Anti-p-tau181 antibodies, methods of making and uses thereof

Abstract

The application relates to the technical field of monoclonal antibodies, and specifically discloses an anti-p-Tau181 antibody and a preparation method and application thereof. The preparation method of the anti-p-Tau181 antibody comprises the following steps: taking a p-Tau181 polypeptide IPAKTPPAPK(pT)PPSSGEPPK as an immunogen, injecting the immunogen into experimental animals to perform an immune response, and obtaining spleen lymphocytes of the experimental animals; taking myeloma cells and the spleen lymphocytes to perform mixed culture to obtain fusion cells; culturing the fusion cells, obtaining supernatant, and performing ELISA detection; the polypeptide used for detection is the p-Tau181 polypeptide; taking the hybridoma cell strain with a positive detection result to perform counter screening with a p-Tau217 polypeptide, a p-Tau231 polypeptide and t-tau protein; screening a positive clone which has a reaction to the p-Tau181 polypeptide and has no reaction to the p-Tau217 polypeptide, the p-Tau231 polypeptide and the t-tau protein; performing multiple cloning screening; obtaining a hybridoma cell strain which can secrete antibodies every time; and taking the antibodies secreted by the hybridoma cell strain as the anti-p-Tau181 antibody. The anti-p-Tau181 antibody can be specifically combined with the p-Tau181, and thus can be used for detecting Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of monoclonal antibodies, and particularly relates to an anti-p-Tau181 antibody and a preparation method and application thereof. BACKGROUND

[0002] Tau is a microtubule-associated protein that can interact with other microtubule proteins under normal physiological conditions, promote microtubule assembly, and regulate microtubule stability and dynamic properties. However, under certain pathological conditions, Tau protein can be modified, mainly through phosphorylation, which can produce abnormal aggregates that are toxic to neurons. When the Tau protein is hyperphosphorylated after translation, it will dissociate from the microtubule, leading to its self-aggregation and microtubule disassembly.

[0003] Alzheimer's disease (AD) is a neurodegenerative disease that seriously affects the health of the elderly, and its pathological features are that amyloid-beta (Aβ) accumulates into plaques outside cells and hyperphosphorylated Tau accumulates into neurofibrillary tangles inside neurons. Although most drug trials for Alzheimer's disease have targeted Aβ or pathways related to Aβ, such as β and γ secretase cleavage, Tau protein is increasingly being recognized as another important target. Current treatments for Alzheimer's disease can only alleviate symptoms, but cannot prevent or cure the disease. Neurofibrillary tangles formed by Tau protein aggregation are one of the defining features of Alzheimer's disease, which makes Tau an attractive therapeutic target. Current drug strategies targeting tau protein can be summarized as inhibiting Tau aggregation, inhibiting Tau phosphorylation, reducing Tau levels, and Tau immunization, and many Tau-specific monoclonal antibodies are in clinical trials. The increased threonine 181 phosphorylation product (p-Tau181) in the early stages of Alzheimer's disease is the most widely used p-Tau biomarker in clinical practice. There is a need for a substance that can specifically bind p-Tau181 to identify Alzheimer's disease. SUMMARY

[0004] In view of the current need for a substance that can specifically bind p-Tau181 to identify Alzheimer's disease, the application proposes an anti-p-Tau181 antibody and a preparation method and application thereof. To this end, the application adopts the following technical solutions.

[0005] In a first aspect, the application proposes a preparation method of an anti-p-Tau181 antibody, and adopts the following technical solutions.

[0006] The preparation method of the anti-p-Tau181 antibody comprises the following steps:

[0007] The immune step: taking a p-Tau181 polypeptide IPAKTPPAPK(pT)PPSSGEPPK as an immunogen, injecting the immunogen into an experimental animal to perform an immune response, and obtaining spleen lymphocytes of the experimental animal;

[0008] The cell fusion step: mixing myeloma cells with the spleen lymphocytes for culture to obtain fusion cells; adding a culture solution to the fusion cells for culture to obtain a primary culture solution;

[0009] The cell screening step: taking supernatant of the primary culture solution for ELISA detection, taking a p-Tau181 polypeptide as a detection polypeptide, taking hybridoma cell strains with positive detection results for counter screening with a p-Tau217 polypeptide, a p-Tau231 polypeptide and a t-tau protein, screening positive clones that have a reaction to the p-Tau181 polypeptide and have no reaction to the p-Tau217 polypeptide, the p-Tau231 polypeptide and the t-tau protein, performing multiple cloning screening to obtain hybridoma cell strains that can secrete antibodies each time, and taking the antibodies secreted by the hybridoma cell strains as the anti-p-Tau181 antibodies.

[0010] By adopting the technical solution, the hybridoma cell strains can secrete the anti-p-Tau181 antibodies, the anti-p-Tau181 antibodies can specifically bind to p-Tau181, and thus can be used for detecting Alzheimer's disease. In the cell fusion step, the myeloma cells and the spleen lymphocytes are uniformly mixed at a ratio of 5:1 to 10:1.

[0011] A preferred solution of the preparation method of the anti-p-Tau181 antibodies further includes a cell culture and monoclonal antibody purification step.

[0012] The hybridoma cell strains are amplified and cultured, the liquid after the amplification and culture is collected, treated with an ammonium sulfate solution to obtain a precipitate, the precipitate is dissolved in PBS, purified, and the anti-p-Tau181 antibodies are obtained.

[0013] By adopting the technical solution, a large amount of anti-p-Tau181 antibodies are obtained.

[0014] A preferred solution of the preparation method of the anti-p-Tau181 antibodies specifically includes the following steps.

[0015] The p-Tau181 polypeptide is mixed with the immunoadjuvant squalene at a mass ratio of 1: (2.5-3) to obtain an immunoreagent, and a plurality of 6-8-week-old BALB / c mice are immunized for the first time by subcutaneous injection, and the injection dose of the first immunoreagent is 40-60 μg per mouse; after an interval of 10-20 days, the second immunization is performed; the second immunization is: the p-Tau181 polypeptide is mixed with the immunoadjuvant squalene at a mass ratio of 1: (2.5-3), and the mixture is injected into the mice after the first immunization by subcutaneous injection, and the injection dose of the second immunoreagent is 40-60 μg per mouse;

[0016] After the second immunization, 5-10 days later, the blood of each mouse is collected, and the antibody titer in the serum is detected: first, a 5-15 μg / ml p-Tau181 polypeptide is coated on a multi-well enzyme plate for 20-30 h, then 0.5%-1.5% bovine serum albumin is used to block the multi-well enzyme plate at 36-38°C for 0.5-1.5 h, then a plurality of dilution multiples of unimmunized mouse serum and twice-immunized mouse serum are added to each well, and incubated at 36-38°C for 0.5-1.5 h, then HRP-labeled secondary antibody is added and incubated at 36-38°C for 0.5-1.5 h, then TMB substrate solution is added, and reacted at room temperature for 10-20 min, and finally, sulfuric acid is used to terminate the reaction, and the OD value at 450 nm is read to screen out the BALB / c mouse with the highest P / N value of the immune titer.

[0017] By using the above technical solution, the experimental mice obtain the ability to be immunized with p-Tau181, and the spleen lymphocytes can secrete anti-p-Tau181 antibodies, and the mice that can stably secrete anti-p-Tau181 antibodies are screened out.

[0018] In a second aspect, the present application also provides an anti-p-Tau181 antibody, and uses the following technical solution.

[0019] An anti-p-Tau181 antibody, the VH gene sequence encoding the anti-p-Tau181 antibody is:

[0020] AACCACCTTTTAGGAACTGGCGGTGTCCACTCCCAGGTCCAACTGCAGCAACCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACATTCACCAGCTACTGGATGCACTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTAGTACTCACTACGATGAAAAGTTCAGGAACAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGTAGTGGGAGACTACTGGGGCCAAGGCAGCACTCTCACAGTCTCCTCCGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATTGTTATCCT.

[0021] By adopting the technical scheme, the anti-p-Tau181 antibody has the property of specifically binding to p-Tau181. The VH is a variable region of a heavy chain.

[0022] In one preferred embodiment of the anti-p-Tau181 antibody, the VL gene sequence encoding the anti-p-Tau181 antibody is as follows:

[0023] GTTGACATTGATGACATTGAGGTCACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGAGCGTACACGTC.

[0024] By adopting the technical scheme, the VH sequence and the VL sequence of the anti-p-Tau181 antibody are the positions combined with the anti-p-Tau181 antibody, and the anti-p-Tau181 antibody has the property of specifically combining with p-Tau181. The VL is a light chain variable region.

[0025] In a third aspect, the application further provides an application of the anti-p-Tau181 antibody, and adopts the following technical scheme.

[0026] The application of the anti-p-Tau181 antibody is that the anti-p-Tau181 antibody is used to improve the Aβ25-35-induced N2a cell damage.

[0027] By adopting the technical scheme, the application field of the anti-p-Tau181 antibody is improved.

[0028] Another application of the anti-p-Tau181 antibody is that the anti-p-Tau181 antibody is used to improve the cognitive function of OA-induced Alzheimer's disease (AD) rats.

[0029] By adopting the technical scheme, the anti-p-Tau181 antibody can be used to improve the cognitive function of Alzheimer's disease animals, and has the prospect of further clinical trials to improve the cognitive function of Alzheimer's disease patients.

[0030] In the application of the anti-p-Tau181 antibody, the cognitive function is the spatial learning and memory ability.

[0031] By adopting the technical scheme, the anti-p-Tau181 antibody has the prospect of further clinical trials to improve the spatial learning and memory ability of Alzheimer's disease patients.

[0032] In summary, the anti-p-Tau181 antibody and the preparation method and application thereof have the following beneficial effects:

[0033] The hybridoma cell strain capable of secreting the anti-p-Tau181 antibody is obtained by immunization, cell fusion and cell screening, and the anti-p-Tau181 monoclonal antibody can be industrially produced by further cell culture and monoclonal antibody purification of the hybridoma cell strain. The anti-p-Tau181 monoclonal antibody can specifically combine with p-Tau181, and thus can be used for detecting Alzheimer's disease.

[0034] The anti-p-Tau181 monoclonal antibody is applied to AD cells and animal models, and can improve the survival rate of AD cells and the behavior and ability of AD rats. The monoclonal antibody of the application has high clinical application value in the detection, prevention and treatment of AD. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Serum titer comparison bar chart for mice immunization of Example 1.

[0036] Figure 2 VH, VL gene electropherogram for retrieving anti-p-Tau181 monoclonal antibody of Example 1.

[0037] Figure 3 Purity identification electropherogram of anti-p-Tau181 monoclonal antibody of Example 2.

[0038] Figure 4 Affinity results chart of test anti-p-Tau181 antibody and p-Tau181 antigen of Example 2.

[0039] Figure 5 Results chart of evaluating protective effect of anti-Tau antibody on Aβ25-35 induced PC12 cells of Example 3.

[0040] Figure 6 Test results chart of evaluating spatial learning and memory ability of rats in water maze experiment of Example 3.

[0041] Figure 7 Y-maze test of Example 3 was used to track the results of latitudinal memory function test. DETAILED DESCRIPTION

[0042] Example 1, preparation of monoclonal antibody specifically recognizing p-Tau181 phosphorylation site

[0043] 1. Immunogen

[0044] The immunogen is IPAKTPPAPK(pT)PPSSGEPPK, which is a p-Tau181 polypeptide. The polypeptides used in the present application are synthesized by Shenguo Biotechnology Co., Ltd.

[0045] 2. Mouse immunization

[0046] 5 six to eight week old BALB / c mice were used as experimental subjects, one of which was not injected with immunogen as a blank control. The p-Tau181 polypeptide was mixed with the immunoadjuvant squalene at a mass ratio of 1:2.8, and 4 six to eight week old BALB / c mice were immunized by subcutaneous injection, with an immunization dose of 50 μg per mouse. Two weeks later, the same method and dose were used for the second immunization. After two immunizations, the tail blood was taken to determine the serum titer by ELISA gradient dilution. According to the results, it was determined whether to boost the immunization. This example was performed twice.

[0047] One week after the last immunization, blood was collected from 5 mice and the antibody titers in 5 serum samples were tested: 10 μg / ml p-Tau181 peptide was first coated on a 96-well ELISA plate at 4°C for 24 h, and then blocked with 1% (wt) BSA (bovine serum albumin) at 37°C for 1 h. Figure 1 Then, five serum samples diluted 1000, 3×1000, 32×1000, 33×1000, 34×1000, 35×1000, 36×1000, and 2×36×1000 were added and incubated at 37°C for 1 hour. HRP (horseradish peroxidase)-labeled secondary antibody was then added and incubated at 37°C for 1 hour. TMB (tetramethylbenzidine) substrate solution was then added and allowed to react at room temperature in the dark for 15 minutes. The reaction was terminated with 2M sulfuric acid and the OD value at 450nm was read. The highest dilution factor with a P / N ≥ 2.1 was used as the serum titer. Results are shown in the table. Figure 1 , Figure 1 Part A shows that after immunization, all four mice produced corresponding immune titers, among which the immune titer P / N value (OD value of immune mice / OD value of non-immunized mice) of mouse 4 was the highest at the same dilution (e.g. Figure 1 Therefore, mouse 4 was selected as the experimental subject in the next step.

[0048] 3. Cell Fusion

[0049] Mouse 4, with the highest P / N value, was sacrificed by cervical dislocation and sterilized by immersion in 75% alcohol for 5 minutes. Mouse 4 was pinned to a foam board. The abdominal skin was cut open, the abdominal cavity was opened, and fat and connective tissue from the spleen were removed. The spleen surface was rinsed with Dulbecco's Modified Eagle Medium (DMEM). A 200-mesh cell sieve was placed in a dish and moistened with 10 mL of DMEM. The spleen was gently crushed and triturated with a syringe plunger. The cells were collected into a 50 mL centrifuge tube and centrifuged at 1500 rpm for 3 minutes at room temperature. The supernatant was discarded. 5 mL of red blood cell lysis buffer, previously brought to room temperature, was added to remove cell debris. 5 mL of DMEM was then added, mixed, and centrifuged at 1500 rpm for 3 minutes at room temperature. The supernatant was discarded to obtain splenic lymphocytes from mouse 4.

[0050] SP2 / 0 cells (mouse myeloma cells) were centrifuged at 1500 r / min for 3 min at room temperature, and the supernatant was discarded. The SP2 / 0 cells were mixed with the spleen lymphocytes of mouse 4 at a ratio of 8:1 in a 50 mL centrifuge tube, 10 mL of DMEM medium was added, and the mixture was centrifuged at 1500 r / min for 3 min at room temperature. After the supernatant was discarded, the following operations were performed in a 37°C water bath: 1 mL of 37°C preheated polyethylene glycol 1500 (PEG1500) was slowly added dropwise into the centrifuge tube while oscillating, 1 mL of DMEM medium was then added, and oscillation was performed for 1 min. Subsequently, 8 mL of DMEM medium was added to terminate the fusion, and the mixture was centrifuged at 1500 r / min for 3 min. The supernatant was discarded, and the fused cells were mixed uniformly in a high-concentration nucleotide precursor medium, and then were added dropwise into a 96-well cell culture plate in which feeder cells had been added.

[0051] 4. Cell screening

[0052] After the cells in the culture plate grew to a medium size, the cell supernatant was removed for ELISA detection. The polypeptide used for detection was p-Tau181 polypeptide. After two days, the detection was performed again, and the hybridoma cell strain with positive results in both detections was subjected to counter-screening with p-Tau217 polypeptide, p-Tau231 polypeptide, and t-tau protein. The positive clone screened with a reaction to p-Tau181 polypeptide and no reaction to p-Tau217 polypeptide, p-Tau231 polypeptide, and t-tau protein was subjected to three times of cloning (each time of cloning was subjected to differential screening), and a hybridoma cell strain capable of stably secreting antibodies was obtained. Finally, the 2-1E cell strain (hybridoma cell strain) against p-Tau181 polypeptide was obtained.

[0053] 5. Cell culture

[0054] The stable 2-1E cell strain was cultured for expansion in a carbon dioxide incubator, and was sequentially transferred from a 96-well plate to a 24-well plate, a 6-well plate, and a 10 cm cell plate. Then, the cells in the cell plates were collected, and were injected into the abdominal cavity of a mouse. After 7 to 10 days, the ascites containing the monoclonal antibody (anti-p-Tau181 antibody) was removed from the abdominal cavity of the mouse.

[0055] 6. Monoclonal antibody purification

[0056] The mouse ascites containing the monoclonal antibody was treated with a 50% saturated ammonium sulfate solution. Then, the obtained precipitate was dissolved in PBS (phosphate buffered saline), and was purified using a Protein A column to obtain a purified monoclonal antibody. The obtained monoclonal antibody was subjected to PCR amplification, and the PCR product was sent to a company for sequencing to obtain a sequence.

[0057] 7. Results and analysis

[0058] 7.1 VH and VL gene retrieval

[0059] Figure 2 The electrophoresis map of PCR products shows that the VH, VL genes of anti-p-Tau181 antibody are successfully retrieved (500bp).

[0060] 7.2 Gene sequencing results

[0061] The VH, VL gene sequences of anti-p-Tau181 antibody obtained by gene sequencing are as follows:

[0062] VH Seq:

[0063] AACCACCTTTTAGGAACTGGCGGTGTCCACTCCCAGGTCCAACTGCAGCAACCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACATTCACCAGCTACTGGATGCACTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTAGTACTCACTACGATGAAAAGTTCAGGAACAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGTAGTGGGAGACTACTGGGGCCAAGGCAGCACTCTCACAGTCTCCTCCGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATTGTTATCCT

[0064] AA1:

[0065] NHLLGTGGVHSQVQLQQPGSELVRPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNIYPGSGSTHYDEKFRNKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTRVVGDYWGQGSTLTVSSAKTTPPSVYPLAPGSAAQTNSIVI

[0066] VL Seq:

[0067] GTTGACATTGATGACATTGAGGTCACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGAGCGTACACGTC

[0068] AA2:

[0069] VDIDDIEVTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRSVHV

[0070] The GENBANK (DNA sequence database established by the National Center for Biotechnology Information of the United States) comparison belongs to the variable regions of the heavy chain and light chain of the mouse-derived antibody, i.e., VH and VL regions.

[0071] Example 2, identification of anti-p-Tau181 monoclonal antibody protein

[0072] 1. Specific identification of anti-p-Tau181 monoclonal antibody

[0073] The p-Tau181 polypeptide, the p-Tau217 polypeptide, the p-Tau231 polypeptide and the t-Tau protein (all 5 ng / μL) were respectively coated on the enzyme-labeled plate, 50 μL of the supernatant of the monoclonal antibody cells to be detected (containing the monoclonal antibody produced by the 2-1E cell strain obtained in Example 1) was added, 37°C incubation for 1 h, PBST (phosphate buffer saline) washing plate 3~5 times, 1:1000 dilution of HRP (horseradish peroxidase) labeled goat anti-mouse IgG secondary antibody (100 μL / well) was added, 37°C incubation for 45 min, PBST washing 3~5 times, 100 μL / well TMB (tetramethyl benzidine) color developing liquid was added, avoiding light for 15 min, 2M sulfuric acid 50 μL / well was added to terminate the reaction, and the OD450nm value of each detection well was read. The OD450nm value of each well was determined as P / N≥2.1 as the positive judgment standard, and the results were as shown in Table 1.

[0074] The identification results in Table 1 show that the monoclonal antibody produced by the 2-1E cell strain is a specific antibody against the p-Tau181 polypeptide, and does not react with the p-Tau217, p-Tau231 polypeptides and t-Tau protein.

[0075] Table 1. Specificity identification of anti-p-Tau181 monoclonal antibody

[0076] Antigen OD value (solution absorbance) P / N Negative control 0.054 / p-Tau181 polypeptide 1.898 35.148 p-Tau217 polypeptide 0.051 1 p-Tau231 polypeptide 0.050 1 t-Tau protein 0.049 1

[0077] The results in Table 1 show that the monoclonal antibody produced by the 2-1E cell strain has specific binding ability to the p-Tau181 polypeptide, and the monoclonal antibody is named as anti-p-Tau181 antibody or anti-p-Tau181 monoclonal antibody.

[0078] 2. Purity identification of anti-p-Tau181 monoclonal antibody

[0079] Prepare 5% (wt) separating gel and 15% (wt) concentrated gel, and then load the purified anti-p-Tau181 antibody and perform electrophoresis under stable pressure. Stop the electrophoresis when the bromophenol blue reaches 1 cm from the lower edge of the separating gel. After removing the gel film, place it in the Coomassie brilliant blue staining solution for 30 min, and finally decolorize it 2-3 times with the decolorizing solution until the background is colorless. Scan the film with a scanner and store the image, and analyze the antibody purity with Gelpro32 software.

[0080] The purity identification results are shown in Figure 3 There are obvious specific bands at 25 kD and 60 kD, and no other bands, and the 25 kD and 60 kD are light chain and heavy chain bands, and the total content of the light chain and heavy chain (antibody purity) is 96.83%, indicating that the obtained antibody has high purity.

[0081] 3. Affinity constant determination of anti-p-Tau181 monoclonal antibody

[0082] The present application uses biofilm interference technology (BLI) to detect the affinity and kinetic parameters of the antibody and antigen (p-Tau181 polypeptide) binding. The antibody is immobilized on the sensor surface using a SA sensor, and then reacts with gradient-diluted antigen, and the information of intermolecular interaction is obtained by analyzing the changes of surface light interference. The specific operation method is referred to the instrument instruction.

[0083] The affinity test results are shown in Figure 4 and Table 2, the affinity of the anti-p-Tau181 antibody to the p-Tau181 antigen is 1.98 nM, indicating that the anti-p-Tau181 monoclonal antibody prepared in Example 1 has high affinity to the antigen p-Tau181.

[0084] Table 2. Affinity test of anti-p-Tau181 monoclonal antibody

[0085] Antibody kon(1 / Ms) koff(1 / s) KD(M) 2-1E 2.02E+006 3.99E-003 1.98E-009

[0086] Example Three, Anti-p-Tau181 Antibody Application

[0087] 1. Protective Effect of Anti-Tau Antibody on Aβ25-35-induced N2a Cells

[0088] 1.1 Cell Culture

[0089] Mouse neuroblastoma cells N2a were cultured in DMEM medium containing 10% (wt) fetal bovine serum and 1% (wt) antibiotics (penicillin 100 IU / ml + streptomycin 100 μg / ml) in an incubator at 37°C, 5% CO2, and cells in the logarithmic growth phase were collected for experiments.

[0090] 1.2 Cell Grouping and Treatment

[0091] N2a cells were divided into three groups: blank control group (N2a cells were normally cultured without treatment), AD group (N2a cells were cultured with 20 μM Aβ25-35 for 24 hours), and anti-p-Tau181 antibody group (N2a cells were treated with Aβ25-35 and anti-p-Tau181 antibody).

[0092] AD group: N2a cells were seeded in a 96-well plate at a concentration of 10 4 / mL. Aβ25-35 at a concentration of 20 μM was added to the cells, and incubated for 24 h.

[0093] Anti-p-Tau181 antibody group: N2a cells were pretreated with 20 μM Aβ25-35 for 24 h, and then 20 μM anti-p-Tau181 antibody was added for continued incubation for 24 h.

[0094] 1.3 Cell Proliferation Experiment

[0095] N2a cells were cultured in a 96-well plate according to the above grouping, 50% (wt) trichloroacetic acid was added to each well, and incubated at 4°C for 1 h. After drying, 50 μL of 0.4% (wt) SRB solution was added and incubated for 15 min, then washed with a small amount of 1% (wt) acetic acid solution for 2-3 times. After drying, 10 mM Tris base solution 100 μL was added to each well to dissolve the crystals, and the absorbance value (OD value) of each well was measured at a wavelength of 540 nm on an enzyme-linked immunosorbent assay instrument.

[0096] 2. Improvement Effect of Anti-p-Tau181 Antibody on Aβ25-35-induced Cognitive Function of AD Rats

[0097] 2.1 Experimental Animals

[0098] 8-week-old, 160-190 g weight, male Sprague-Dawley (SD) rats (n=15) were bred in sterile independent air supply isolation cages in an air laminar flow purification room, kept at constant temperature (26-28℃), constant humidity (relative humidity 40%-60%), and the feed, drinking water, and bedding were sterilized before use.

[0099] 2.2 AD rat model construction and grouping

[0100] Aβ25-35-induced establishment of AD rat model

[0101] 1 mg of Aβ25-35 was dissolved in 200 μL of sterilized normal saline, sealed and placed in a 37℃ cell incubator for 96 h to form Aβ25-35 condensed state.

[0102] The rats were divided into three groups: blank control group (no treatment), AD model group (intra-cerebral injection of Aβ25-35 condensed state to establish AD rat model), and anti-p-Tau181 antibody group (intra-cerebral injection of Aβ25-35 condensed state to establish AD rat model and anti-p-Tau181 antibody treatment).

[0103] AD model rats: After 4% sodium pentobarbital intraperitoneal injection anesthesia, the rat was fixed in the flat skull position with a brain stereotaxic apparatus, and the right side of the hippocampus was determined as 3.8 mm behind the right fontanel, 2.5 mm beside the midline, and 3.0 mm below the skull surface. Aβ25-35 was slowly injected within 5 min using a 1 μL microsyringe, and the needle was left for 10 min. After the operation, 50,000 units of sodium penicillin were intramuscularly injected once a day for 3 consecutive days.

[0104] Anti-p-Tau181 antibody group rats: A portion of the AD model rats were intraperitoneally injected with anti-p-Tau181 antibody at a dose of 10 mg / kg daily for 28 consecutive days.

[0105] 2.3 Water maze test

[0106] 2.3.1 Experimental device

[0107] The Morris water maze was a circular pool with a diameter of 100 cm, a depth of 50 cm, a water depth of 30 cm, a water temperature of (24±2)℃, and a water surface covered with a layer of plastic foam. The pool was divided into four quadrants at four equally spaced points on the pool wall, and a platform was placed in the center of the third quadrant. The platform was colorless and transparent, with a diameter of 10 cm and a height of 28 cm, and was submerged 2 cm below the water surface. The pool perimeter remained unchanged.

[0108] 2.3.2 Positioning navigation experiment

[0109] Each rat was placed in the water facing the wall of the pool, and the starting position was randomly selected from the east, west, south, and north. The time (s) from entering the water to finding the platform (escape latency) was recorded. If the rat did not find the platform within 90 s, it was guided to the platform position. After the rat stayed on the platform for 15 s, it was removed from the pool and dried (if necessary, the rat was placed under a 150 W incandescent lamp for 5 min and returned to the cage). Each rat was trained 4 times a day, with an interval of 15-20 min, and the average value was taken as the daily performance. Training was conducted for 5 consecutive days.

[0110] 2.3.3 Spatial exploration experiment

[0111] On the second day after the training of the place navigation experiment, the platform was removed, and the spatial exploration training of 90 s was started. The rat was placed in the water from the first quadrant. The number of times the rat crossed the original platform within 90 s was recorded.

[0112] 2.4 Y maze test

[0113] The floor and walls of the three-arm maze (40 cm high and 4 cm wide) were made of polyethylene plastic. The rat was placed in one of the arms and allowed to freely move in the maze for 8 minutes. The number of entries into the maze was counted after the rat's front paws all touched the ground. The rat's entry into the three different arms in turn (e.g., I II III, I III II, II III I, II I III, III I II, III II I) was an alternation. After each test, the maze was cleaned with a 10% ethanol solution. The spontaneous alternation rate (%) = (number of spontaneous alternations / [total number of arm entries - 2]) x 100.

[0114] 3. Experimental results

[0115] 3.1 MTT results

[0116] Figure 5 It is shown that, compared with the blank control group, the survival rate of N2a cells in the AD group was significantly reduced (p<0.05); compared with the AD group, the survival rate of N2a cells in the anti-p-Tau181 antibody group was significantly increased (p<0.05). This indicates that the anti-p-Tau181 antibody has an improving effect on the N2a cell damage induced by Aβ25-35.

[0117] 3.2 Water maze test results

[0118] Figure 6 The spatial learning and memory ability of the rat was evaluated by the water maze experiment. Compared with the blank control group, the AD model rat had a significant memory defect in learning performance, as shown by the prolonged escape latency and reduced number of crossings through the platform; compared with the AD group, the anti-p-Tau181 antibody treatment significantly increased the number of crossings through the platform and reduced the escape latency, reflecting the improvement in the spatial memory of the AD model rat.

[0119] 3.3 Y-maze test results

[0120] Y-maze test was used to track the results of the latitudinal memory function test Figure 7 Compared with the control group, the spontaneous alternation times and the total arm entries of the AD model group rats were significantly reduced (p<0.05). In addition, compared with the OA group, the spontaneous alternation times and the total arm entries of the antibody group were significantly increased (p<0.05). The results showed that the p-Tau181 antibody had an improvement effect on the behavioral disorders of AD rats.

[0121] The above are only some embodiments of the present application, and the protection scope of the present application is not limited to the above embodiments. For ordinary skilled persons in the technical field, some improvements and refinements without departing from the creative design of the present application should also fall within the protection scope of the present application.

Claims

1. An anti-p-Tau181 antibody, characterized in that The VH sequence encoding the anti-p-Tau181 antibody gene is as follows: AACCACCTTTTAGGAACTGGCGGTGTCCACTCCCAGGTCCAACTGCAGCAACCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACATTCACCAGCTACTGGATGCACTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTAGTACTCACTACGATGAAAAGTTCAGGAACAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGTAGTGGGAGACTACTGGGGCCAAGGCAGCACTCTCACAGTCTCCTCCGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATTGTTATCCT; The VL sequence encoding the anti-p-Tau181 antibody gene is as follows: GTTGACATTGATGACATTGAGGTCACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGAGCGTACACGTC。

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