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a chimeric body for promoting tau protein dephosphorylation, a coding gene thereof and application thereof.

Background

Alzheimer's Disease (AD) is the most common type of dementia, accounting for about 50-70% of dementia. It is counted that about 1000 ten thousand in 2016 and 4400 ten thousand AD patients worldwide. With aging population, the incidence of AD will rise further, with the expectation that about 4000 tens of thousands of AD patients in china in 2050. All current drugs for treating AD are characterized drugs (Symptomatic drugs) which only temporarily relieve symptoms but cannot delay the progression of the disease. There is a strong global expectation of new drugs (diseases-modifying drugs) that can actually alter the progression of AD.

Two characteristic pathological changes in AD are Senile Plaques (SPs) and neurofibrillary tangles (Neurofibrillary tangles, NFTs), which are polymers formed from β -amyloid (aβ) and hyperphosphorylated tau protein, respectively, where tau lesions, but not aβ lesions, are positively correlated with the degree of dementia in AD. Recent studies have shown that highly phosphorylated tau mediates aβ -induced neurotoxicity, necessary for aβ neurotoxicity; and highly phosphorylated tau is a Prion-like protein (Prion-like protein) that can spread between neurons, resulting in the spread of tau lesions, suggesting that it may be a necessary drug target for AD.

In addition to AD, aggregation of highly phosphorylated tau is also seen in a range of neurodegenerative diseases linked to the frontotemporal dementia of chromosome 17 with Parkinson's Disease (frontotemporal dementia linked to chromosome-17parkinsonism, FTDP-17), pick's Disease (PiD), progressive supranuclear palsy (progressive supranuclear palsy, PSP), corticobasal degeneration (corticobasal degeneration, CBD), primary age-related tauopathy (primary age-related tauopathy, PART), silver-philic granulomatosis (argyrophilic grain Disease, AGD), age-related tauopathies (imaging-related tau astrogliopathy, ARTAG), chronic traumatic encephalopathy (chronic traumatic encephalopathy, CTE), spherical glial tauopathy (Globular glial tauopathy, GGT), parkinson's Disease (PD), huntington's Disease (HD), and the like. Such diseases include AD, which is collectively known as tauopathies (tauopathies). Highly phosphorylated Tau protein is an important cause of this disease and is therefore an important therapeutic target for this disease.

Various therapeutic proposals based on tau protein have been proposed to date mainly focusing on degradation of tau protein. This solution is favored mainly for the following reasons: (1) A great deal of evidence suggests that decreasing intracellular tau protein levels causes little side effects in animal models; (2) Reducing the amount of tau protein inhibits tau protein aggregation, which is a significant cause of neuronal degeneration; (3) Reducing tau protein levels reduces neuronal excitatory neurotoxic effects caused by a variety of factors, such as aβ. Thus, reduction of tau protein is also considered a new potential treatment for epilepsy and stroke.

There are two common technical approaches to reduce intracellular target proteins. (1) The expression of target proteins is reduced by siRNA, miRNA or antisense oligonucleotides, and the oligonucleotides have poor pharmacokinetics due to poor distribution in tissues and possibility of off-target, so that the application of the oligonucleotides in clinic is limited at present and needs to be further improved; (2) enhancing degradation of the target protein: a common approach is to enhance the activity of protein degradation systems, including protease systems and autophagy systems. However, the activity of the protein degradation system is not specifically enhanced, and other non-target proteins are easily degraded to cause serious side effects, so that no medicine for activating the protein degradation system is clinically applied at present.

Disclosure of Invention

In view of the above drawbacks of the prior art, the object of the present invention is to construct a chimera (DEP hosoropolarization-TArgeting Chimaeras, abbreviated as DEPTAC chimera) that promotes dephosphorylation of tau proteins to dephosphorylate highly phosphorylated tau proteins, thereby inhibiting their function. To achieve this object, the inventors of the present invention have found through intensive studies that, based on a similar principle of the protein degradation targeting chimera (PROteolysis TArgeting Chimeras, DEPTAC) technology, a bifunctional protein chimera can be constructed, which has one end capable of specifically binding to a target protein (i.e., tau protein) and the other end specifically binding to a specific phosphatase, which are linked via a Linker sequence (Linker), and to which a transmembrane peptide sequence is further linked at one end specifically binding to a specific phosphatase. The protein chimera thus constructed can bind to both the target protein and the phosphatase, and thus, the target protein is adjacent to the phosphatase, and the dephosphorylation of tau protein is selectively induced. Moreover, the DEPTAC technique has the following advantages in addition to selectivity for target proteins: (1) can act on a plurality of targets which are difficult to prepare in traditional medicines. Many traditional small molecule drugs must act on specific binding pockets (binding pockets) of the target protein to exert their inhibitory effect. The DEPTAC technique is not so limited, and can act on many targets that are traditionally difficult to formulate, as long as it interacts with any segment of the target protein and does not require a high affinity, resulting in rapid degradation of the target protein (dephosphorylation of the target protein in the present application) and thus inhibition of the function of the target protein. (2) The DEPTAC technology can be reused in cells and can have a catalytic-like effect, so that the therapeutic effect can be achieved without a high concentration.

To this end, in a first aspect, the invention provides a chimera for promoting dephosphorylation of a tau protein, the chimera comprising a tau protein binding sequence, a linker sequence, a phosphatase recruiting sequence and a transmembrane peptide sequence, the tau protein binding sequence being linked to the phosphatase recruiting sequence by the linker sequence.

In a preferred embodiment, the phosphatase is AB αc phosphatase 2A.

In a preferred embodiment, the tau protein binding sequence is fragments 422-434 of beta tubulin; preferably, the amino acid sequence of the tau protein binding sequence is shown in SEQ ID NO. 1.

In a preferred embodiment, the amino acid sequence of the linker sequence is shown in SEQ ID NO. 2.

In a preferred embodiment, the phosphatase recruitment sequence is fragments 224-236 of full-length human tau protein; preferably, the amino acid sequence of the phosphatase recruitment sequence is shown in SEQ ID NO. 3.

In a preferred embodiment, the amino acid sequence of the transmembrane peptide sequence is shown in SEQ ID No. 4.

In a preferred embodiment, the amino acid sequence of the chimera is shown in SEQ ID No. 5.

In a second aspect, the invention provides a gene encoding a chimera as described above that promotes tau dephosphorylation.

In a preferred embodiment, the nucleotide sequence of the gene is shown in SEQ ID No. 6.

In a third aspect, the present invention provides a recombinant vector comprising a vector and a gene as described above linked to said vector.

In a preferred embodiment, the vector is a plasmid, preferably a pIRES2-EGFP plasmid, or an adenovirus vector, preferably an adenovirus AOV002 vector; preferably, the gene is linked in-frame between EcoRI and BamHI cleavage sites of the adenovirus vector.

In a fourth aspect, the present invention provides the use of a chimera as described above, a gene as described above or a recombinant vector as described above in the manufacture of a medicament for the treatment and/or prophylaxis of a tau protein associated disease.

Alternatively, the Disease is at least one of Alzheimer's Disease (AD), frontotemporal dementia (Frontotemporal dementia linked to chromosome-17park insonism, FTDP-17), pick's Disease (PiD), progressive supranuclear palsy (progressive supranuclear palsy, PSP), corticobasal degeneration (corticobasal degeneration, CBD), primary age-related tauopathy (primary age-related tauopathy, PART), silver-philic granulomatosis (argyrophilic grain Disease, AGD), aging-related tau astrocytopathy (imaging-related tau astrogliopathy, ARTAG), chronic traumatic encephalopathy (chronic traumatic encephalopathy, CTE), spherical glial tauopathy (Globular glial tauopathy, GGT), parkinson's Disease (PD), huntington's Disease (HD), cerebral stroke, and epilepsy.

In a fifth aspect, the invention also provides a method of promoting tau protein dephosphorylation in a patient in need thereof, the method comprising administering to the patient an effective amount of a chimera as described above, a gene as described above or a recombinant vector as described above;

preferably, the mode of administration is selected from at least one of nasal, inhalation, topical, oral, intramuscular, subcutaneous, transdermal, intraperitoneal, epidural, intrathecal and intravenous routes.

The recombinant plasmid obtained by cloning the gene for encoding the chimeric body for promoting tau protein dephosphorylation provided by the invention into the plasmid can be efficiently expressed in cells. In addition, the chimera of the invention is used for treating SD rat primary neurons or HEK293 cells transfected with htau-GFP, so that the phosphorylation level of tau protein AT Ser396 and AT8 epitopes can be effectively reduced. Thus, it has been shown that the chimeras of the present invention that promote tau protein dephosphorylation may play a role in the prevention and treatment of a range of tau diseases including Alzheimer's disease.

Drawings

FIG. 1 is a schematic representation of the dephosphorylation of tau by a chimera provided by the present invention.

FIG. 2 shows dephosphorylation of tau protein by the chimeric DEPTAC in rat primary neuronal cells;

wherein "DEPTAC" is a chimeric provided by the invention, and "AT8" is a phosphorylated tau protein antibody, which can detect human or mouse tau protein phosphorylated AT Ser202 and Thr205 sites, and the phosphorylation level of tau detected by AT8 is reduced after dephosphorylation; "pS396" is a phosphorylated tau protein antibody, and can detect human or mouse tau protein phosphorylated at Ser396 site, and the level of tau phosphorylation detected by pS396 is reduced after dephosphorylation; "Tau5" is a Tau antibody that detects the overall levels of human and mouse-derived Tau.

FIG. 3 shows dephosphorylation of tau protein by the chimeric DEPTAC in HEK293 cells;

FIG. 4 shows expression of a gene expressing the chimeric DEPTAC in HEK293 cells; wherein the expression level of the flag tag is positively correlated with the expression level of DEPTAC.

FIG. 5 is a schematic representation of pIRES2-EGFP plasmid.

Detailed Description

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In a first aspect, the invention provides a chimera for promoting dephosphorylation of a tau protein, the chimera comprising a tau protein binding sequence, a linker sequence, a phosphatase recruiting sequence, and a transmembrane peptide sequence, the tau protein binding sequence being linked to the phosphatase recruiting sequence by the linker sequence.

According to the present invention, the phosphatase may be any phosphatase capable of dephosphorylating a target protein, and in a preferred embodiment of the present invention, the phosphatase is phosphatase 2A, more preferably AB.alpha.C type phosphatase 2A.

According to the invention, the tau protein binding sequence may be any sequence capable of specifically binding to tau protein, preferably the tau protein binding sequence is a 422-434 contiguous fragment of beta tubulin (beta-tubulin), which specifically recognizes and binds tau protein (e.g. tau 1); further preferably, the amino acid sequence of the tau protein binding sequence is shown in SEQ ID NO. 1 (YQQYQDATADEQG).

According to the present invention, the Linker sequence may be a Linker capable of linking two amino acid sequences, which is conventionally used in the art, preferably, the amino acid sequence of the Linker sequence is shown as SEQ ID NO. 2 (GSGS).

According to the invention, the phosphatase recruiting sequence may be any phosphatase recruiting sequence capable of recruiting phosphatases to dephosphorylate a target protein. In a preferred aspect of the invention, the phosphatase recruiting sequence is a sequence capable of recruiting AB oc C phosphatase 2A (whose B subunit is 55 kD); more preferably, the phosphatase recruitment sequence is fragments 224-236 of full length human tau protein, which recruits AB.alpha.C-type phosphatase 2A, selectively inducing dephosphorylation of tau protein, and the amino acid sequence of the phosphatase recruitment sequence is shown in SEQ ID NO. 3 (KKVAVVRTPPKSP).

According to the invention, the membrane-penetrating peptide can be any peptide which can carry a target fragment into cells so as to realize the function of the target fragment, and according to a preferred embodiment of the invention, the amino acid sequence of the membrane-penetrating peptide is shown as SEQ.ID.NO. 4 (RRRRRRRRRR).

According to a particularly preferred embodiment of the invention, the amino acid sequence of the chimera is shown in SEQ ID No. 5, which includes a tau protein specific binding sequence (YQQYQDATADEQG), a junction sequence (GSGS), a phosphatase recruiting sequence (KKVAVVRTPPKSP) and a transmembrane sequence (RRRRRRRRRR) to enable recruitment of AB.alpha.C-type phosphatase 2A for dephosphorylation of specifically bound tau protein (as shown in FIG. 1).

SEQ.ID.NO:5：

YQQYQDATADEQGGSGSKKVAVVRTPPKSPRRRRRRRR

The chimeras may also be linked to other sequences conventional in the art, for example, marker genes (e.g., genes encoding beta-galactosidase, luciferase, green fluorescent protein, or other fluorescent proteins), expression tags (e.g., flag tags), related transcriptional regulatory sequences associated with the transcribed sequences (e.g., promoters, enhancers, etc.), according to the invention. These are well known to those skilled in the art and the present invention is not described in detail herein.

It is well known in the art that of the 20 different amino acids that make up a protein, other than Met (ATG) or Trp (TGG) are each encoded by a single codon, the 18 other amino acids are each encoded by 2-6 codons (Sambrook et al, molecular cloning, cold spring harbor laboratory Press, new York, U.S. second edition, 1989, see page 950 appendix D). That is, due to the degeneracy of the genetic code, the nucleotide sequence of the gene encoding the same protein may differ, since the substitution of the third nucleotide in the triplet codon, which determines most of the codons of one amino acid, does not change the composition of the amino acid. The nucleotide sequences of the genes encoding them can be deduced entirely from the amino acid sequences disclosed in the present invention according to known codon tables by those skilled in the art, and they can be obtained by biological methods (e.g., PCR methods, mutation methods) or chemical synthesis methods, and thus, all such partial nucleotide sequences are intended to be included in the scope of the present invention. Conversely, by using the DNA sequences disclosed herein, amino acid sequences consistent with the xylanase activities described herein can also be obtained by modifying the nucleic acid sequences provided herein by methods well known in the art, such as the method of Sambrook et al (molecular cloning, cold spring harbor laboratory Press, new York, U.S. second edition, 1989).

Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO: shown at 6.

SEQ ID NO：6

TACCAGCAGTACCAGGACGCCACGGCCGACGAACAAGGGGGCAGCGGCAGCAAGAAGGTGGCAGTGGTCCGTACTCCACCCAAGTCGCCGCGTCGTCGTCGTCGTCGTCGTCGT

In a third aspect, the present invention also provides a recombinant vector comprising a vector and a gene as described above linked to the vector.

According to a preferred embodiment of the invention, the vector may be a plasmid, for example, pIRES2-EGFP plasmid, the specific pattern of which is shown in FIG. 5. The insertion site of the gene of interest may be between CMV and PRES.

According to another preferred embodiment of the present invention, the vector is an adenovirus vector, which is a large molecule (36 kb) double-stranded non-enveloped DNA virus that enters the cell by receptor-mediated endocytosis, and then the adenovirus genome is transferred into the nucleus, remains extrachromosomally, and is not integrated into the host cell genome, and thus, the gene of interest can be efficiently delivered into the cell by integrating the gene of interest into the adenovirus vector, and does not affect the chromosome of the cell. Further preferably, the adenovirus vector is an adenovirus AOV002 vector.

Wherein the gene of interest can be inserted into the conventional insertion site of the adenovirus vector, preferably the gene of interest is inserted in-frame between EcoRI and BamHI cleavage sites of the adenovirus vector. Specifically, an Adeno-associated virus (AAV) capable of stably expressing the chimera provided by the present invention can be obtained by subjecting an adenovirus vector and a target gene to cleavage with EcoRI and BamHI, respectively, and then ligating the two with a ligase.

In a fourth aspect, the invention also provides the use of a chimera as described above, a gene as described above or a recombinant vector as described above in the manufacture of a medicament for the treatment and/or prophylaxis of a disease associated with tau protein.

According to the invention, the disease may be at least one of alzheimer's disease, frontotemporal dementia linked to chromosome 17 with parkinson's disease, pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathy, silver-philic granulomatosis, age-related tauastrocytopathy, chronic traumatic encephalopathy, globoid tauopathy, parkinson's disease, huntington's disease, stroke and epilepsy.

According to the invention, the medicine can contain the chimera of the invention as an active ingredient, can also contain other medicines for treating related diseases known in the art for matching use, and can also contain pharmaceutically acceptable carriers, excipients and other components which are suitable for different dosage forms.

according to the invention, said administration may be achieved by at least one means selected from the group consisting of: nasal, inhalation, topical, oral, intramuscular, subcutaneous, transdermal, intraperitoneal, epidural, intrathecal and intravenous routes.

The present invention will be described in detail by examples.

In the following examples;

htau-GFP plasmid commissioned and metabiotechnology (Shanghai) incorporated, which transfects cells not expressing human tau protein to express human tau protein, and which expresses human tau protein in cells with a high level of phosphorylation after expression, which can be detected with T8 and pS396 antibodies;

HEK293 cells were purchased from the chinese collection of typical cultures cell bank (university of martial arts), accession number GDC0187;

rat primary neurons are derived from about 15 day SD fetal mouse hippocampus and cortical neurons in the embryo phase of primary culture, and have the expression of endogenous mouse tau per se, and the basic phosphorylation level can be detected by using AT8 and pS396 antibodies;

other biochemical reagents are available from general biochemical reagent company.

Examples

The pIRES2-EGFP plasmid expressing the chimeras of the present application was constructed by the company, inc. of the entrusted and metabiotechnology (Shanghai), and was designated as DEPTAC-Flag plasmid.

In order to facilitate the measurement of the expression of the chimeric antibody, a flag tag is further added at the tail end of SEQ ID No. 5, the sequence is shown as SEQ ID No. 7 (DYKDDDDK), and the coding sequence is shown as SEQ ID No. 8 (GATTACAAGGATGACGACGATAAG).

The marnhao Biotechnology Co., ltd was commissioned to synthesize the application chimera (SEQ. ID. NO: 5) with a molecular weight of about 4.443kD and a purity of 95%.

Comparative example

The pIRES2-EGFP plasmid of the chimera expressing the reference was constructed by the commission and metabiotechnology (Shanghai) Inc., and did not contain a phosphatase recruiting sequence relative to the chimeras of the examples.

Reference chimeras were synthesized by the entrusted wunhao biotechnology company at 95% purity and did not contain phosphatase recruiting sequences relative to SEQ ID No. 5.

Test example 1

200. Mu.L/1 mL Tip each of the cartridges was autoclaved. 1mL of Trypsin solution was added to a flask in which HEK293 cells were cultured, and the mixture was digested for 1 minute (37 ℃ C., 5% CO) ₂ ). When the wall of the flask was tapped by hand to observe complete detachment of cells from the wall, the reaction was stopped by adding 1mL of serum-containing medium. Adding culture solution to blow cells, transferring the cells into a culture plate for culture, transfecting DEPTAC-Flag plasmid when the cell density is about 60% -80% after cell wall attachment, continuing to culture for 24 hours after transfection, lysing the cells by using RIPA solution, collecting cell extract, and performing Western blot detection by using anti-Flag antibody to determine the expression of DEPTAC-Flag. As shown in FIG. 4, it can be seen from FIG. 4 that the chimeric substance of the present application can be efficiently expressed in HEK293 cells.

Test example 2

Taking brain from SD fetal mice at embryo stage of 12-15 days under aseptic condition, separating and removing soft membrane and blood vessel, collecting cortex and Hippocampus tissue, rinsing, repeatedly cutting cortex into pieces with ophthalmic scissors, transferring into culture dish, sucking dissecting solution, adding 0.25% trypsin 2mL, digesting in 37 deg.C incubator for 20min, discarding the residual digestive juice, inoculating with inoculating solution (90% DME/F12, 10% fetal bovine serum, 10) ⁵ Washing with U/L penicillin and 0.1g/L streptomycin solution for 3 times, settling tissue block to the bottom of test tube after each washing, discarding supernatant, and inoculating with inoculating solution (90% DME/F12, 10% fetal bovine serum, 10%) ⁵ U/L penicillin, 01g/L streptomycin solution) 1mL suspension, repeatedly blowing the tissue blocks in the suspension until turbidity occurs, and transferring the supernatant into a culture flask for later use. After 1mL of inoculation liquid is added, the mixture is blown again, the process is repeated for 3 to 4 times, and after the tissue blocks are gradually digested, the final residual blocks are discarded. About 4-5mL of the supernatant containing the single cell suspension was collected in a flask to inoculate the solution (90% DME/F12, 10% fetal bovine serum, 10) ⁵ U/L penicillin, 0.1g/L streptomycin solution) and cell counting was performed. According to about 1x10 ⁷ Density of individual cells/well cells were inoculated into 6-well plates and cultured for 4 hours until the cells were attached to the wall, and then the whole culture medium was changed (97% Neurobasal, 2% B27, 1% glutamine, 10) ⁵ After 3 days of culture with U/L penicillin and 0.1g/L streptomycin solution, cytarabine broth (97% Neurobasal, 2% B27, 1% glutamine, 10% was used instead ⁵ U/L penicillin, 0.1g/L streptomycin solution and cytarabine 2.5 ug/mL) for 3 days to inhibit the growth of glial cells and other cells, and obtain the primary neural cells which are simply cultured. And finally, using the whole culture solution for 1 time every 3 days, and changing the culture solution half at each time. On the fifth day of primary neuron culture, the primary neurons were further cultured for 24 hours after the polypeptide shown in SEQ.ID.NO. 5 was diluted to a target concentration (10 nM, 100 nM) with complete medium. Cells were lysed with RIPA solution and cell extracts were collected, western blot was performed with tau5 (a tau antibody that detects the total levels of human and mouse tau), AT8 (a phosphorylated tau antibody that detects the decrease in the level of tau phosphorylation detected by AT8 after dephosphorylation of Ser202 and Thr 205), pS396 antibody (a phosphorylated tau antibody that detects the decrease in the level of tau phosphorylation detected by pS396 after dephosphorylation of Ser 396) and the changes in tau phosphorylation and total tau protein were quantified.

As shown in fig. 2, it can be seen from fig. 2 that the chimera was effective in reducing the phosphorylation levels of tau protein AT Ser396 and AT8 epitopes in rat primary neuronal cells, and was dose dependent.

Test example 3

1mL of Trypsin solution was added to a HEK293 cell culture flask and digested for 1 min (37 ℃,5% CO 2). When the wall of the flask was tapped by hand to observe complete detachment of cells from the wall, the reaction was stopped by adding 1mL of serum-containing medium. Adding culture solution to blow cells, transferring to a culture plate for culturing, transfecting htau-GFP plasmid when the cells are attached and the cell density is about 60% -80%, observing the growth state of the cells after continuous culturing for 24 hours after transfection, adding polypeptide (0, 10nM, 20nM, 50nM, 100 nM) shown in SEQ ID.NO. 5 into the culture medium when more than 60% of the cells express GFP, and culturing the cells for 24 hours. Cells were lysed with RIPA solution and cell extracts were collected, immunoblotted with tau5 (a tau antibody that detects the total levels of human and mouse tau), AT8 (a phosphorylated tau antibody that detects the level of tau phosphorylation detected by AT8 after dephosphorylation of either human or mouse tau protein) and pS396 antibody (a phosphorylated tau antibody that detects the level of tau phosphorylation detected by pS396 after dephosphorylation of either human or mouse tau protein) and tau phosphorylation and total protein content was quantitatively analyzed.

As shown in FIG. 3, it can be seen from FIG. 3 that the chimera was effective in reducing the phosphorylation levels of tau protein AT Ser396 and AT8 epitopes in HEK293 cells transfected with htau-GFP, and was dose dependent.

Wherein: 1. the preparation method of the cell extract comprises the following steps:

(1) Observing the growth condition and density of cells in the cell culture plate under a microscope;

(2) Pre-cooling the 1 XPBS and the cell culture plate on ice, adding 80 μl/6cm dish per well of the 6-well plate, and adding 150 μl to prepare a 1 XPBS+PMSF (1:100) +proteinase mixed inhibitor (1:1000);

(3) Sucking out the culture medium by using a 1ml sample gun to cling to the bottom wall of a cell culture plate, adding 1ml of 1 XPBS to cling to the wall, gently flushing for 1-3 times according to the cell density, sucking out the PBS by using 1ml and 200 mu l of sample guns to cling to the bottom wall of the cell culture plate, adding the mixed solution, then cleaning the cell scraper by using ultrapure water, slightly scraping the bottom of the pore plate by using the cell scraper, sucking out the cell suspension to a 1.5ml EP tube, and cleaning the cell scraper between different samples;

(4) Boiling for 10min (boiling at maximum temperature and then maintaining boiling at 150deg.C, opening antioxidant epitope);

(5) After centrifugation, 20kHz ultrasonic wave is carried out for 20 times (in order to open a DNA chain, an ultrasonic machine is opened and closed according to I, after each time one sample is exceeded, the ultrasonic machine is firstly wiped by toilet paper in double distilled water for three times, and an ultrasonic gun head cannot contact air bubbles and the bottom of an EP tube);

(6) Mixing under shaking, and placing in a refrigerator at-20deg.C.

2. Process for quantitative analysis of tau protein content by immunoblotting

1. Set up rack (two glass plates, three bottles, five reagents, filter paper, toilet paper, dustbin, gun head, comb teeth)

(1) Cleaning the table top and the underframe, cleaning comb teeth, a glass plate, a distilled water bottle, an upper rubber bottle and a lower rubber bottle, drying the upper rubber bottle and the lower rubber bottle, taking out a reagent for preparing the electrophoresis gel, and recovering to room temperature;

(2) The higher glass plates are folded inwards, the upper part is pressed to enable the lower part to be clung to the tabletop to be flush, the clamp is outwards turned and clamped, and the clamp is placed on the underframe to be buckled.

(3) And (5) injecting double distilled water to check whether liquid leaks, and if so, reinstalling and then detecting the leakage.

2. Preparation of electrophoresis gel (see Table 1, AP and TEMED can be added 1.5 times during the day cold)

TABLE 1

(1) Sequentially adding 20% arc/Bis, tris buffer solution, TEMED and 10% AP, and blowing and uniformly mixing by a liquid shifter, wherein the whole process prevents bubbles from being mixed in the mixed solution;

(2) Slowly injecting the separation glue into the glue film along two corners (the glue is sucked deep under the liquid surface and gently blown and evenly mixed, a small amount of liquid is reserved at each gun head to prevent bubbles), the dosage of each glue is 3 multiplied by 900 mu l, gaps of the glue film are filled with double distilled water along the two corners after the glue is not leaked, and the oxygen inhibition polymerization is prevented and the glue at the lower part is kept flat for a period of time;

(3) After the gel is solidified for about 30min, pouring out double distilled water, sucking the residual water by using filter paper, and marking the upper edge of the lower gel by using a marker;

(4) Slowly injecting concentrated gel into the adhesive film along two corners, obliquely inserting comb teeth (sample amount is smaller than 20 μl and larger comb teeth are larger than 20 μl) of required specification from left to right, and feeding gel between lanes to avoid gel shrinkage, and waiting for gel for 50min.

3. Sample processing

Adding ready-prepared bromophenol blue and beta-mercaptoethanol (reducer) into the cell extract, wherein the bromophenol blue is as follows: beta-mercaptoethanol=1:3, mixed liquor: sample = 1:10 Boiling water at 100deg.C for 10min, shaking on a shaker for 20 seconds, packaging, and if the result is bad, boiling water at 100deg.C for 10min before loading.

4. Loading and electrophoretic separation of proteins (loading needle, sample, row plug, marker, electrophoresis liquid, electrophoresis tank, distilled water bottle)

Washing the conductive wire below the electrophoresis frame, transferring to the electrophoresis frame, marking lanes and numbers by using a Marker pen, slowly and vertically pulling out comb teeth, filling a gel groove with electrophoresis liquid, adding each lane by using a microsampler sample (1 μl of Marker is added to lanes 1 and 8, and adding bromophenol blue and 1 Xbuffer mixed liquid to lane 15 for balancing). After the sample is applied, transferring the electrophoresis frame to an electrophoresis tank, adding electrophoresis liquid, then covering a cover to enable red to be red and black to be black, after the sample is applied, firstly, performing electrophoresis for about 30min by using constant current 10 mA/block glue (according to two times of starting), and when the bromophenol blue indicator is electrophoresed until the junction of the concentrated glue and the separation glue is linear, changing the electrophoresis into constant voltage 100V (if the constant voltage can be adjusted to high current) electrophoresis for about 60min until bromophenol blue reaches the bottom of the gel, and completely running the Marker.

5. Transfer film (NC film, transfer film liquid, filter paper, ice box, basin, dish, transfer film groove, plastic plate, cleaning forceps)

(1) The NC film is marked by a marker pen and then immersed in the recovered film transferring liquid for 10min-20min (which is beneficial to fixing protein, balancing gel and removing SDS), the gel groove is taken down by pressing the bayonets at two sides, and the glass plate and the middle part of the right side of the white porcelain plate are pried by a small plate, so that the electrophoresis of the residual gel is kept during the period.

(2) Cutting the gel with glass plate vertically slightly inclined and slightly left and right according to the molecular weight range to be displayed, sticking three layers of filter paper immersed in the transfer membrane liquid on the gel with tweezers, carefully prying the gel on a sponge (with the filter paper facing downwards) with a small plate, sticking an inverted NC membrane on the other side, immersing the gel and the NC membrane in the transfer membrane liquid (with the gel on) to remove bubbles with a glass rod, carefully holding the gel on the hand (with the gel on) with tweezers, sticking the three layers of filter paper immersed in the transfer membrane liquid on the gel with tweezers, reversely putting the sponge, and sticking three layers of filter paper. Placing a black plastic plate, a layer of sponge, three layers of filter paper, glue, an NC film, three layers of filter paper, a layer of sponge and a transparent plastic plate from bottom to top, and fixing the transparent plastic plate by using rubber bands if the transparent plastic plate is not tight.

(3) The red, white and black are put into ice bath (gel is not required to be soaked in the film transferring liquid for a long time before electrifying, so that protein is prevented from being diffused and decomposed), the transferring current is constant current 276mA, the voltage is generally 140V (methanol can be properly supplemented to improve the voltage), the specific transferring time is determined according to the molecular weight of the protein to be transferred, the time is 1h when the molecular weight of the protein to be transferred is less than 100kDa, and the time is 1.5h when the molecular weight of the protein to be transferred is more than 100 kDa.

6. Immunoblotting (cleaning tweezers, box with double distilled water, milk, fresh-keeping bag, toilet paper, primary antibody, ice box, plate, transparent adhesive tape, TBST, black plastic bag, secondary antibody)

(1) Closing: after completion of transfer, NC membrane was carefully blocked with TBS blocking solution containing 5% skimmed milk powder at room temperature for 1h or overnight at 4℃with shaking, and filter paper not contacted with gum was recovered.

(2) Incubation resistance: taking out NC film, rinsing with 1 XTBS to remove residual milk stain on the surface of the film, standing on toilet paper with forceps to remove excessive water, and placing NC film Marker side outside into fresh-keeping bag, and draining with toilet paper. A primary antibody (0.1% Tween 20 added to reduce background) was added and the seal was attached to the plate (Marker and protein side facing up), and the scotch tape was incubated overnight at 4℃without pressing against the target strip.

(3) Secondary antibody incubation: the next day the NC film was removed from the incubation bag and the primary antibody was recovered, rinsed with TBST buffer for 3X 5min, rinsed with 1 XTBS to remove residual salt ions on the surface of the film, and the NC film was placed in a fresh-keeping bag with forceps standing on toilet paper to remove excess water, and drained with toilet paper. Luciferin-labeled goat anti-rabbit or goat anti-mouse Odyssey secondary antibody (0.1% tween 20 can be added to reduce background) is added in the dark, the seal is attached to a flat plate (the side with Marker and protein is upward), scotch tape is not pressed on a target strip, incubation is carried out at room temperature for 1h (more than 1h can increase background, sky cold can be prolonged to 2 h), NC membrane is taken out from incubation bag, secondary antibody is recovered, and rinsing is carried out for 3×5min with TBST buffer. After the rinsing, the residual salt ions on the surface of the film were removed by rinsing with 1 XTBS.

(4) Color development: the glass plate is wiped clean by using mirror wiping paper dipped with absolute ethyl alcohol. The Marker side is downward, and the Marker is placed in the scanner from top to bottom according to the molecular weight from small to large, covered with a plastic film and exhausted. Opening Odyssey software, clicking File, new, browse, inputting date, clicking Obtain image to select the scanning film length and width, scanning, imaging and storing original image, clicking Alter image to adjust image, marking sample and antibody type by text box, clicking Export image File, save, collecting film, and wiping glass plate with toilet paper.

It follows that the chimeras of the present invention that specifically degrade tau protein can degrade tau protein content in cells. Since abnormal accumulation of tau protein in cells is involved in more than 20 neurodegenerative diseases, the amount of accumulation of tau protein is positively correlated with neurodegeneration and memory impairment of these degenerative diseases, degradation of tau protein can achieve prevention or/and treatment of tau-related neurodegenerative diseases such as Alzheimer's disease, frontotemporal dementia linked to chromosome 17 with Parkinson's disease, pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathy, silver-philic granulomatosis, age-related tau astrocytopathy, chronic traumatic encephalopathy, globoid tauopathy, parkinson's disease, huntington's disease, cerebral stroke and epilepsy.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

SEQUENCE LISTING

<110> university of science and technology in China

<120> chimeras promoting tau protein dephosphorylation, genes encoding same, and uses thereof

<130> HBI0184HUST

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 13

<212> PRT

<213> amino acid sequence of tau protein binding sequence

<400> 1

Tyr Gln Gln Tyr Gln Asp Ala Thr Ala Asp Glu Gln Gly

1 5 10

<210> 2

<211> 4

<212> PRT

<213> amino acid sequence of linker sequence

<400> 2

Gly Ser Gly Ser

1

<210> 3

<211> 13

<212> PRT

<213> amino acid sequence of phosphatase recruiting sequence

<400> 3

Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro

1 5 10

<210> 4

<211> 8

<212> PRT

<213> amino acid sequence of transmembrane peptide

<400> 4

Arg Arg Arg Arg Arg Arg Arg Arg

1 5

<210> 5

<211> 38

<212> PRT

<213> amino acid sequence of chimera

<400> 5

Tyr Gln Gln Tyr Gln Asp Ala Thr Ala Asp Glu Gln Gly Gly Ser Gly

1 5 10 15

Ser Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Arg Arg

20 25 30

Arg Arg Arg Arg Arg Arg

35

<210> 6

<211> 114

<212> DNA

<213> coding sequence of chimera

<400> 6

taccagcagt accaggacgc cacggccgac gaacaagggg gcagcggcag caagaaggtg 60

gcagtggtcc gtactccacc caagtcgccg cgtcgtcgtc gtcgtcgtcg tcgt 114

<210> 7

<211> 8

<212> PRT

<213> amino acid sequence of flag tag

<400> 7

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 8

<211> 24

<212> DNA

<213> coding sequence of flag tag

<400> 8

gattacaagg atgacgacga taag 24

Claims

1. A chimeric body for promoting tau protein dephosphorylation, which is characterized in that the amino acid sequence of the chimeric body is shown as SEQ ID No. 5.

2. A gene encoding the chimera of claim 1 that promotes dephosphorylation of tau protein.

3. The gene according to claim 2, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 6.