LU501908B1 - Preparation Method of Adeno-associated Virus with Epigenetic Modification Function and Application Thereof - Google Patents

Abstract

The invention relates to the application of a recombinant single-stranded DNA virus vector with heterologous nucleic acid sequence in preparing drugs for treating stress cognitive impairment, which comprises the virus vector, a method for preparing the virus vector, and the application of the virus vector in the research of epigenetic regulation mechanism and the treatment of cognitive impairment caused by stress, wherein, the recombinant single-stranded DNA virus vector is an adeno-associated virus vector, and the heterologous nucleic acid encodes therapeutic protein; the therapeutic protein is TET1(1418-2136aa). According to the present invention, the adeno-associated virus with epigenetic modification function is artificially prepared, and after the adeno-associated virus infects the organism, it can induce the expression of neurotrophic factors such as BDNF through epigenetic modification, maintain the survival of neurons and nerve development, and promote the maturation of dendritic spines, thus playing the role of resisting and treating stress cognitive impairment.

Description

DESCRIPTION LU501908 Preparation Method of Adeno-associated Virus with Epigenetic Modification Function and Application Thereof

TECHNICAL FIELD The invention belongs to the field of biotechnology, in particular to a preparation method of adeno-associated virus with epigenetic modification function and its application in stress cognitive impairment.

BACKGROUND With the quickening pace of modern social life and the intensification of social competition, most people are under different levels of stress load, which is characterized by excessive activation of sympathetic nervous system and hyperactivity of HPA axis. Epidemiological survey shows that the prevalence of mild cognitive impairment and dementia among people who have been in stress for a long time is more than twice that of non-stressed people of the same age. Among the confirmed patients with cognitive dysfunction, more than 70% are accompanied by dysfunction of HPA axis. High levels of stress hormones can lead to structural and functional changes in many brain regions, including shrinking of hippocampus, reduction of dendritic spines in vertebral cells, abnormal synaptic plasticity, blockage of dentate gyrus nerve, remodeling of neural circuit in prefrontal cortex and reduction of spontaneous activity. However, clinically, drugs targeting stress hormones cannot effectively prevent and treat stress-related cognitive dysfunction.

Neurotrophic factors include many different members and are widely distributed in the central nervous system. They play an important role in regulating the survival and differentiation of neurons, neurogenesis, dendritic structure remodeling, synapse formation and long-term enhancement. Under stress, the expression of neurotrophic factors such as BDNF in hippocampus decreased significantly, which was related to stress cognitive impairment in adults. The expression and regulation mechanisms of neurotrophic factors are different. Take BDNF as an example, its promoter region has glucocorticoid response elements and several CpG island regions, which can be regulated by hormone nuclear receptor transcription factors and epigenetic modification of DNA at the same time. At present, the regulation mechanism of the expression of neurotrophic factors is still not completely clear, and there is no report of treating stress cognitivé/501908 impairment by regulating its expression through epigenetic modification.

Gene therapy refers to the treatment method of transferring specific genetic material into specific target cells of patients, so as to finally prevent or change the state of special diseases. Adeno-associated virus vector is a common virus vector in gene therapy, which has the characteristics of good safety, wide host range, high infection efficiency, convenient preparation and easy purification and concentration. Drugs with adeno-associated virus as carrier have been widely used in the treatment of many diseases including malignant tumors.

According to the present invention, the artificially prepared adeno-associated virus can induce the expression of neurotrophic factors such as BDNF through epigenetic modification, maintain the survival of neurons and neurogenesis, and promote the maturity of dendritic spines, thus playing a role in resisting and treating stress cognitive impairment, and solving the technical problem of treating stress cognitive impairment by regulating gene expression in brain through epigenetic modification for the first time.

SUMMARY In view of the above problems, the first aspect of the present invention provides the application of recombinant single-stranded DNA virus vector containing a heterologous nucleic acid sequence in preparing drugs for treating stress cognitive impairment.

The recombinant single-stranded DNA virus vector is an adeno-associated virus vector, and the heterologous nucleic acid encodes a therapeutic protein.

The therapeutic protein is TET1(1418-2136aa), and its amino acid sequence is shown in SEQ ID NO:3.

On the other hand, the invention provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition comprises the recombinant single-stranded DNA virus vector.

The invention also provides a method for preparing the recombinant single-stranded DNA virus vector, which is characterized by comprising the following steps: S1: construction and identification of eukaryotic expression vector containing catalytic activity domain of TET1 enzyme;

S2: detection of recombinant plasmid expression: transfect the identified positiké}501908 recombinant plasmid into 293T cells, collect cells to extract protein, and detect the expression of recombinant plasmid by western blot experiment; S3: packaging adeno-associated virus, co-transfecting the recombinant expression plasmid pAAV-TET1 (1418-2136aa), pAAV-RC and pHelper into AAV-293 cells, collecting cell supernatant and cells after 3 days, cracking to obtain AAV particles, concentrating and purifying the virus by CsCl density gradient centrifugation and ultrafiltration, and finally detecting the titer of the virus by real-time PCR.

In S1, the coding gene of catalytic activity domain of Tet] enzyme is obtained by PCR, and then cloned into adeno-associated expression vector; Particularly, the primer sequences used in the PCR reaction are shown in SEQ ID NO:3 and SEQ ID NO:4; Particularly, the adeno-associated expression vector is GV467, and the element sequence is CMV-beta globin-MCS-EGFP-3flag-SV40polya; In the S1, the peptide segment generated by the translation of the amplification product obtained by PCR is TET11418- 2136aa, and its amino acid sequence is shown in SEQ ID NO:2 Finally, the invention provides the application of the virus vector in the research of epigenetic regulation mechanism and the treatment of cognitive disorder caused by stress.

The invention has the advantages that:

1. The present invention has prepared adeno-associated virus containing the expression sequence of human methylcytosine dioxygenase (Tetl), which can efficiently produce Tetl protein after infecting mammals and induce epigenetic modification of DNA in infected areas. The virus has a high efficiency of expressing Tet enzyme, can achieve obvious epigenetic modification on local tissues of animals at the in vivo level, and can be used for epigenetic regulation research of various diseases.

2. In the research of epigenetic regulation mechanism, compared with the current commonly used methyltransferase inhibitors, the present invention has low cost, especially in the long-term administration at the whole animal level, only a single local injection of 3 x10? viruses is needed, and the cost 1s one third to one half of the continuous intraperitoneal injection of 5-aza and other methyltransferase inhibitors. At the same time, the adeno-associated virus of the present invention can induce the active DNA demethylation process, which is independent 68501908 DNA replication. Compared with the methyltransferase inhibitor, the adeno-associated virus has a faster effect and a wider application range, and can be used for neurons and other non-dividing cells (Figures 10 and 11). In addition, the adeno-associated virus of the present invention can induce DNA demethylation in local tissues, thus avoiding the nonspecific effect caused by systemic administration.

3. The injection of adeno-associated virus constructed by the invention can obviously inhibit methylation of BDNF gene promoter in hippocampus of stressed rats, and increase BDNF level, so as to improve new object recognition ability and enhance learning and memory ability. The adeno-associated virus of the invention has a wide host range, and the high homology of Tetl gene carried by virus makes the application cover from rats and mice to other mammals including humans. The method for constructing Tetl adeno-associated virus has potential application value in treating cognitive decline caused by various stress pressures.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1: Structure of human Tet] gene and the position of amplification primers; Fig. 2: Electrophoretic identification of Tetl target gene obtained by PCR, in which 1 is PCR product and 2 is Marker; Fig. 3: Plasmid structure for constructing recombinant expression vector of target gene; Fig. 4: Transformation identification of recombinant plasmid, in which 1: negative control (ddH20), 2: negative control (no-load self-linking control group), 3: positive control (GAPDH), 4: Marker, the order from top to bottom is Skb, 3kb, 2kb, 1 .5kb, 1Kb, 750bp, S00bp, 250bp and 100bp, 5-12: 11-18 transformants; Fig. 5: Results of plasmid transfection of 293T cells under phase-contrast and fluorescence microscope; Fig. 6: Identification of recombinant Tet] expressed after plasmid transfection, in which 1: molecular weight Marker, 2: survivin-3 flag-GFP standard positive control (molecular weight 48KDa), 3: untransfected 293T cell control, 4: target gene fusion protein expressed by transfection (molecular weight 108 kDa); Fig. 7: Changes of methylation of BDNF gene promoter in hippocampus under stress (*: P<0.05 compared with control);

Fig. 8: Changes of expression level of neurotrophic factor BDNF gene in hippocampti$)501908 under stress (*: P<0.05 compared with control); Fig. 9: Effect of stress on cognitive function of rats (*: P<0.05 compared with control); Fig. 10: Effect of Tetl adeno-associated virus transfection on methylation of BDNF gene promoter in hippocampal neurons of stressed rats (*: P<0.05 compared with stressed group); Fig. 11: Effect of Tet] gene adeno-associated virus transfection on BDNF gene expression in hippocampal neurons of stressed rats (*: P<0.05); Fig. 12: Improvement of cognitive function in stressed rats by transfection of tet] gene adeno-associated virus (*: p<0 .05 compared with stressed rats).

DESCRIPTION OF THE INVENTION The embodiment will be described in detail with reference to the accompanying drawings.

Example 1 construction and identification of eukaryotic expression vector containing catalytic activity domain of TET1 enzyme

1.1 Cloning and purification of target gene (1) CDS region sequence of 1)Tet]l mRNA (NM_030625.3) >NM 030625.3:529-6939 homo sapiens Tet methylcytosine dioxygenase 1 (Tetl), mRNA. The specific sequence is shown in SEQ ID No: 1, where the underline is the position of PCR primer.

(2) Designing and synthesizing primers for cloning.

The full-length gene of Tetl cannot be cloned into adeno-associated expression vector because of its long length, so the designed primer was used to obtain the coding gene of catalytic activity domain of Tetl enzyme by PCR, and then cloned into adeno-associated expression vector. The primer sequence used for cloning is as follows: SEQ ID NO:3TET1-Forward primer: GGAGGTAGTGGAATGGATCCCGCCACCATGGAACTGCCCACCTGCAGCTGTC; SEQ ID NO:4TET1-Reverse primer: TCACCATGGTGGCGGGATCGACCCAATGGTTATAGGGCCCCGCAAC.

Among them, the underlined sequence is the cleavage site (BamHI). The length of the product is 2207bp, the sequence is shown in SEQ ID NO:5, and the annealing temperature is

60°C. The peptide generated by translation of the product is TET1 1418-2136aa (Figure 1), and)501908 the amino acid sequence is shown in Seq ID No.2.

(3) Using plasmid containing the full length of human-TET1 as template, PCR was performed with high fidelity enzyme (Takara Company), and the TET1 fragment was amplified. The PCR reaction solution was prepared as follows: $x PS Buffer 19 nil dNTPs {2,5 mM each} 4 pul PrimeSTAR HS DNA polymerase (4.5 pl Forward primer {10 pM) | nl Reverse primer (10 gd) tu Form {10 nel} tel SAHA ih Total Volume SO nl PCR reaction was carried out in PCR instrument according to the following procedure: Pre-denaturation at 98°C for Smin, 35 cycles of denaturation at 98°C for 10sec, annealing at 60°C for 10sec and extension at 72°C for 2 min and 30 sec; finally, the reaction was carried out at 72°C for 8min.

(4) Add 10 pL of 6xloading buffer to the PCR product, and perform agarose gel electrophoresis after mixing, so as to confirm the size of the product (Figure 2). The objective band was cut off under ultraviolet lamp and recovered and purified according to the instructions of gel recovery kit (Tiangen Biological Co, Ltd.).

1.2 Enzymatic digestion of fragments and vectors (1) The adeno-associated expression vector used is GV467; element sequence is CM V-beta globin-MCS-EGFP-3flag-SV40polya; clone site: BamHI/BamHI. (Figure 3) (2) Cut the recovered fragment and vector with restriction endonuclease BamHI (NEB Company) respectively. The reaction solution was prepared according to the following system, mixed evenly, and digested overnight in a water bath at 37°C.

Fragment enzyme digestion system:

He Cutämart Buffer Sal LUS01908 Fragment A nt Dam (10 Weld à ut Total Volume ME pl. Vector enzyme digestion system: (0x CutSmart Bufter Sal vector £1 uœul) = ul BamHI {10 Wal) i ul dathbO Auk Total Volume Spl (3) Add 10 pL of 6x loading buffer to the enzyme digestion products of fragment and vector respectively, perform agarose gel electrophoresis, cut the target band under ultraviolet lamp, and recycle it according to the instructions of gel recycling kit (Tiangen).

1.3 Ligation of fragment and vector (1) Take 1 pL of recovered fragment and carrier for agarose gel electrophoresis, carry out gray-scale quantification, calculate the molar mass, and connect the fragment and carrier according to the molar ratio of 3:1. (2) Prepare the reaction solution according to the following system, and place at room temperature for 1-2 hours after mixing evenly. T4 Powerful ligase (Invitrogen Company} inl Fragment 5 ul Total Volume EL 1 .4 Transformation and screening of recombinant vectors (1) Take a competent cell of E .coli DH5a (Takara Company) and put it on ice to naturally melt, add 10 pL ligation product to the competent cell, stand for 20 minutes on ice, heat shock for 90sec in a 42°C water bath, immediately insert it into ice, put it for 2 minutes, add 700 (IU501908 LB medium, and shake the bacteria for 45 minutes at 37°C.

(2) Apply the bacterial solution evenly to the agar plate containing ampicillin. Firstly, it was placed in an oven at 37°C for 30min, and then it was cultured upside down for 12-16 hours.

(3) Put the monoclones into a shake tube, add 3mL of LB medium containing ampicillin, and shake the bacteria for 12-16 hours at 37°C.

1 .5 Identification of positive recombinants (1) Design and synthesize primers for PCR identification, with the sequence as follows: Pl: CACTAAAACTTATTCGCTGATG; P2: CGTCGCCGTCCAGCTCGACCAG The length of the product is 1007bp and the annealing temperature is 60°C.

(2) PCR is carried out with the bacterial solution as the template, and the reaction system is as follows: Iv POR mx (Tiangen Company) ID nt Pt primar CID phd) tt.

BY primer (10 pa t ut. Bacterial liquid tut Gell Fak Total volo 26 pa.

PCR reaction was carried out in PCR instrument according to the following procedure: Pre-denaturation at 95°C for Smin; 35 cycles of denaturation at 95°C for 30sec, annealing at 60°C for 30sec and extension at 72°C for 1min; at last, react at 72°C for 10 min.

(3) Take 6 ul of PCR products for agarose gel electrophoresis to confirm the product size (Figure 4).

(4) Sequencing the positive bacteria group preliminarily identified by PCR (Shanghai Jikai Biological Co., Ltd.), and comparing with the target gene sequence, it is confirmed that the recombinant plasmid sequence is correct and there is no gene mutation.

(5) Add the correctly sequenced bacteria solution into 10 mL LB medium containing ampicillin antibiotics, and shake the bacteria for 12-16hr hours at 37°C. The recombinant plasmid was extracted by plasmid small extraction medium amount kit (Tiangen Biological Co., Ltd.) according to the instructions, and pAAV-TET1 (1418-2136aa) was obtained.

Example 2 Expression Detection of Recombinant Plasmid

2 .1 Cell transfection LU501908 (1) 293T cells in logarithmic growth phase were inoculated into 6-well culture plates at 50% density, and cultured in 5% CO; incubator at 37°C until the cell density reached about 80%.

(2) According to the instructions of lipofectamine 2000 transfection reagent (invitrogen company), prepare the mixture of plasmid and transfection reagent, and add the mixture into cells drop by drop.

(3) After 4-6 hours, observe the cell state and replace it with fresh complete medium. After transfection for 24-48 hours, the fluorescence expression of cells was observed under fluorescence microscope and photographed (Figure 5).

2.2 Extract total cell protein (1) Discard the culture medium after cell growth, wash them twice with PBS, add 100 pL RIPA lysate containing protease inhibitor (Biyuntian Company) to each well. Scrape off the cells and transfer them to EP tube on ice for 15 min.

(2) Ultrasonic disruption of cells (200 W for 4 times, each time for 5 seconds, with an interval of 2 seconds), centrifuge at 4°C, 12000 g for 15 min, and take the supernatant.

(3) Detect protein concentration by BCA method according to BCA protein quantitative kit instructions (Biyuntian Company). Adjust the protein concentration of each sample to 2 ug/ul, store at -80°C for later use.

2.3 SDS-PAGE electrophoresis and membrane transfer (1) SDS-PAGE adhesive was prepared, the separation adhesive concentration was 10%, and the lamination adhesive concentration was 5%. After the gel is solidified, remove the comb, assemble the electrophoresis device, and add the electrophoresis buffer.

(2) Take 20 pg protein sample, add 6.5 pL of 4xloading buffer, and slowly add it into the loading well after mixing.

(3) First, keep the sample at a constant voltage of 80V until it enters the separation gel, and then change it to 120V electric voltage until bromophenol blue runs to about 0.5cm from the bottom of the gel. Electrophoresis usually takes 2-3 hours.

(4) After the electrophoresis, use the wet membrane transfer device, and put it in the order of filter paper-membrane-gel-filter paper from the positive electrode to the negative electrode,

with no bubbles in the middle of each layer. At 4°C and 300 mA constant current, the protel4/501908 was transferred to PVDF membrane by electrotransfer for 150 min.

2.4 Antibody incubation and detection (1) After the film transfer, take out the PVDF film, put it in the blocking solution of 5% skim milk, and put it on a shaking table at room temperature for 1 hour.

(2) Dilute the Flag antibody (sigma) with antibody diluent at 1:3000. The membrane was washed with TBST solution for three times, after 8min/ time, the primary antibody was put in and incubated at room temperature for 2 hours or 4°C overnight.

(3) Dilute the mouse-derived second antibody (santa-cruz) with antibody diluent at a ratio of 1:4000. The membrane was washed three times with TBST solution the second antibody was added and incubated for 1.5 hours at room temperature.

(4) Washing the membrane with TBST solution for three times, 8min/time. The PVDF film was placed on the spread plastic wrap. According to the ECL substrate kit instructions (Thermo), liquid A and liquid B were mixed at a ratio of 1:40, evenly dropped on the PVDF film, and reacted in the dark for 5 minutes.

(5) Take out the film, drain the excess ECL substrate reaction solution slightly, put it into a cassette, spread a plastic wrap (to avoid bubbles), put in an X-ray film (to avoid X-ray film moving), close the cassette, and expose for 1-2 min.

(6) Take out the X-ray film, put it into developer, take it out after about 1min, rinse it in clean water for a few seconds, and then put it into fixer for at least 2 min (the exposure time needs to be tried several times, and the exposure time is not properly adjusted according to whether the fluorescence can be seen by naked eyes and the intensity of fluorescence).

(7) Take out the X-ray film, air-dry, analyze and take photos (Figure 6).

Example 3 Packaging Adeno-associated Virus

3.1 AAV-293 cell culture (1) Resuscitate AAV-293 cells a. DMEM medium containing 10% FBS (called complete medium) was prepared for the cultivation of AAV-293 cells.

b. Add 3 mL of complete culture medium into a 10 mL glass centrifuge tube.

c. Take the cells out of the liquid nitrogen tank or -80°C refrigerator, quickly put them int&J501908 a 37°C water bath, and gently shake them for 1-2 min to completely melt them.

d. Take the frozen storage tube to the ultra-clean table, wipe the surface with alcohol cotton ball for disinfection, and add the cell suspension into the centrifuge tube prepared in advance.

e. Centrifuge for 800 gx3 min, discard the supernatant, add 2 mL of new complete medium, gently blow with dropper to make the cells float, inoculate into a 10 cm culture dish containing 8 mL of fresh complete medium, and place in an incubator with 5% CO; at 37°C for culture.

3.2 Passage of AAV-293 cells a. Observe the growth state and density of gentry cells every day, and subculture when the cell density reaches 50%.

b. Suck up the original culture medium, wash the cells twice with 10 mL of normal saline, add 1 ml of 0. 5% trypsin solution, and put it in an incubator at 37°C for digestion for 1-3 minutes until the cells just fall off from the culture dish.

c. Add 3 mL complete medium to stop digestion, and transfer the cell suspension to 10mL glass centrifuge tube.

d. Centrifuge for 800 gx3 min, and discard the supernatant, add 5 mL of fresh complete medium, gently blow with dropper to make the cells float, take 1 mL and inoculate it into a 10 cm culture dish containing 8 mL of fresh complete medium, totally inoculate 5 bottles, and put them in a 5% CO; incubator at 37°C for culture.

3.3 Frozen AAV-293 cells a. Take AAV-293 cells in logarithmic phase, suck out the original culture medium, wash the cells twice with 10mL of normal saline, add 1 ml of 0.5% trypsin solution, and put them in an incubator at 37°C for digestion for 1-3 minutes until the cells just fall off from the Petri dish.

b. Add 3 mL complete culture medium to stop digestion, and transfer the cell suspension to 10mL glass centrifuge tube.

c. Centrifuge for 800 gx3 min, and discard the supernatant, add 3 mL of cell cryopreservation solution (Suzhou Xinsaimei Co., Ltd.) to resuspend the fine cells, subpackage them into cryopreservation tubes with 1 mL/tube, put them in the -80°C refrigerator, and put them into the liquid nitrogen tank for long-term storage the next day.

3.4 Cell transfection

(1) AAV-293 cells in logarithmic growth stage were inoculated into a 10 cm Petri dish501908 cultured in a 5% CO» incubator at 37°C, and transfected when the cell density reached 70-80%.

(2) Take a 1.5 mL EP tube, add 500 ul. CaCl, solution (0.3M), then add 10 pg pAAV-TET1(1418-2136aa), pAAV-RC and pHelper, and mix gently.

(3) Take a new 1.5 mL EP tube, add 500 pL of 2xHBS solution, add DNA/CaCl, mixture drop by drop, and mix it upside down.

(4) Dropping the mixed DNA/CaCl,/HBS solution on the cell culture dish, while gently shaking the culture dish to make the solution evenly distributed in the culture medium, and placing it in an incubator with 5% CO; and 37°C for culture.

(5) After 6 hours, change the solution, add 10 mL fresh complete medium, and continue to cultivate for 66-72 hours.

3 .5 Collecting adeno-associated virus (1) Observe the changes of cell morphology and culture medium color. When some cells become round and fall off, and the color of culture medium changes from red to orange or yellow, the virus packaging is successful. Generally, viruses are collected three days after transfection.

(2) Gently blow down the cells with a dropper, and transfer the cells together with the culture medium to a 15 mL centrifuge tube.

(3) Centrifuge for 800 gx3 min, transfer the supernatant to a new 15 mL centrifuge tube, and add 1 mL PBS to the cell precipitate for resuspension.

(4) Placing the cell suspension in liquid nitrogen and 37°C water bath repeatedly, freezing and thawing for 4 times.

(5) Centrifuge for 10000 gx3 min, and transfer the supernatant to a new EP tube.

3.6 Virus concentration (1) Add appropriate amount of PEG8000 (40%) to the supernatant of 3.5-(3) to make its final concentration 8%, place it on ice for 2 hours, and mix it upside down every 15 minutes.

(2) Centrifuge for 2500 gx30 min, discard the supernatant, add PBS to resuspend, and combine with the supernatant collected in 3.5-(5).

(3) Centrifuge for 3000 gx30 min, and transfer the supernatant to a new EP tube. Benzonase nuclease (final concentration of 50 U/mL)(Merck Company) was added to digest and remove residual plasmid DNA, mixed upside down and incubated at 37°C for 30 min.

(4) Use 0.45 um filter to filter out impurities in the solution. LU501908 3 .7 virus purification (1) Add solid CsCl (about 6.5 g/10 mL, concentration 1.41 g/mL) to virus concentrated solution, and shake and dissolve.

(2) Add the sample into the ultracentrifuge tube and fill the remaining space of the centrifuge tube with the pre-configured CsCl solution (1.41 g/mL).

(3) Centrifuge for 175000 gx24 hr to form a density gradient, collect solutions with different densities in sequence, measure the drop degree and collect the composition of AAV particles.

(4) Repeat the above steps once.

3.8 Ultrafiltration desalination (1) Add 4 mL deionized water to Amicon-15 ultrafiltration device to soak the membrane.

(2) Add the purified virus solution to the ultrafiltration device, make up the total volume to 4 mL with PBS, and cover it.

(3) Centrifuge at 1500 g, observe the remaining solution volume every 5 minutes until the final volume is 200-250 pL.

(4) Add PBS to the remaining solution to make up the volume to 4 mL.

(5) Repeat the above steps for 3 times.

(6) Centrifuge the ultrafiltration tube to make the final volume of virus solution 0.5 mL.

(7) Add appropriate amount of glycerin (final concentration 5%), subpackage and store at -80°C.

3.9 Determination of virus titer (1) Zfcas9 plasmid (2.45E+13 Copies/mL) was used and diluted with ddHzO gradient to obtain the standard, with the concentrations of 2.45E+10, 2.45E+9, 2.45E+8, 2.45E+7, 2.45E+6 Copies/ml, respectively.

(2) Dilute gradient diluted virus solution as follows:

(3) Prepare real-time PCR reaction solution, and the reaction system of each well is 44501908 follows: IXSYRB Green Master ROX (Taka) 15 ul Forward Primer {5 ph Sal Total Volume IS al (4) Add it to 96-well plate, 15 uL per well, then add 5 uL standard or sample, and set up a re-well. (5) After blocking the 96-well plate, put it into a real-time PCR instrument, and carry out the reaction according to the following procedures: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 min for 40 cycles; reaction at 95°C for 15 sec, 60°C for 60°C 60 sec, and 95°C for 15 sec to obtain the dissolution curve. (6) Draw the standard curve according to the logarithmic value of the standard concentration and the average Ct value: Y =-3. 345 * LOG (X)+39.74, R° = 0. 999. (7) Calculate the concentration according to the Ct value of each Sample.

Since the standard is double-stranded DNA and AAV virus particles are single-stranded DNA, the concentration of virus stock solution is equal to the concentration of each Sample divided by the dilution and multiplied by 2. (8) Averaging the concentration of virus stock solution calculated by each Sample, the titer of AAV virus was 3.07 E+13 Copies/mL. | Assayed Semple Cimean (Calovlaion | Dilution | Original | nooo | Red Sed TIRE ese soe SIENS |

Example 4 Application of overexpression of TET1 in the treatment of stress cognitik&éJ501908 impairment Take the chronic unpredictable temperature and stimulation (CUMS) stress model as an example, but the application of AAV-TET1 (141 8-2136aa) is not limited to this stress model, but also includes the treatment of cognitive impairment caused by other stress models.

4.1 Effects of stress on bdnf DNA methylation and cognitive ability (1) Chronic unpredictable mild stimulation (CUMS) was given to rats to establish a stress animal model. The tissue of hippocampus was taken, DNA was extracted, and methylation-specific PCR experiment was carried out to detect the effect of stress on methylation of bdnf gene promoter in hippocampus. The results showed that the methylation level of bdnf gene promoter in hippocampus of stressed rats increased (Figure 7).

(3) The effects of stress on cognitive ability of rats were detected by open field experiment, object recognition experiment and water maze experiment. The results showed that, compared with the control group, the open-field score and cognitive index of object recognition of rats in the stress group decreased, and the searching time of water maze increased, which indicated that stress could reduce the cognitive ability of rats (Figure 9).

4.2 The regulatory effect of AAV-TET1 (1418-2136aa) injected into hippocampus on bdnf DNA methylation and cognitive ability.

(1) 1 uL AAV-TET1 (1418-2136aa) and control virus (3x109TU/uL) were injected into the hippocampus of rats in stress group. Then, the hippocampus tissue was taken, DNA was extracted, and methylation-specific PCR experiment was carried out to detect the effect of TET1 expression on the methylation of bdnf gene promoter. The results showed that TET1 could reduce the methylation of bdnf gene promoter (Figure 10).

(3) The effects of overexpression of TET1 on cognitive ability of stressed rats were detected by open field experiment, object recognition experiment and water maze experiment. The results showed that compared with the injected control virus, the open-field score and cognitive index of object recognition of rats overexpressing TET1 increased, and the searching time of water maze decreased, which indicated that overexpression of TET1 could promote the recovery of cognitive ability of stressed rats, suggesting that TET1 might be a potential target for the treatment of stress cognitive impairment (Figure 12).

This example is only a preferred embodiment of the present invention, but the scope 68501908 protection of the present invention is not limited to this.

Any changes or substitutions that can be easily thought of by those familiar with the technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Therefore, the scope of protection of the present invention should be subject to the scope of protection of the claims.

Sequence Listing LU501908 <110> Academy of Military Medical Sciences, Academy of Military Science of Chinese PLA <120> Preparation Method of Adeno-associated Virus with Epigenetic Modification Function and Application Thereof <130> PT1988 <160> 5 <170> PatentIn version 3.3 <210> 1 <211> 6411 <212>DNA <213> NM _030625.3:529-6939 Homo sapiens tet methylcytosine dioxygenase 1 (TET1) <400> 1 ATGTCTCGATCCCGCCATGCAAGGCCTTCCAGATTAGTCAGGAAGGAAGA 50 TGTAAACAAAAAAAAGAAAAACAGCCAACTACGAAAGACAACCAAGGGAG 100 CCAACAAAAATGTGGCATCAGTCAAGACTTTAAGCCCTGGAAAATTAAAG 150 CAATTAATTCAAGAAAGAGATGTTAAGAAAAAAACAGAACCTAAACCACC 200 CGTGCCAGTCAGAAGCCTTCTGACAAGAGCTGGAGCAGCACGCATGAATT 250 TGGATAGGACTGAGGTTCTTTTTCAGAACCCAGAGTCCTTAACCTGCAAT 300 GGGTTTACAATGGCGCTACGAAGCACCTCTCTTAGCAGGCGACTCTCCCA 350 ACCCCCACTGGTCGTAGCCAAATCCAAAAAGGTTCCACTTTCTAAGGGTT 400 TAGAAAAGCAACATGATTGTGATTATAAGATACTCCCTGCTTTGGGAGTA 450 AAGCACTCAGAAAATGATTCGGTTCCAATGCAAGACACCCAAGTCCTTCC 500 TGATATAGAGACTCTAATTGGTGTACAAAATCCCTCTTTACTTAAAGGTA 550 AGAGCCAAGAGACAACTCAGTTTTGGTCCCAAAGAGTTGAGGATTCCAAG 600 ATCAATATCCCTACCCACAGTGGCCCTGCAGCTGAGATCCTTCCTGGGCC650 ACTGGAAGGGACACGCTGTGGTGAAGGACTATTCTCTGAAGAGACATTGA 700 ATGATACCAGTGGTTCCCCAAAAATGTTTGCTCAGGACACAGTGTGTGCT 750 CCTTTTCCCCAAAGAGCAACCCCCAAAGTTACCTCTCAAGGAAACCCCAG 800 CATTCAGTTAGAAGAGTTGGGTTCACGAGTAGAATCTCTTAAGTTATCTG850

ATTCTTACCTGGATCCCATTAAAAGTGAACATGATTGCTACCCCACCTCC 900 LU501908 AGTCTTAATAAGGTTATACCTGACTTGAACCTTAGAAACTGCTTGGCTCT 950 TGGTGGGTCTACGTCTCCTACCTCTGTAATAAAATTCCTCTTGGCAGGCT 1000 CAAAACAAGCGACCCTTGGTGCTAAACCAGATCATCAAGAGGCCTTCGAA 1050 GCTACTGCAAATCAACAGGAAGTTTCTGATACCACCTCTTTCCTAGGACA 1100 GGCCTTTGGTGCTATCCCACATCAATGGGAACTTCCTGGTGCTGACCCAG 1150 TTCATGGTGAGGCCCTGGGTGAGACCCCAGATCTACCAGAGATTCCTGGT 1200 GCTATTCCAGTCCAAGGAGAGGTCTTTGGTACTATTTTAGACCAACAAGA 1250 AACTCTTGGTATGAGTGGGAGTGTTGTCCCAGACTTGCCTGTCTTCCTTC 1300 CTGTTCCTCCAAATCCAATTGCTACCTTTAATGCTCCTTCCAAATGGCCT 1350 GAGCCCCAAAGCACTGTCTCATATGGACTTGCAGTCCAGGGTGCTATACA 1400 GATTTTGCCTTTGGGCTCAGGACACACTCCTCAATCATCATCAAACTCAG 1450 AGAAAAATTCATTACCTCCAGTAATGGCTATAAGCAATGTAGAAAATGAG 1500 AAGCAGGTTCATATAAGCTTCCTGCCAGCTAACACTCAGGGGTTCCCATT 1550 AGCCCCTGAGAGAGGACTCTTCCATGCTTCACTGGGTATAGCCCAACTCT 1600 CTCAGGCTGGTCCTAGCAAATCAGACAGAGGGAGCTCCCAGGTCAGTGTA 1650 ACCAGCACAGTTCATGTTGTCAACACCACAGTGGTGACTATGCCAGTGCC 1700 AATGGTCAGTACCTCCTCTTCTTCCTATACCACTTTGCTACCGACTTTGG 1750 AAAAGAAGAAAAGAAAGCGATGTGGGGTCTGTGAACCCTGCCAGCAGAAG 1800 ACCAACTGTGGTGAATGCACTTACTGCAAGAACAGAAAGAACAGCCATCA 1850 GATCTGTAAGAAAAGAAAATGTGAGGAGCTGAAAAAGAAACCATCTGTTG 1900 TTGTGCCTCTGGAGGTTATAAAGGAAAACAAGAGGCCCCAGAGGGAAAAG 1950 AAGCCCAAAGTTTTAAAGGCAGATTTTGACAACAAACCAGTAAATGGCCC 2000 CAAGTCAGAATCCATGGACTACAGTAGATGTGGTCATGGGGAAGAACAAA 2050 AATTGGAATTGAACCCACATACTGTTGAAAATGTAACTAAAAATGAAGAC 2100 AGCATGACAGGCATCGAGGTGGAGAAGTGGACACAAAACAAGAAATCACA 2150 GTTAACTGATCACGTGAAAGGAGATTTTAGTGCTAATGTCCCAGAAGCTG 2200 AAAAATCGAAAAACTCTGAAGTTGACAAGAAACGAACCAAATCTCCAAAA 2250 TTGTTTGTACAAACCGTAAGAAATGGCATTAAACATGTACACTGTTTACC 2300

AGCTGAAACAAATGTTTCATTTAAAAAATTCAATATTGAAGAATTCGGCA 2350 LU501908 AGACATTGGAAAACAATTCTTATAAATTCCTAAAAGACACTGCAAACCAT 2400 AAAAACGCTATGAGCTCTGTTGCTACTGATATGAGTTGTGATCATCTCAA 2450 GGGGAGAAGTAACGTTTTAGTATTCCAGCAGCCTGGCTTTAACTGCAGTT 2500 CCATTCCACATTCTTCACACTCCATCATAAATCATCATGCTAGTATACAC 2550 AATGAAGGTGATCAACCAAAAACTCCTGAGAATATACCAAGTAAAGAACC 2600 AAAAGATGGATCTCCCGTTCAACCAAGTCTCTTATCGTTAATGAAAGATA2650 GGAGATTAACATTGGAGCAAGTGGTAGCCATAGAGGCCCTGACTCAACTC 2700 TCAGAAGCCCCATCAGAGAATTCCTCCCCATCAAAGTCAGAGAAGGATGA 2750 GGAATCAGAGCAGAGAACAGCCAGTTTGCTTAATAGCTGCAAAGCTATCC 2800 TCTACACTGTAAGAAAAGACCTCCAAGACCCAAACTTACAGGGAGAGCCA 2850 CCAAAACTTAATCACTGTCCATCTTTGGAAAAACAAAGTTCATGCAACAC 2900 GGTGGTTTTCAATGGGCAAACTACTACCCTTTCCAACTCACATATCAACT 2950 CAGCTACTAACCAAGCATCCACAAAGTCACATGAATATTCAAAAGTCACA 3000 AATTCATTATCTCTTTTTATACCAAAATCAAATTCATCCAAGATTGACAC 3050 CAATAAAAGTATTGCTCAAGGGATAATTACTCTTGACAATTGTTCCAATG 3100 ATTTGCATCAGTTGCCACCAAGAAATAATGAAGTGGAGTATTGCAACCAG 3150 TTACTGGACAGCAGCAAAAAATTGGACTCAGATGATCTATCATGTCAGGA 3200 TGCAACCCATACCCAAATTGAGGAAGATGTTGCAACACAGTTGACACAAC 3250 TTGCTTCGATAATTAAGATCAATTATATAAAACCAGAGGACAAAAAAGTT3300 GAAAGTACACCAACAAGCCTTGTCACATGTAATGTACAGCAAAAATACAA 3350 TCAGGAGAAGGGCACAATACAACAGAAACCACCTTCAAGTGTACACAATA 3400 ATCATGGTTCATCATTAACAAAACAAAAGAACCCAACCCAGAAAAAGACA 3450 AAATCCACCCCATCAAGAGATCGGCGGAAAAAGAAGCCCACAGTTGTAAG 3500 TTATCAAGAAAATGATCGGCAGAAGTGGGAAAAGTTGTCCTATATGTATG 3550 GCACAATATGCGACATTTGGATAGCATCGAAATTTCAAAATTTTGGGCAA3600 TTTTGTCCACATGATTTTCCTACTGTATTTGGGAAAATTTCTTCCTCGAC 3650 CAAAATATGGAAACCACTGGCTCAAACGAGGTCCATTATGCAACCCAAAA 3700 CAGTATTTCCACCACTCACTCAGATAAAATTACAGAGATATCCTGAATCA 3750

GCAGAGGAAAAGGTGAAGGTTGAACCATTGGATTCACTCAGCTTATTTCA 3800 LUS01908 TCTTAAAACGGAATCCAACGGGAAGGCATTCACTGATAAAGCTTATAATT 3850 CTCAGGTACAGTTAACGGTGAATGCCAATCAGAAAGCCCATCCTTTGACC 3900 CAGCCCTCCTCTCCACCTAACCAGTGTGCTAACGTGATGGCAGGCGATGA 3950 CCAAATACGGTTTCAGCAGGTTGTTAAGGAGCAACTCATGCATCAGAGAC 4000 TGCCAACATTGCCTGGTATCTCTCATGAAACACCCTTACCGGAGTCAGCA 4050 CTAACTCTCAGGAATGTAAATGTAGTGTGTTCAGGTGGAATTACAGTGGT 4100 TTCTACCAAAAGTGAAGAGGAAGTCTGTTCATCCAGTTTTGGAACATCAG 4150 AATTTTCCACAGTGGACAGTGCACAGAAAAATTTTAATGATTATGCCATG4200 AACTTCTTTACTAACCCTACAAAAAACCTAGTGTCTATAACTAAAGATTC 4250 TGAACTGCCCACCTGCAGCTGTCTTGATCGAGTTATACAAAAAGACAAAG 4300 GCCCATATTATACACACCTTGGGGCAGGACCAAGTGTTGCTGCTGTCAGG 14350 GAAATCATGGAGAATAGGTATGGTCAAAAAGGAAACGCAATAAGGATAGA 4400 AATAGTAGTGTACACCGGTAAAGAAGGGAAAAGCTCTCATGGGTGTCCAA 4450 TTGCTAAGTGGGTTTTAAGAAGAAGCAGTGATGAAGAAAAAGTTCTTTGT 4500 TTGGTCCGGCAGCGTACAGGCCACCACTGTCCAACTGCTGTGATGGTGGT 4550 GCTCATCATGGTGTGGGATGGCATCCCTCTTCCAATGGCCGACCGGCTAT 4600 ACACAGAGCTCACAGAGAATCTAAAGTCATACAATGGGCACCCTACCGAC 4650 AGAAGATGCACCCTCAATGAAAATCGTACCTGTACATGTCAAGGAATTGA 4700 TCCAGAGACTTGTGGAGCTTCATTCTCTTTTGGCTGTTCATGGAGTATGT 4750 ACTTTAATGGCTGTAAGTTTGGTAGAAGCCCAAGCCCCAGAAGATTTAGA 14800 ATTGATCCAAGCTCTCCCTTACATGAAAAAAACCTTGAAGATAACTTACA 4850 GAGTTTGGCTACACGATTAGCTCCAATTTATAAGCAGTATGCTCCAGTAG 4900 CTTACCAAAATCAGGTGGAATATGAAAATGTTGCCCGAGAATGTCGGCTT 4950 GGCAGCAAGGAAGGTCGTCCCTTCTCTGGGGTCACTGCTTGCCTGGACTT 5000 CTGTGCTCATCCCCACAGGGACATTCACAACATGAATAATGGAAGCACTG 5050 TGGTTTGTACCTTAACTCGAGAAGATAACCGCTCTTTGGGTGTTATTCCT 5100 CAAGATGAGCAGCTCCATGTGCTACCTCTTTATAAGCTTTCAGACACAGA 5150 TGAGTTTGGCTCCAAGGAAGGAATGGAAGCCAAGATCAAATCTGGGGCCA 5200 TCGAGGTCCTGGCACCCCGCCGCAAAAAAAGAACGTGTTTCACTCAGCCT 5250

GTTCCCCGTTCTGGAAAGAAGAGGGCTGCGATGATGACAGAGGTTCTTGC 5300 LU501908 ACATAAGATAAGGGCAGTGGAAAAGAAACCTATTCCCCGAATCAAGCGGA 5350 AGAATAACTCAACAACAACAAACAACAGTAAGCCTTCGTCACTGCCAACC 5400 TTAGGGAGTAACACTGAGACCGTGCAACCTGAAGTAAAAAGTGAAACCGA 5450 ACCCCATTTTATCTTAAAAAGTTCAGACAACACTAAAACTTATTCGCTGA 5500 TGCCATCCGCTCCTCACCCAGTGAAAGAGGCATCTCCAGGCTTCTCCTGG 5550 TCCCCGAAGACTGCTTCAGCCACACCAGCTCCACTGAAGAATGACGCAAC 5600 AGCCTCATGCGGGTTTTCAGAAAGAAGCAGCACTCCCCACTGTACGATGC 5650 CTTCGGGAAGACTCAGTGGTGCCAATGCAGCTGCTGCTGATGGCCCTGGC 5700 ATTTCACAGCTTGGCGAAGTGGCTCCTCTCCCCACCCTGTCTGCTCCTGT 5750 GATGGAGCCCCTCATTAATTCTGAGCCTTCCACTGGTGTGACTGAGCCGC 5800 TAACGCCTCATCAGCCAAACCACCAGCCCTCCTTCCTCACCTCTCCTCAA 5850 GACCTTGCCTCTTCTCCAATGGAAGAAGATGAGCAGCATTCTGAAGCAGA 5900 TGAGCCTCCATCAGACGAACCCCTATCTGATGACCCCCTGTCACCTGCTG 5950 AGGAGAAATTGCCCCACATTGATGAGTATTGGTCAGACAGTGAGCACATC 6000 TTTTTGGATGCAAATATTGGTGGGGTGGCCATCGCACCTGCTCACGGCTC 6050 GGTTTTGATTGAGTGTGCCCGGCGAGAGCTGCACGCTACCACTCCTGTTG 6100 AGCACCCCAACCGTAATCATCCAACCCGCCTCTCCCTTGTCTTTTACCAG6150 CACAAAAACCTAAATAAGCCCCAACATGGTTTTGAACTAAACAAGATTAA 6200 GTTTGAGGCTAAAGAAGCTAAGAATAAGAAAATGAAGGCCTCAGAGCAAA 6250 AAGACCAGGCAGCTAATGAAGGTCCAGAACAGTCCTCTGAAGTAAATGAA 6300 TTGAACCAAATTCCTTCTCATAAAGCATTAACATTAACCCATGACAATGT 6350 TGTCACCGTGTCCCCTTATGCTCTCACACACGTTGCGGGGCCCTATAACC 6400 ATTGGGTCTGA 6411 <210>2 <211> 719 <212> Amino acid <213> TET1 (1418-2136aa) <400> 2

ELPTCSCLDRVIQKDKGPYYTHLGAGPSVAAVREIMENRY GQKGNAIRIEIVVYTGKEGKU501908

SSHGCPIAK WVLRRSSDEEKVLCLVRQRTGHHCPTAVMV VLIMVWDGIPLPMADRLYTELTENLKSY NGHPTDRRC TLNENRTCTCQGIDPETCGASFSFGCSWSMYFNGCKFGRSPSPRRFRIDPSSPLHEKNLED NLQSLATRL APIYKQYAPVAYQNQVEYENVARECRLGSKEGRPFSGVTACLDFCAHPHRDIHNMNNGS TVVCTLTRE DNRSLGVIPQDEQLHVLPLYKLSDTDEFGSKEGMEAKIKSGAIEVLAPRRKKRTCFTQPV PRSGKKRAA MMTEVLAHKIRAVEKKPIPRIKRKNNSTTTNNSKPSSLPTLGSNTETVQPEVKSETEPHFI LKSSDNTKT YSLMPSAPHPVKEASPGFSWSPKTASATPAPLKNDATASCGFSERSSTPHCTMPSGRLSG ANAAAADGP GISQLGEVAPLPTLSAPVMEPLINSEPSTGVTEPLTPHQPNHQPSFLTSPQDLASSPMEEDE QHSEADEPP SDEPLSDDPLSPAEEKLPHIDEYWSDSEHIFLDANIGGVAIAPAHGSVLIECARRELHATTP VEHPNRNHP TRLSLVFYQHKNLNKPQHGFELNKIKFEAKEAKNKKMKASEQKDQAANEGPEQSSEVN ELNQIPSHK

ALTLTHDNVVTVSPYALTHVAGPYNHWV <210> 3 <211> 52 <212> DNA <213> TET1-Forward primer <400> 3

GGAGGTAGTGGAATGGATCCCGCCACCATGGAACTGCCCACCTGCAGCTGTC <210> 4 <211> 46 <212> DNA

<213> TET1-Reverse primer LU501908 <400> 4

TCACCATGGTGGCGGGATCGACCCAATGGTTATAGGGCCCCGCAAC <210>5 <211> 2157 <212> DNA <213> tet] gene mRNA coding for TET1 protein 1418-2136aa <400> 5 GAACTGCCCACCTGCAGCTGTCTTGATCGAGTTATACAAAAAGACAAAGG 50 CCCATATTATACACACCTTGGGGCAGGACCAAGTGTTGCTGCTGTCAGGG 100 AAATCATGGAGAATAGGTATGGTCAAAAAGGAAACGCAATAAGGATAGAA 150 ATAGTAGTGTACACCGGTAAAGAAGGGAAAAGCTCTCATGGGTGTCCAAT 200 TGCTAAGTGGGTTTTAAGAAGAAGCAGTGATGAAGAAAAAGTTCTTTGTT 250 TGGTCCGGCAGCGTACAGGCCACCACTGTCCAACTGCTGTGATGGTGGTG 300 CTCATCATGGTGTGGGATGGCATCCCTCTTCCAATGGCCGACCGGCTATA 350 CACAGAGCTCACAGAGAATCTAAAGTCATACAATGGGCACCCTACCGACA 400 GAAGATGCACCCTCAATGAAAATCGTACCTGTACATGTCAAGGAATTGAT 450 CCAGAGACTTGTGGAGCTTCATTCTCTTTTGGCTGTTCATGGAGTATGTA 500 CTTTAATGGCTGTAAGTTTGGTAGAAGCCCAAGCCCCAGAAGATTTAGAA 550 TTGATCCAAGCTCTCCCTTACATGAAAAAAACCTTGAAGATAACTTACAG 600 AGTTTGGCTACACGATTAGCTCCAATTTATAAGCAGTATGCTCCAGTAGC 650 TTACCAAAATCAGGTGGAATATGAAAATGTTGCCCGAGAATGTCGGCTTG 700 GCAGCAAGGAAGGTCGTCCCTTCTCTGGGGTCACTGCTTGCCTGGACTTC 750 TGTGCTCATCCCCACAGGGACATTCACAACATGAATAATGGAAGCACTGT 800 GGTTTGTACCTTAACTCGAGAAGATAACCGCTCTTTGGGTGTTATTCCTC 850 AAGATGAGCAGCTCCATGTGCTACCTCTTTATAAGCTTTCAGACACAGAT 900 GAGTTTGGCTCCAAGGAAGGAATGGAAGCCAAGATCAAATCTGGGGCCAT 950 CGAGGTCCTGGCACCCCGCCGCAAAAAAAGAACGTGTTTCACTCAGCCTG 1000 TTCCCCGTTCTGGAAAGAAGAGGGCTGCGATGATGACAGAGGTTCTTGCA 1050

CATAAGATAAGGGCAGTGGAAAAGAAACCTATTCCCCGAATCAAGCGGAA 1100 LU501908 GAATAACTCAACAACAACAAACAACAGTAAGCCTTCGTCACTGCCAACCT 1150 TAGGGAGTAACACTGAGACCGTGCAACCTGAAGTAAAAAGTGAAACCGAA 1200 CCCCATTTTATCTTAAAAAGTTCAGACAACACTAAAACTTATTCGCTGAT 1250 GCCATCCGCTCCTCACCCAGTGAAAGAGGCATCTCCAGGCTTCTCCTGGT 1300 CCCCGAAGACTGCTTCAGCCACACCAGCTCCACTGAAGAATGACGCAACA 1350 GCCTCATGCGGGTTTTCAGAAAGAAGCAGCACTCCCCACTGTACGATGCC 1400 TTCGGGAAGACTCAGTGGTGCCAATGCAGCTGCTGCTGATGGCCCTGGCA 1450 TTTCACAGCTTGGCGAAGTGGCTCCTCTCCCCACCCTGTCTGCTCCTGTG 1500 ATGGAGCCCCTCATTAATTCTGAGCCTTCCACTGGTGTGACTGAGCCGCT 1550 AACGCCTCATCAGCCAAACCACCAGCCCTCCTTCCTCACCTCTCCTCAAG 1600 ACCTTGCCTCTTCTCCAATGGAAGAAGATGAGCAGCATTCTGAAGCAGAT 1650 GAGCCTCCATCAGACGAACCCCTATCTGATGACCCCCTGTCACCTGCTGA 1700 GGAGAAATTGCCCCACATTGATGAGTATTGGTCAGACAGTGAGCACATCT 1750 TTTTGGATGCAAATATTGGTGGGGTGGCCATCGCACCTGCTCACGGCTCG 1800 GTTTTGATTGAGTGTGCCCGGCGAGAGCTGCACGCTACCACTCCTGTTGA 1850 GCACCCCAACCGTAATCATCCAACCCGCCTCTCCCTTGTCTTTTACCAGC 1900 ACAAAAACCTAAATAAGCCCCAACATGGTTTTGAACTAAACAAGATTAAG 1950 TTTGAGGCTAAAGAAGCTAAGAATAAGAAAATGAAGGCCTCAGAGCAAAA 2000 AGACCAGGCAGCTAATGAAGGTCCAGAACAGTCCTCTGAAGTAAATGAAT 2050 TGAACCAAATTCCTTCTCATAAAGCATTAACATTAACCCATGACAATGTT 2100 GTCACCGTGTCCCCTTATGCTCTCACACACGTTGCGGGGCCCTATAACCA 2150 TTGGGTC 2157

Claims (9)

CLAIMS LU501908

1. An application of recombinant single-stranded DNA virus vector containing a heterologous nucleic acid sequence in preparing medicine for treating stress cognitive impairment, wherein the recombinant single-stranded DNA virus vector is an adeno-associated virus vector, and the heterologous nucleic acid encodes therapeutic protein.

2. The application according to claim 1, characterized in that the therapeutic protein is TET1(1418-2136aa), and its amino acid sequence is shown in SEQ ID NO 2.

3. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the recombinant single-stranded DNA virus vector according to any one of claims 1 to

2.

4. À method for preparing the recombinant single-stranded DNA virus vector according to claim 1, characterized by comprising the following steps: S1: construction and identification of eukaryotic expression vector containing catalytic activity domain of TET1 enzyme; S2, detect recombinant plasmid expression, transfect the identified positive recombinant plasmid into 293T cells, collect cells, extract protein, and detect the expression of recombinant plasmid by western blot experiment; S3: packaging adeno-associated virus, co-transfecting the recombinant expression plasmid pAAV-TET1(1418-2136aa), pAAV-RC and pHelper into AAV-293 cells, collecting cell supernatant and cells after 3 days, cracking to obtain AAV particles, concentrating and purifying the virus by CsCl density gradient centrifugation and ultrafiltration, and finally detecting the titer of the virus by real-time PCR.

5. The preparation method according to claim 4, wherein in S1, the coding gene of the catalytic activity domain of Tetl enzyme is obtained by PCR, and then cloned into the adeno-associated expression vector.

6. The preparation method according to any one of claims 4-5, wherein the primer sequences used in the PCR reaction are shown in SEQ ID NO:3 and SEQ ID NO:4.

7. The preparation method according to claims 4-5, characterized in that th&J501908 adeno-associated expression vector is GV467, and the element sequence is CMV-betaGlobin-MCS-EGFP-3Flag-SV40 PolyA.

8. The preparation method according to any one of claims 4-7, characterized in that in S1, the peptide segment generated by translation of amplification products obtained by PCR is TET1 1418-2136aa, and its amino acid sequence is shown in SEQ ID No.2.

9. An application of the viral vector according to any one of claims 1-3 in the research of epigenetic regulation mechanism and the treatment of cognitive impairment caused by stress.