Modified E. coli strain expressing the fusion protein Tau-4R

Abstract

The present invention discloses a fusion protein Tau-4R that can be recombinantly expressed in E. coli. Experiments have demonstrated that the tau protein produced in this strain is applicable to Alzheimer's disease treatment research.

Description

[Technical Field]

[0001] This application claims priority to a prior application, filed with the State Intellectual Property Office of China on January 29, 2022, under patent application number 2022101129048, entitled "Escherichia coli modified strain expressing fusion-type Tau-4R protein," which is incorporated herein by reference in its entirety.

[0002] [Technical field] The present invention relates to the field of biomedicine, and specifically, the present invention relates to a modified Escherichia coli strain that expresses a fusion protein, Tau-4R. [Background technology]

[0003] Alzheimer's disease (AD) is a neurodegenerative disease with a significantly higher incidence among the elderly population, and the number of people affected is increasing. In 2020, there were approximately 60 million AD patients worldwide, and by 2050, the number of AD patients is expected to reach approximately 150 million.

[0004] Alzheimer's disease (AD) is the most common form of dementia, accounting for approximately 55% of all dementia cases. It exhibits two major pathological features: amyloid plaques and neurofibrillary tangles. Extracellular amyloid plaques are primarily composed of beta-amyloid peptides, while intracellular neurofibrillary tangles are primarily composed of tau protein. Furthermore, AD manifests as synaptic dystrophy, astrocyte proliferation, microglial activation, and alterations of mature neuronal markers. Clinically, AD manifests as cognitive impairment and memory loss. Currently, the main drugs used to treat AD are NMDA receptor antagonists (memantine) and acetylcholinesterase inhibitors (donepezil, galantamine). However, these two drugs only delay disease symptoms and cannot reverse disease progression.

[0005] The β-amyloid cascade hypothesis of Alzheimer's disease posits that the accumulation of Aβ drives the onset of AD. While Aβ-related mutations and the neurotoxicity of Aβ oligomers strongly support this hypothesis, Aβ plaques can also appear in the brains of normal individuals, raising doubts about the hypothesis. On the other hand, the hypothesis that tau protein aggregation is the primary driver of neurodegenerative disease has recently gained widespread acceptance. Hyperphosphorylated tau protein aggregates propagate between cells, and tau pathology is more closely associated with the clinical symptoms of dementia than Aβ pathology. The β-amyloid antibody aducanumab (Biogen), approved under accelerated US FDA approval, is used to treat Alzheimer's disease and reduces the burden of amyloid β plaques in patients, but also causes side effects such as temporary swelling of brain regions and headache, confusion, dizziness, vision changes, nausea, angioedema, hives, amyloid protein-associated imaging abnormalities, headache, falls, diarrhea, and mental confusion.

[0006] Tau protein is a microtubule-associated protein involved in nervous system development. It is concentrated around neuronal axons, binds to tubulin to form microtubules, stabilizes microtubules, and facilitates axonal transport. Normally, the tau gene encodes 16 exons, translating into tau proteins with lengths of 352–441 amino acids and molecular weights of 45–65 kDa. Tau proteins can be divided into an N-terminal projection functional domain (N-terminus), a proline-rich domain, a microtubule-binding domain (MBD), and a C-terminal functional domain. The length of the N-terminus is not fixed, and additional fragments can be inserted via exons 2 (E2) and 3 (E3). The proline-rich domain contains multiple phosphorylation sites, and the C-terminus provides partial phosphorylation sites. Repeat sequences (R1–R4) located in the MBD have the strongest microtubule-binding ability, promote microtubule self-assembly, and also serve as the core structure of double-helical filaments (PHFs). Human tau expresses six isoforms, Tau352, 381, 383, 410, 412, and 441, due to differences in mRNA editing. These isoforms differ in whether they contain three or four repeats at the C-terminus and one or two inserts at the N-terminus. Exon 10 (E10) encodes the R2 fragment. Tau that retains E10 and has four repeats is collectively referred to as 4RTau, while tau that does not retain E10 and has only three repeats is collectively referred to as 3RTau. While the expression levels of 3RTau and 4RTau are comparable in adult brains, only 4RTau is expressed in adult mice.

[0007] Under pathological conditions, Tau undergoes conformational changes, aberrant mRNA splicing, dissociates from microtubules, undergoes a series of post-translational modifications, and forms pathological aggregates itself, which further induces neurodegenerative lesions and ultimately results in Tau pathology. Summary of the Invention

[0008] To prevent or reverse the oligomerization of tau protein, we designed a fusion protein, Tau-4R, which can be recombinantly expressed in E. coli. Experiments have demonstrated that the tau protein produced in this strain is applicable to Alzheimer's disease treatment research. Therefore, the technical solutions used in the present invention are as follows:

[0009] The present invention provides a Tau-4R protein, the amino acid sequence of which is set forth in SEQ ID NO.1.

[0010] The present invention further provides a fusion protein comprising the Tau-4R protein of the present invention. Preferably, the fusion protein comprises maltose binding protein (MBP) and the Tau-4R protein of the present invention.

[0011] According to the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO.3.

[0012] The present invention further provides a derivative protein of the fusion protein, characterized in that a linker sequence is added or partially replaced based on the fusion protein. Preferably, the linker sequence is GGGGSGGGSGGGGGS.

[0013] The linker sequence of the present invention is a flexible short peptide sequence that improves flexibility and allows fragments of the reporter protein to easily come close to each other and form complementarity.

[0014] According to the present invention, the amino acid sequence of the derivative protein of the above fusion protein is shown in SEQ ID NO.4.

[0015] The present invention further provides polynucleotides encoding the Tau-4R proteins, fusion proteins and / or derivative proteins of the fusion proteins of the present invention.

[0016] Preferably, the polynucleotide sequence is shown in SEQ ID NO.2.

[0017] The present invention further provides a recombinant expression vector comprising a Tau-4R protein and / or a fusion protein thereof, or a derivative protein of the fusion protein.

[0018] The present invention further provides a host cell characterized in that it contains a Tau-4R protein and / or a fusion protein thereof, or a derivative protein of said fusion protein.

[0019] According to the present invention, the fusion protein is obtained by inducing an expression strain of the Tau-4R protein with an MBP tag.

[0020] Preferably, the fusion protein is induced by IPTG, and the strain is transformed with a substance containing the Tau-4 gene of the present invention via a vector carrying an MBP tag. The substance containing the Tau-4 gene of the present invention may be a PCR product or a plasmid. The MBP-tagged vector is one or more selected from pMal-p5x, pMAL-c2G, pMAL-c2x, pMAL-c4x, and pET-28b-MBP-His.

[0021] The present invention provides pharmaceutical compositions comprising the Tau-4R protein of the present invention and / or a fusion protein thereof, or a derivative protein of the fusion protein.

[0022] The present invention further provides a method for producing the Tau-4R protein.

[0023] The present invention provides a method for producing the above-mentioned fusion protein or a derivative thereof, comprising the steps of: (1) obtaining a substance containing the Tau-4R protein gene of the present invention; (2) ligating the vector having the MBP tag to the substance containing the Tau-4R protein obtained in step (1), transforming the substance, inducing expression, and obtaining the fusion protein.

[0024] According to the present invention, the substance containing the Tau-4 gene of the present invention may be a PCR product or a plasmid. Preferably, the step (1) is obtained by a molecular biological method or a chemical synthesis method. In the step (2), the MBP-tagged vector is one or more selected from pMal-p5x, pMAL-c2G, pMAL-c2x, pMAL-c4x, and pET-28b-MBP-His. The fusion protein is induced by IPTG.

[0025] The present invention further provides a method for purifying a fusion protein, comprising using the fusion protein purified by an amylose-resin affinity column, preferably followed by further elution on an ion exchange column.

[0026] According to the present invention, proteins purified using an amylose-resin affinity column are eluted with 20 mM Tris-HCl (pH 7.4), then applied to a Q-Sepharose FF ion exchange column and further eluted with 20 mM Tris-HCl, 200 mM NaCl (pH 7.4).

[0027] The present invention further provides the use of a Tau-4R protein, a fusion protein, or a derivative protein of said fusion protein in the manufacture of a medicament for a neurological disease.

[0028] According to the present invention, the neurological disease is a neurodegenerative disease, more preferably Alzheimer's disease.

[0029] The present invention further provides the use of a Tau-4R protein, a fusion protein, or a derivative of said fusion protein in a drug for extending the lifespan of patients with a neurological disease, preferably a neurodegenerative disease, more preferably Alzheimer's disease.

[0030] According to the use of the present invention, the amino acid sequence of said Tau-4R protein is shown in SEQ ID NO.1.

[0031] According to the use of the present invention, the amino acid sequence of said fusion protein is shown in SEQ ID NO.3.

[0032] According to the use of the present invention, the amino acid sequence of the derivative protein of the above fusion protein is shown in SEQ ID NO.4.

[0033] Beneficial effects of the present invention: 1. The inventors have designed a fusion protein, Tau-4R, that can be expressed in E. coli through recombinant DNA technology. After purification using affinity columns and ion exchange columns, the purity is significantly improved and production costs are significantly reduced. 2. Experiments demonstrate that the tau protein produced by this strain is applicable to Alzheimer's disease treatment research. [Brief explanation of the drawings]

[0034] [Figure 1] Double enzyme digestion identification of the Tau-4R gene (1. Double enzyme digestion products, M:Marker 10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp). [Figure 2] FIG. 1 is a sequence diagram of the Tau-4R gene. [Figure 3]Identification of E. coli strains expressing the Tau-4R fusion protein (M: low molecular weight protein standard 97.4, 66.2, 43, 31, 22, 14.4 kD; 1: DH5α-pMal-p5x-Tau-4-1 strain before IPTG induction; 2: DH5α-pMal-p5x-Tau-4-1 strain after IPTG induction; 3: DH5α-pMal-p5x-Tau-4-2 strain before IPTG induction; 4: DH5α-pMal-p5x-Tau-4-2 strain after IPTG induction; 5: DH5α-pMal-p5x strain before IPTG induction; 6: DH5α-pMal-p5x strain after IPTG induction). [Figure 4] Purification of bacterial cells expressing the fusion protein Tau-4R (1: before induction, 2: after induction, 3: precipitation, 4: precipitation after sonication, 5: before injection of bacterial cell supernatant, 6: bacterial cell supernatant elution, 7-8: bacterial cell supernatant elution, 9: low molecular weight protein marker, 97.4, 66.2, 43, 31, 22.0, 14.4 KD). [Figure 5] Purification of the medium expressing the fusion protein Tau-4R (1: before medium injection, 2: after injection, 3: Marker, 97.4, 66.2, 43, 31, 22.0, 14.4 KD, 4: eluate). [Figure 6] Flow optimization for fusion-expressed Tau-4R protein (1: Marker, 97.4, 66.2, 43, 31, 22.0, 14.4 KD, 2: Eluent). [Figure 7] These are stored in a fusion with the Tau-4R protein (1: Marker, 97.4, 66.2, 43, 31, 22.0, 14.4 KD, 2: Stored at 4°C after lyophilization, 3: Stored at -20°C, 4: Stored at -80°C, 5: Before lyophilization). [Figure 8] TauVax immunization of triple transgenic AD mice shortens the latency period (stage-walking time) of the fourth water maze exploration. [Figure 9] This shows the effect of TauVax immunization on the latency period (stage-on time) for stage jumping and stage-on in triple transgenic AD mice. [Figure 10]TauVax immunization of triple transgenic AD mice can reduce amyloid plaque burden in the hippocampus (A. Wild-type mice; B. Triple transgenic mice treated with saline; C. Triple transgenic mice immunotreated with TauVax protein). [Figure 11] Tau-4R immunization reduces Tau protein phosphorylation in the hippocampus of triple transgenic mice. DETAILED DESCRIPTION OF THE INVENTION

[0035] The technical solutions of the present invention will be described in more detail below with reference to specific examples. It should be understood that the following examples are merely for illustrative purposes and should not be construed as limiting the scope of the claims of the present invention. Any technology realized based on the above content of the present invention is included within the scope of the claims of the present invention.

[0036] Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0037] Example 1. Gene design The amino acid sequence of the recombinant Tau-4R protein, SEQ ID NO. 1, is TENLKHQPGGGKGSKDNIKHV It was designed to be PGGGSGSLGNIHHKPGGGQGSLDNITHVPGGGN.

[0038] By optimizing the preferred codons of E. coli, the following gene was designed, and SEQ ID NO. 2 is as follows:

[0039] The gene contained an NdeI digestion site at the 5' end and an NcoI digestion site at the 3' end. The gene was synthesized by multi-PCR, transformed into pUCm-T plasmid, and screened on ampicillin-LB plates. White colonies were selected by IPTG / X-gal staining and identified by PCR. The plasmid was then extracted and double-digested with NdeI and NcoI. The fragment size was confirmed to be between 100 and 200 bp, close to the theoretical value of 168 bp (Figure 1).

[0040] Plasmids correctly identified by enzyme digestion were sequenced. The results showed that the base sequence was completely correct, the length was correct, and there was an NdeI enzyme digestion site at the 5' end and an NcoI enzyme digestion site at the 3' end (Figure 2).

[0041] Example 2. Establishment of an E. coli strain expressing a fusion protein, Tau-4R The pUCm-T-Tau-4R and pMal-p5x plasmids were double-digested with NdeI and NcoI. The small and large plasmid fragments were collected, ligated with T4 ligase, and transformed into DH5α competent bacteria. Screening was performed on ampicillin-LB plates and further identified by PCR. The plasmid was extracted and double-digested with NdeI and NcoI to confirm that the fragment size was between 100 and 200 bp. This resulted in the pMal-p5x-Tau-4R plasmid. Two clones were randomly selected and transferred to ampicillin-LB medium and grown overnight at 37°C and 220 rpm. The next day, the clones were transferred to 5 mL of ampicillin-LB medium at a 1:1000 ratio and grown for 2.5 hours at 37°C and 220 rpm. After overnight induction with 1 mM IPTG, the pMal-p5x empty plasmid was transformed into competent DH5α bacteria as a negative control. SDS-PAGE analysis was then performed.

[0042] The results showed that the DH5α-pMal-p5x-Tau-4-1 and DH5α-pMal-p5x-Tau-4-2 strains expressed nascent proteins at around 50 KD, while DH5α-pMal-p5x expressed nascent proteins at 43 KD. Thus, we established the E. coli strains DH5α-pMal-p5x-Tau-4-1 and DH5α-pMal-p5x-Tau-4-2, which successfully expressed the fusion protein Tau-4R.

[0043] Example 3. Purification of MBP-Tau-4R protein DH5α-pMal-p5x-Tau-4-1 was induced with 1 mM IPTG to obtain a bacterial sludge containing the MBP-Tau-4R protein. The sludge was resuspended in 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4, and then sonicated. The sludge was centrifuged three times at 12,000 rpm for 10 min, then passed through 0.45 μm and 0.22 μm filters and applied to an amylose-resin column. The eluate was then loaded into 10 mM maltose, 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4, and the resulting protein was subjected to SDS-PAGE analysis.

[0044] As a result, a protein of approximately 50 kD was purified, which matched the theoretical molecular weight of the MBP-Tau-4R protein, but the purity of the target protein was only 34% (Figure 4).

[0045] The bacterial culture supernatant was filtered through a membrane (first 0.45 μm, then 0.22 μm), applied to an amylose-resin column, and eluted with 10 mM maltose, 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4. SDS-PAGE analysis of the purified protein revealed a protein of approximately 50 kDa (Figure 5). The purity of the target protein obtained in the medium was relatively high, at 60%.

[0046] The amino acid sequence of MBP-Tau-4R, SEQ ID NO. 3, is as follows: KIEEGKLVIWINGDKG YNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPK TWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAA SGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISHMTENLKHQPGGGKGSKDNIKHVPGGGSGSLGNIHHKPGGGQGSLDNITHVPPGGGNPWAAAISSTDPNSLQVIK.

[0047] Example 4. Derivatization of MBP-Tau-4R protein The new derivative molecule was obtained by replacing NNNNNNNNNN with GGGGSGGGGS, the sequence of which is SEQ ID NO. 4:

[0048] KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVE ALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNI PQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSGGGGSGGGGSLGIEGRISHMTENLKHQPGGGKGSKDNIKHVPGGGGSGSLGNIHHKPGGGQGSLDNITHVPGGGN Example 5. Optimization of MBP-Tau-4R protein purification flow The purified MBP-Tau-4R was dialyzed against 20 mM Tris-HCl (pH 7.4) and applied to a Q-Sepharose FF ion-exchange column. The purified protein was further purified by elution with 20 mM Tris-HCl, 200 mM NaCl (pH 7.4) (Fig. 6).

[0049] Example 6. Screening of MBP-Tau-4R protein storage conditions The MBP-Tau-4R protein was freeze-dried and stored at 4°C for one month. SDS-PAGE analysis was performed together with samples that had not been freeze-dried and were stored at -20 and -80°C for one month to determine the purity of the target protein.

[0050] [Table 1]

[0051] As shown in FIG. 7 and Table 1, no obvious decrease in the purity of the MBP-Tau-4R protein was observed.

[0052] Example 7. Immunization of triple transgenic mice with MBP-Tau-4R protein and water maze behavioral testing The MBP-Tau-4R protein concentration was adjusted to 1 mg / mL and mixed with an equal volume of 2% aluminum hydroxide adjuvant to obtain TauVax. Fifteen-month-old triple transgenic mice (APP / PS1 / Tau) were immunized with TauVax by intramuscular injection of 100 μL twice weekly for three consecutive weeks. The experiment was divided into three groups: Group 1 consisted of six 15-month-old normal C57BL / 6J mice injected with saline; Group 2 consisted of six 15-month-old triple transgenic mice injected with saline; and Group 3 consisted of six 15-month-old triple transgenic mice (APP / PS1 / Tau) injected with TauVax. After the experiment, behavioral testing in the water maze was performed. The results showed that TauVax immunization of the triple transgenic AD mice significantly improved the latency of the second water maze exploration, but not the latency of the fourth water maze exploration, from 46.7 ± 3.2 to 34.8 ± 7.9 s (p < 0.05) (Figure 8). This suggests that TauVax injection significantly improved the learning ability of the triple transgenic mice.

[0053] Example 8. Stage jumping test after immunization of triple transgenic mice with MBP-Tau-4R protein The four-stage mouse reaction chambers measured 25 cm x 25 cm x 30 cm, with a copper grid at the bottom, through which a 36 V continuous electrical stimulus was applied. A 12 cm diameter, 4.5 cm high rubber pad was placed in the center of each stage as a safe haven to protect the mice from electric shock. Before the experiment, mice were allowed to adapt to the environment for 3 min, and then a 36 V AC current was applied to the copper grid at the bottom. The reaction time for the mice to jump onto the rubber pad after receiving the electrical stimulus was recorded as learning performance. After 24 h, the mice were placed back on the stage for 3 min of adaptation, then placed on the rubber pad. The latency to jump onto the rubber pad after jumping off the stage for the first time was recorded as memory performance. Results showed that the latency to jump onto the stage in the model group mice was significantly longer than that in the control group mice in the first and second memory recall tests. After TauVax treatment, the stage-mounting latency of the treated mice was shortened (Figure 9), suggesting that the learning and memory abilities of the triple transgenic mice were impaired, while TauVax treatment improved the learning and memory abilities of the triple transgenic mice.

[0054] Example 9. Immunohistochemical staining after immunization of triple transgenic mice with MBP-Tau-4R protein After behavioral detection was completed, mice were perfused. On day 1, the heart was perfused, the brain tissue was extracted, and fixed in 4% paraformaldehyde for 24 h in a refrigerator at 4°C. On day 2, the tissue was removed from the 4% paraformaldehyde and rinsed overnight in tap water. On day 3, the tissue was cut into appropriate sizes, marked with a pencil on an embedding box, and passed through alcohol cylinders (concentrations: 50%, 75%, 85%, 95%, 100%, TO clearing agent) for 1 h per cylinder, then through paraffin cylinders (I wax-II wax-III wax, I wax is the oldest, III wax is the newest; the wax is placed in a dry box at 65°C for 6-8 h in advance). Finally, the tissue was embedded in paraffin and stored in a refrigerator at 4°C. On day 4, the tissue was sectioned (4 μm), baked (65°C, 2 h), cleared in TO clearing agent (100%, 95%, 85%, 75%, 50%, dissolve in alcohol, 100% per cylinder). The slides were then incubated at 97.5°C for 40 min for antigen retrieval (EDTA citrate repair solution), followed by adding an endogenous peroxidase blocker (10% hydrogen peroxide) to cover the slides and blocking by incubating at room temperature for 10 min. The slides were then washed three times with PBS for 5 min each. The PBS was then removed, and the slides were placed on a glass slide. Normal goat serum working solution (1% normal goat serum) was added dropwise to each section and incubated at room temperature for 10-15 min. The serum was then removed, and the primary antibodies 6E10 (amyloid 1-16) or AT8 (S 202 T 205) was added dropwise and incubated overnight at 4°C. On the fifth day, the sections were washed three times with PBS, and a secondary antibody (biotin-labeled goat anti-mouse IgG) was added and incubated for 60 min at 37°C. After washing three times with PBS, horseradish enzyme-labeled streptavidin (conjugated to the secondary antibody) was added dropwise and incubated for 10 min at room temperature. Then, the sections were washed three times with PBS for 5 min each. The PBS was removed, and fresh DAB solution was added dropwise (1–2 min; the incubation was stopped as soon as the sections turned yellow). The sections were then immersed in tap water for 1–2 min and counterstained with hematoxylin for 1 min. The sections were then immersed in 1% HCl alcohol for 2–3 s (to remove nonspecific staining), and then immersed in tap water for approximately 15–30 min to restore the blue color. The sections were dehydrated through a gradient of alcohol (50%, 75%, 85%, 95%, 100%), sealed with TO transparency agent, and dried on the sixth day. The sections were then observed and photographed under a microscope. The results showed that numerous amyloid plaques appeared in the hippocampus of triple transgenic mice, and that amyloid plaques were significantly reduced after TauVax treatment (Figure 10). At the same time, compared with wild-type mice, the triple transgenic mice exhibited a large amount of phosphorylated tau in the hippocampus, with the DAB staining area increasing from 3.27±1.07% to 18.47±0.92% (p<0.0001). After TauVax treatment, phosphorylated tau was significantly reduced, with the DAB staining area decreasing from 18.47±0.92% to 10.63±1.36% (p<0.01) (Figure 11).

[0055] The technical features of the embodiments described above can be combined in any manner. For the sake of brevity, not all possible combinations of the technical features in the embodiments described above are described. However, as long as no contradiction arises in the combination of these technical features, any combination should be considered to be within the scope described in this specification.

Claims

1. A Tau-4R protein, Its amino acid sequence is shown in SEQ ID NO.

1. A Tau-4R protein characterized by:

2. A fusion protein comprising: The fusion protein comprises the Tau-4R protein of claim 1. A fusion protein characterized by:

3. The fusion protein comprises MBP and the Tau-4R protein of claim 1. The fusion protein according to claim 2 .

4. The fusion protein comprises a linker sequence GGGGSGGGGS, The fusion protein according to claim 3 .

5. The amino acid sequence of the fusion protein is shown in SEQ ID NO. 3 or SEQ ID NO.

4. The fusion protein according to claim 3 .

6. A gene encoding the Tau-4R protein of claim 1. A polynucleotide characterized by:

7. The sequence of the polynucleotide is shown in SEQ ID NO.

2. The polynucleotide of claim 6.

8. Encoding the fusion protein of claim 2 A polynucleotide characterized by:

9. A method for expressing the gene encoding the Tau-4R protein of claim 1. Recombinant expression vector or host cell.

10. Expressing the gene for the fusion protein according to claim 2. Recombinant expression vector or host cell.

11. 10. A method for treating a Tau-4R infection comprising administering to a subject a Tau-4R protein ... A pharmaceutical composition comprising:

12. 3. The fusion protein of claim 2, A pharmaceutical composition comprising:

13. Use of the Tau-4R protein of claim 1 in the manufacture of a drug for treating a neurodegenerative disease.

14. 10. Use of the fusion protein of claim 2 in the manufacture of a medicament for treating a neurodegenerative disease.

15. The neurodegenerative disease is Alzheimer's disease.

15. The use according to claim 14.

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