E. coli engineered strain expressing 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 the People's Republic of China on January 29, 2022, with patent application number 2022101129048 and titled "Escherichia coli modified strain expressing fusion with Tau-4R protein," which is incorporated herein by reference in its entirety.

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

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

[0004] Alzheimer's disease is the most typical disease of dementia, accounting for about 55% of all dementia cases. Alzheimer's disease shows two major pathological features, amyloid plaques and neurofibrillary tangles, where the extracellular amyloid plaques are mainly composed of β-amyloid peptides, and the intracellular neurofibrillary tangles are mainly composed of Tau protein. In addition, it further includes synaptic dystrophy, astrocyte proliferation, microglial activation, and alteration of mature neuron markers. Clinically, it appears as accompanied by impaired cognitive ability and reduced memory ability. Currently, the drugs applied to treat AD mainly include two drugs, NMDA receptor antagonists (memantine) and acetylcholinesterase inhibitors (donepezil, galantamine), but these two drugs can only delay the symptoms of the disease and cannot reverse the progression of the disease.

[0005] The β-amyloid cascade hypothesis of Alzheimer's disease is believed to be driven by the accumulation of Aβ, which drives the onset of AD. Although Aβ-related mutations and the neurotoxicity of Aβ oligomers strongly support this hypothesis, Aβ plaques can also appear in the brains of normal cognitive subjects, raising doubts about the Aβ cascade hypothesis. On the other hand, the hypothesis that tau protein aggregation is the main factor in neurodegenerative diseases has recently become widely accepted, and hyperphosphorylated tau protein aggregates grow and spread between cells, and tau pathology is more closely related to the clinical symptoms of dementia than Aβ pathology. The β-amyloid antibody aducanumab (Biogen), which has been approved by the US FDA for accelerated approval, is used to treat Alzheimer's and reduces the burden of amyloid β plaques in patients, but also causes side effects such as temporary swelling of brain regions and headaches, confusion, dizziness, vision changes, nausea, angioedema, urticaria, amyloid protein-related imaging abnormalities, headaches, falls, diarrhea, and mental confusion.

[0006] Tau protein is a microtubule-associated protein involved in the development of the nervous system. It is concentrated around neuronal axons, binds to tubulin to form microtubules, stabilizes microtubules, and can promote axonal transport. Normally, the Tau gene encodes 16 exons and is translated into Tau with a length of 352 to 441 amino acids and a molecular weight of 45 to 65 kDa. Tau protein can be classified into the N-terminal projection functional region (N-terminus), proline-rich region, microtubule-binding region (MBD) and C-terminal functional region. Among them, the length of the N-terminus is not determined, and additional fragments can be inserted by exons 2 (E2) and 3 (E3). The proline-rich region contains multiple phosphorylation sites, and the C-terminus provides partial phosphorylation sites. The repeat sequence (R1-R4) is located in the MBD, which has the strongest microtubule-binding ability and promotes microtubule self-aggregation, and is also the core structure of double-helical filaments (PHFs). Human Tau is expressed in six isoforms, Tau352, 381, 383, 410, 412 and 441, due to differences in the way mRNA is edited, and the difference is whether there are three or four repeat sequences at the C-terminus and whether there are one or two inserts at the N-terminus. Exon 10 (E10) codes for the R2 fragment, and Tau that can retain E10 and has four repeat sequences is collectively called 4RTau, and Tau that does not retain E10 and has only three repeat sequences is collectively called 3RTau. In the adult brain, the expression levels of 3RTau and 4RTau are equivalent, while only 4RTau is expressed in adult mice.

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

[0008] In order to prevent or reverse the oligomerization of tau protein, the present inventors designed a fusion protein, Tau-4R, which can be recombinantly transformed into E. coli to achieve fusion expression. Experiments demonstrate that the tau protein produced in this strain is applicable to Alzheimer's treatment research. To this end, 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 shown in SEQ ID NO.1.

[0010] The present invention further provides a fusion protein, characterized in that it comprises the Tau-4R protein of the present invention. Preferably, said 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 said fusion protein is shown in SEQ ID NO.3.

[0012] The present invention further provides a derivative of the above 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 increases flexibility and allows fragments of the reporter protein to easily come into close proximity with each other to form complementarity.

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

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

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

[0017] The present invention further provides a recombinant expression vector, characterized in that it comprises a Tau-4R protein and / or a fusion protein thereof, or a derivative protein of said 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 of said fusion protein.

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

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

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

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

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

[0024] According to the present invention, preferably, 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 vector having the MBP tag 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, the protein purified using an amylose-resin affinity column is 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 derived 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 the Tau-4R protein, the fusion protein or a derivative of said fusion protein in a drug for extending the life span of patients with a neurological disease, preferably said neurological disease being 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 derived protein of said fusion protein is shown in SEQ ID NO.4.

[0033] Beneficial Effects of the Invention: 1. The inventors have designed a fusion protein Tau-4R that can be recombinantly expressed in E. coli. After purification by affinity column and ion exchange column, the purity is greatly improved and the production cost is greatly reduced. 2. Experiments have demonstrated that the tau protein produced by this strain can be applied to Alzheimer's treatment research. [Brief description of the drawings]

[0034] [Figure 1] Double enzyme digestion identification of 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). [Diagram 2] FIG. 1 is a sequence diagram of the Tau-4R gene. [Diagram 3]Identification of E. coli strains expressing fusion proteins with Tau-4R (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 fusion protein Tau-4R (1: before induction, 2: after induction, 3: precipitation, 4: precipitation after ultrasonication, 5: before injection of bacterial cell supernatant, 6: bacterial cell supernatant removal, 7-8: bacterial cell supernatant elution, 9: low molecular weight protein marker, 97.4, 66.2, 43, 31, 22.0, 14.4 KD). [Diagram 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] Optimization of flow for fusion expression of Tau-4R protein (1: Marker, 97.4, 66.2, 43, 31, 22.0, 14.4 KD, 2: Eluent). [Figure 7] These are the storages of fusion-expressed Tau-4R protein (1: Marker, 97.4, 66.2, 43, 31, 22.0, 14.4 KD, 2: after freeze-drying, stored at 4°C, 3: stored at -20°C, 4: stored at -80°C, 5: before freeze-drying). [Figure 8] TauVax immunization of triple transgenic AD mice shortened the latency period (stage-stepping time) of the fourth water maze exploration. [Figure 9] Effect of TauVax immunization on the stage jumping and stage-mounting latency (stage-mounting time) 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 PREFERRED EMBODIMENTS

[0035] The technical solution 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 illustrative and explanatory of the present invention 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 in the scope of the claims of the present invention.

[0036] Unless otherwise specified, all 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 protein Tau-4R, 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] CATATGACTGAAAATCTGAAACATCAACCTGGCGGCGGTAAAGGCTCTAAAGATAACATCAAGCACGTTCCTGGTGGCGGTTCTGGTTCCCTGGGTAACATCCATCACAAACCGGGTGGCGGTCAAGGTAGCCTGGATAATATCACTCATGTGCCAGGTGGTGGCAATCCATGG. The gene had an NdeI enzyme digestion site at the 5' end and an NcoI enzyme digestion site at the 3' end. It was synthesized by multi-PCR method, transformed into pUCm-T plasmid, screened on ampicillin LB plate, white colonies were selected by IPTG / X-gal staining and PCR identification was performed, and further identified by PCR, the plasmid was extracted and NdeI, NcoI double enzyme digestion was performed, and it was confirmed that the fragment size was between 100 and 200 bp, close to the theoretical value of 168 bp (Figure 1).

[0040] The plasmids correctly identified by enzyme digestion were sequenced, and the results showed that the base sequence was completely correct, the length size was correct, and it had 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 E. coli strain expressing fusion protein Tau-4R The pUCm-T-Tau-4R and pMal-p5x plasmids were double-digested with NdeI and NcoI, and the small and large fragment plasmids were collected, ligated with T4 ligase, transformed into DH5α competent bacteria, screened using 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, and the pMal-p5x-Tau-4R plasmid was obtained. Two clones were randomly selected and transferred to ampicillin LB medium and cultured overnight at 37 °C and 220 rpm. The next day, the clones were transferred to 5 mL of ampicillin LB medium at 1:1000 and cultured at 37 °C and 220 rpm for 2.5 hours, and then induced overnight by adding 1 mM IPTG. The pMal-p5x empty plasmid was used to transform a competent DH5α strain as a negative control. Then, SDS-PAGE analysis was performed.

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

[0043] Example 3. Purification of MBP-Tau-4R protein DH5α-pMal-p5x-Tau-4-1 strain was induced with 1 mM IPTG to obtain a bacterial sludge containing MBP-Tau-4R protein. The sludge was resuspended in 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4, and then disrupted by sonication. The sludge was centrifuged three times at 12000 rpm for 10 min, and then passed through 0.45 μm and 0.22 μm filters. The sludge was 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. The obtained protein was subjected to SDS-PAGE analysis.

[0044] As a result, a protein of approximately 50 KD was purified, which was consistent with 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 passed through a filter (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. The obtained protein was subjected to SDS-PAGE analysis, and the result showed that a protein of about 50 KD was purified (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 PQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSGGGGSGGGGSLGIEGRISHMTENLKHQPGGGKGSKDNIKHVPGGGSGSLGNIHHKPGGGQGSLDNITHVPGGGN Example 5. Optimization of MBP-Tau-4R protein purification flow The purified MBP-Tau-4R from the amylose-resin affinity column was dialyzed against 20 mM Tris-HCl (pH 7.4), applied to a Q-Sepharose FF ion-exchange column, and further eluted with 20 mM Tris-HCl, 200 mM NaCl (pH 7.4), resulting in a protein of even higher purity (Figure 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 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 concentration of MBP-Tau-4R protein was adjusted to 1 mg / mL and mixed with an equal volume of 2% aluminum hydroxide adjuvant to obtain TauVax. 15-month-old triple transgenic mice (APP / PS1 / Tau) were immunized with TauVax by intramuscular injection of 100 μL twice a week for three consecutive weeks. The experiment was divided into three groups, the first group was six 15-month-old normal mice C57BL / 6J injected with saline, the second group was six 15-month-old triple transgenic mice injected with saline, and the third group was six 15-month-old triple transgenic mice (APP / PS1 / Tau) injected with TauVax. After the experiment, water maze behavioral detection was performed, and the results showed that TauVax immunization of triple transgenic AD mice had little effect on the latency of the second water maze exploration, but could shorten the latency of the fourth water maze exploration, decreasing from 46.7±3.2 to 34.8±7.9 s (p<0.05) (Figure 8). This suggested that the learning ability of triple transgenic mice was significantly improved by TauVax injection.

[0053] Example 8. Stage jump test after immunization of triple transgenic mice with MBP-Tau-4R protein The four-stage reaction box for mice had a size of 25 cm × 25 cm × 30 cm, and the bottom of the box was a copper grid through which 36 V of continuous electrical stimulation was passed. In the center of each stage, a rubber pad with a diameter of 12 cm and a height of 4.5 cm was installed as a safe place to avoid electric shock of the mice. Before the experiment, the mice were allowed to adapt to the environment for 3 min, and then a 36 V alternating current was passed through the copper grid at the bottom. The reaction time when the mice jumped onto the rubber pad after receiving the electrical stimulation was recorded as the learning performance. After 24 h, the mice were placed on the stage again and allowed to adapt for 3 min, then placed on the rubber pad, and the latency period when they jumped onto the rubber pad after jumping off the stage for the first time was recorded as the memory performance. The results showed that the stage-climbing latency period of the model group mice was obviously longer than that of the normal 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 in a refrigerator at 4 °C for 24 h; on day 2, the tissue was removed from 4% paraformaldehyde and rinsed overnight in tap water; on day 3, the tissue was cut into appropriate sizes, marked with a pencil in an embedding box, passed through alcohol cylinders (concentrations 50%-75%-85%-95%-100%-TO clearing agent) at 1 h per cylinder, passed through paraffin cylinders (I wax-II wax-III wax, I wax is the oldest and III wax is the newest, put the wax in a drying box at 65 °C for 6-8 h in advance), and finally embedded in paraffin and stored in a refrigerator at 4 °C; on day 4, sectioned (4 μm)-baked (65 °C, 2 h), cleared with TO clearing agent (100%-95%-85%-75%-50%, dissolved in alcohol, 10 min per cylinder). The slides were then covered with an endogenous peroxidase blocker (10% hydrogen peroxide) and incubated at room temperature for 10 min to block, then washed three times with PBS for 5 min each time. The PBS was removed, the slides were placed on a glass slide, and normal goat serum working solution (1% normal goat serum) was dropped onto each section and incubated at room temperature for 10-15 min. The serum was removed, and primary antibodies 6E10 (amyloid1-16) or AT8 (S 202 T 205) was dropped onto the sections, left overnight at 4°C, and 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 at 37°C for 60 min. The sections were washed three times with PBS, and horseradish enzyme-labeled streptavidin (bound with the secondary antibody) was dropped onto the sections, incubated at room temperature for 10 min, and washed three times with PBS for 5 min each time. The PBS was removed, and fresh DAB solution was dropped onto the sections (completed in 1-2 min, and ended as soon as the sections turned yellow). The sections were immersed in tap water for 1-2 min, and counterstained with hematoxylin for 1 min. The sections were immersed in 1% hydrochloric acid alcohol for 2-3 s (to remove nonspecific staining), and then immersed in tap water for about 15-30 min to return them to blue. The sections were dehydrated with gradient alcohol (50%-75%-85%-95%-100%)-TO transparent agent-sealant, dried on the sixth day, and then observed under a microscope and photographed. The results showed that a large number of amyloid plaques appeared in the hippocampus of triple transgenic mice, and after TauVax treatment, the amyloid plaques were obviously reduced (Figure 10). At the same time, compared with wild-type mice, a large amount of phosphorylated Tau appeared in the hippocampus of triple transgenic mice, and the DAB staining area increased from 3.27±1.07% to 18.47±0.92% (p<0.0001), and after TauVax treatment, phosphorylated Tau was significantly reduced, and the DAB staining area decreased 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 within the scope described in this specification.

Claims

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

1.

2. A Tau-4R protein characterized in that:

2. A fusion protein or a derived protein thereof, The fusion protein comprises the Tau-4R protein of claim 1, Preferably, the fusion protein comprises MBP and a Tau-4R protein according to claim 1, Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.3, The derivative of the fusion protein adds or partially replaces a linker sequence based on the fusion protein, preferably the linker sequence is GGGGSGGGSGGGGGS; Preferably, the derived protein of the fusion protein has the amino acid sequence shown in SEQ ID NO.

4. A fusion protein or a derived protein thereof.

3. A polynucleotide comprising: The polynucleotide encodes the Tau-4R protein according to claim 1, the fusion protein according to claim 2 or a derived protein thereof, Preferably, the polynucleotide sequence encoding the Tau-4R protein according to claim 1 is as shown in SEQ ID NO. 2: A polynucleotide characterized by:

4. A recombinant expression vector or host cell expressing a gene for the Tau-4R protein according to claim 1, or a gene for the fusion protein or a derived protein thereof according to claim 2, Preferably, the gene sequence of the Tau-4R protein is shown in SEQ ID NO.

2. Recombinant expression vectors or host cells.

5. 1. A pharmaceutical composition comprising: The Tau-4R protein according to claim 1, or the fusion protein according to claim 2, or a protein derived therefrom, Preferably, the pharmaceutical composition comprises a pharma- ceutically acceptable vector or adjuvant, More preferably, the adjuvant is preferably an aluminium hydroxide adjuvant. A pharmaceutical composition comprising:

6. A method for producing the Tau-4R protein according to claim 1, the fusion protein according to claim 2 and / or a derivative protein of the fusion protein according to claim 3.

7. The method for producing the fusion protein includes the steps of: (1) obtaining a substance containing the Tau-4R protein gene according to claim 1; (2) ligating a vector carrying an MBP tag gene to a substance containing the Tau-4R protein gene obtained in step (1), transforming the substance, inducing expression, and obtaining the fusion protein; Preferably, the material containing the Tau-4R protein gene according to claim 1 may be a PCR product or a plasmid, among which, preferably, the step (1) comprises obtaining the material of the Tau-4R protein gene by a molecular biology method or a chemical synthesis method, Preferably, in the step (2), the vector having the MBP tag is one or more selected from pMal-p5x, pMAL-c2G, pMAL-c2x, pMAL-c4x, and pET-28b-MBP-His; Preferably, the fusion protein is inducibly expressed by IPTG. The method according to claim 6.

8. A method for purifying the fusion protein or a derived protein thereof according to claim 2, comprising the steps of: using the fusion protein purified by an amylose-resin affinity column; Preferably, after elution, the protein is eluted through an ion exchange column. More preferably, the protein purified through the amylose-resin affinity column is 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). Purification method.

9. Use of the Tau-4R protein according to claim 1, the fusion protein according to claim 2 or a derived protein thereof in the manufacture of a drug for treating a neurological disease, Preferably, the neurological disease is a neurodegenerative disease, more preferably Alzheimer's disease. use.

10. Use of the Tau-4R protein according to claim 1, the fusion protein according to claim 2 or a derived protein thereof in the manufacture of a drug for extending the life span of a patient with a neurological disease, Preferably, the neurological disease is a neurodegenerative disease, more preferably Alzheimer's disease. use.

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