CN114507693A

CN114507693A - Recombinant adeno-associated virus expression vector and application thereof

Info

Publication number: CN114507693A
Application number: CN202210122025.3A
Authority: CN
Inventors: 袁野; 汪黎鸿; 王姣; 韦森; 罗韬; 卞修武
Original assignee: First Affiliated Hospital of Army Medical University
Current assignee: First Affiliated Hospital of Army Medical University
Priority date: 2022-02-09
Filing date: 2022-02-09
Publication date: 2022-05-17

Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant adeno-associated virus expression vector and application thereof. The recombinant adeno-associated virus expression vector hGFAP-promoter-dsacas9-VP64-3flag-polyA viral vector, U6-sagRNA (HUWE1) × 3 viral vector, U6-sagRNA (NC) viral vector, pAAV-RC vector and pHelper plasmid. The recombinant adeno-associated virus expression vector can realize endogenous overexpression of HUWE1 in glioblastoma by utilizing SadCas9, has strong specificity and small side effect, and provides a good theoretical and practical basis for treatment of glioblastoma.

Description

Recombinant adeno-associated virus expression vector and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant adeno-associated virus expression vector and application thereof.

Background

Tumors that grow in the cranium are known as brain tumors, and include primary brain tumors that arise from the parenchyma of the brain and secondary brain tumors that metastasize to the cranium from other parts of the body, the etiology of which is unknown, and tumors that arise from brain, meninges, pituitary, cranial nerves, cerebral vessels, and embryonic residual tissue, known as primary intracranial tumors. Malignant tumors from other organs and tissues of the body metastasize to the intracranial, called secondary intracranial tumors.

Primary brain tumors can be classified as benign and malignant according to their biological properties. Among them, gliomas are the most common malignant brain tumors of the central nervous system, and are classified into grades II-IV according to the classification standard of the World Health Organization (WHO) for gliomas, and among them, glioblastoma (WHO grade IV) is extremely high in malignancy. Glioblastoma has fast growth, no envelope, no obvious boundary, infiltrative growth and poor differentiation. Clinical symptoms of glioblastoma include increased intracranial pressure (specifically, headache, nausea, vomiting, papilledema, hypopsia, dizziness, diplopia, amaurosis, cataplexy, confusion, restlessness or apathy), delayed response, loose life, hypomnesis, splenic qi irritability, easy excitation or euphoria, seizures, cone beam impairment symptoms, two-point discrimination, disturbance of pattern, quality and material sensation, aphasia, visual field defect, hemianopsia, endocrine dysfunction and the like.

The prognosis of patients with glioblastoma is poor, and the quality of life and cognitive functions of the patients are seriously affected. Worldwide, the annual incidence of malignant gliomas is 5.25/100000, and about 17000 people per year are diagnosed with glioblastoma. In spite of the fact that in recent years, a combined treatment scheme of surgery, radiotherapy and chemotherapy is adopted for malignant glioma. However, median survival in patients did not improve significantly.

Currently, Temozolomide (TMZ) is used clinically as a first-line chemotherapeutic for the treatment of glioblastoma. However, the use of Temozolomide (TMZ) for the treatment of glioblastoma has problems such as low methylation of the promoter of the O6-methylguanine-DNA methyltransferase (MGMT) gene in the Blood Brain Barrier (BBB). Obviously, the effect of glioblastoma chemotherapy with temozolomide as the basic drug is not ideal.

Therefore, new approaches to glioblastoma treatment are urgently sought.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant adeno-associated virus expression vector.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention aims to protect a recombinant adeno-associated virus expression vector, which comprises an hGFAP-promoter-dsacas9-VP64-3flag-polyA virus vector, a U6-sagRNA (HUWE1)3 virus vector, a U6-sagRNA (NC) virus vector, a pAAV-RC vector and a pHelper plasmid.

The invention also aims to protect the preparation method of the recombinant adeno-associated virus expression vector, which comprises the following steps:

constructing hGFAP-promoter-dsacas9-VP64-3flag-polyA virus vector, U6-sagRNA (HUWE1)3 virus vector and U6-sagRNA (NC) virus vector;

the pHelper plasmid and the pAAV-RC plasmid are respectively transfected into AAV-293 cells together with hGFAP-promoter-dsacas9-VP64-3flag-polyA viral vectors, U6-sarRNA (HUWE1) × 3 viral vectors and U6-sarRNA (NC) viral vectors, and the recombinant adeno-associated virus expression vector is obtained after 2-3 days of transfection and assembly.

Further, the construction of the hGFAP-promoter-dsacas9-VP64-3flag-polyA viral vector comprises the following steps: and (3) carrying out gel electrophoresis on the enzyme digestion product by using the XbaI/PmlI enzyme digestion CV182 vector, recovering a target band, carrying out PCR amplification, exchanging the PCR amplification product with the enzyme digestion vector, and placing the exchange reaction product in a competent cell for conversion to obtain the recombinant plasmid.

Furthermore, the nucleotide sequence of the upstream amplification primer amplified by the PCR is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream amplification primer is shown as SEQ ID NO. 2.

Further, the construction of the U6-saGRNA (HUWE1) × 3 viral vector includes: and (3) carrying out gel electrophoresis on the enzyme digestion product by using the XbaI/PmlI enzyme digestion CV182 vector, recovering a target band, carrying out PCR amplification, exchanging the PCR amplification product with the enzyme digestion vector, and placing the exchange reaction product in a competent cell for conversion to obtain the recombinant plasmid.

Further, the nucleotide sequence of the upstream amplification primer amplified by the PCR is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream amplification primer is shown as SEQ ID NO.4

The present invention also aims to protect the application of the recombinant adeno-associated virus expression vector in the preparation of a medicament for treating glioblastoma.

The invention has the beneficial effects that:

the recombinant adeno-associated virus expression vector can realize endogenous overexpression of HUWE1 in glioblastoma by utilizing SadCas9, and has strong specificity and small side effect.

The invention provides a good theoretical and practical basis for the treatment of the glioblastoma.

Drawings

FIG. 1 is a schematic diagram of the structure of hGFAP-promoter-dsacas9-VP64-3flag-polyA vector;

FIG. 2 is an agarose gel electrophoresis of the digestion product, in which 1# is 10kb Marker (bands are from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250 bp); the No.2 is a carrier enzyme digestion product 3 is an un-enzyme digestion carrier;

FIG. 3 is an agarose gel electrophoresis image of the PCR amplification product, wherein the Marker comprises, from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250 bp;

FIG. 4 is an agarose gel electrophoresis chart of PCR-identified amplification products, in which # 1 is a negative control (ddH)₂O); 2# is negative control (no-load self-connected control group); 3# is yangA sexual control (GAPDH); the No.4 is Marker, and sequentially comprises 5Kb, 3Kb, 2Kb, 1.5Kb, 1Kb, 750bp, 500bp, 250bp and 100bp from top to bottom; 5-12# are 1-8 transformants, respectively;

FIG. 5 is a U6-sagRNA (HUWE1) × 3 vector map;

FIG. 6 is an agarose gel electrophoresis of the digestion product, in which 1# is 10kb Marker (bands are, from top to bottom, 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250 bp); 2# is a carrier enzyme digestion product; 3# is a vector which is not subjected to enzyme digestion;

FIG. 7 is an agarose gel electrophoresis image of the PCR amplification product, wherein the Marker comprises, from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250 bp;

FIG. 8 is an agarose gel electrophoresis image of the PCR-identified amplification product, in which # 1 is a negative control (ddH 2O); 2# is negative control (no-load self-connected control group); # 3 is positive control (GAPDH); the No.4 is Marker, and the 5Kb, the 3Kb, the 2Kb, the 1.5Kb, the 1Kb, the 750bp, the 500bp, the 250bp and the 100bp5-12# are respectively 1-8 transformants from top to bottom;

FIG. 9 is a schematic structural diagram of a constructed U6-sagRNA (NC) vector;

FIG. 10 is an agarose gel electrophoresis of the digestion product, in which 1# is 10kb Marker (bands are, from top to bottom, 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250 bp); 2# is a carrier enzyme digestion product; 3# is a vector which is not subjected to enzyme digestion;

FIG. 11 is an agarose gel electrophoresis of the PCR amplification product, wherein the Marker comprises, from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250 bp;

FIG. 12 is an agarose gel electrophoresis image of the PCR-identified amplification product, in which # 1 is a negative control (ddH 2O); 2# is negative control (no-load self-connected control group); 3# is positive control (GAPDH); the No.4 is Marker, and the 5Kb, the 3Kb, the 2Kb, the 1.5Kb, the 1Kb, the 750bp, the 500bp, the 250bp and the 100bp5-12# are respectively 1-8 transformants from top to bottom;

FIG. 13 is a schematic diagram of the structure of pAAV-RC9 plasmid;

FIG. 14 is a schematic diagram of the structure of the pHelper plasmid;

FIG. 15 is a flow chart of a treatment experiment performed on a glioblastoma in situ graft tumor model;

FIG. 16 is a fluoroscopic image;

FIG. 17 is a graph showing the results of immunohistochemical staining with Flag-tagged proteins;

FIG. 18 is a life cycle test result chart;

FIG. 19 is a photograph of HUWE1 and Ki67 protein immunohistochemical staining.

Detailed Description

The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

(one) constructing hGFAP-promoter-dsacas9-VP64-3flag-polyA vector, which specifically comprises the following steps:

experimental Material

1.1 Primary reagents

pAAV-RC plasmid (plasmid concentration adjusted to 1mg/ml using pH7.5TE buffer) Yunzhu Biotechnology (Guangzhou) Ltd

pHelper plasmid (plasmid concentration adjusted to 1mg/ml using pH7.5TE buffer) Yunzhu Biotechnology (Guangzhou) Ltd

1.2 Main instruments and Equipment

1.3 Gene of interest and information on tool vector

The target gene is as follows: hGFAP-promoter-dsacas9-VP64-3flag-polyA

Carrier name: CV182

The element sequence is as follows: MCS (modulation and coding scheme)

Cloning site: XbaI/PmlI

Vector mapping:

the target gene is as follows: hGFAP-promoter-dsacas9-VP64-3flag-polyA

Carrier name: CV182

The element sequence is as follows: MCS (modulation coding scheme)

Cloning site: XbaI/PmlI

The map of the constructed vector is shown in FIG. 1.

2.1 vector cleavage

Prepare 50 μ l enzyme system as shown in Table 1, add various reagents in sequence according to the list order, gently blow and mix with a pipette, centrifuge briefly, and react at 37 ℃ for 3 h. Carrying out agarose gel electrophoresis on the vector enzyme digestion product, and recovering a target band;

the agarose gel electrophoresis comprises the following specific steps:

(1) to the beaker was added 100ml of 1 × TAE buffer, followed by 2% agarose powder, and the powder was mixed well with the buffer and shaken.

(2) And (3) putting the beaker filled with the suspension into a microwave oven, heating until the dry powder is dissolved, and taking out the beaker by using a heat-insulating glove.

(3) Add 2. mu.g of ethidium bromide to the beaker and shake the liquid in the beaker evenly.

(4) And adding the agarose solution into a dry plastic tray, inserting the sample comb teeth according to the sample loading amount, standing until the sample comb teeth are cooled and shaped, and finishing the preparation of the electrophoresis gel.

(5) The gel was placed horizontally in an electrophoresis tank containing TAE buffer, the comb was carefully pulled out, and TAE buffer was added just above the top of the gel.

(6) Adding a sample mixed with DNA and Loading buffer solution or a DNA sample added with restriction enzyme and buffer solution into the Loading holes respectively, and adding DNA ladders into at least two Loading holes to display the position of the DNA in the glue running process.

(7) And closing the electrophoresis tank cover, measuring the negative pole of the tank cover to the upper sample hole, measuring the positive pole of the tank cover to the DNA electrophoresis side, inserting the positive pole of the tank cover into the positive pole hole of the electrophoresis apparatus, and inserting the negative pole of the tank cover into the negative pole hole.

(8) The voltage of the electrophoresis apparatus is adjusted to 100V, the time is set to be 20-40 minutes according to the size of DNA, the electrophoresis tank is observed for many times during the electrophoresis period, and the sample is prevented from running into the electrophoresis liquid and separating from the DNA gel.

(9) And when the DNA sample runs out of a sufficient distance, the electrophoresis power supply is closed, the gel is taken out, and the gel is placed into a developing instrument for developing and photographing.

TABLE 1 enzyme digestion System

Reagent	Volume (μ l)
		ddH₂O	42
10×CutSmart Buffer2	5
		Purified plasmid DNA (1. mu.g/. mu.l)	2
AgeI(10U/μl)	1
		Total	50

2.2 the electrophoretogram results of the cleavage products are shown in FIG. 2.

3. Obtaining a target Gene fragment

3.1 primers

TABLE 2 primers

ID	seq
		SEQ ID NO.1	GCCGCACGCGTGTGTCTAGAGTCTGTAAGCTGAAGACCTGGCAGTGC
SEQ ID NO.2	GGCCGCTCGGTCCGCACGTGGAACAAACGACCCAACACCCGTGCG

3.2PCR amplification of Gene fragments of interest

Preparing a reaction system, lightly blowing, uniformly mixing, centrifuging for a short time, and placing in a PCR instrument for reaction.

The reaction system is shown in table 3:

TABLE 3 PCR amplification reaction System

The PCR amplification reaction conditions are shown in Table 4:

TABLE 4 PCR amplification reaction conditions

3.3 PCR amplification results

The PCR amplification products were subjected to agarose gel electrophoresis in the same manner as described above, and the results are shown in FIG. 3.

As is clear from FIG. 3, the size of the PCR amplification product was 5309 bp.

Exchanging PCR amplification product with carrier

The reaction system shown in Table 5 was prepared in an ice-water bath. And lightly blowing and beating the mixture by using a pipettor, and centrifuging the mixture for a short time to avoid generating bubbles. The reaction was carried out at 37 ℃ for 30min, followed by cooling in an ice-water bath for 5min and then immediately followed by conversion.

Reaction system:

TABLE 5 reaction System

5. Transformation of

Add 10. mu.L of the exchange reaction product to 100. mu.L of competent cells, flick the tube wall and mix well, and leave on ice for 30 min. Heat shock at 42 deg.C for 90s, and incubating in ice water bath for 2 min. Adding 500. mu.L LB medium, and shaking-culturing at 37 deg.C for 1 h. Taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and carrying out inverted culture in a constant-temperature incubator for 12-16 h.

6. Colony PCR identification

6.1 PCR-identified primers for the colonies are shown in Table 6.

TABLE 6 PCR primers for colony identification

ID	seq
		SEQ ID NO.3	GAGAGAATGAGGGGTACCCAG
SEQ ID NO.4	GTGGGCCATTTCTCCCTGATC

6.2 PCR identification

Prepare the reaction system shown in Table 7, shake and mix, and centrifuge briefly. Picking single colony in a super clean bench by using a sterile gun head to a 20 mu L identification system, blowing, uniformly mixing, and placing in a PCR instrument for reaction.

TABLE 7 PCR identification reaction System

The PCR reaction conditions are shown in Table 8.

TABLE 8 PCR identification reaction conditions

6.3 identification results

The PCR-identified product was subjected to agarose gel electrophoresis in the same manner as described above, and the results are shown in FIG. 4.

As can be seen from FIG. 4, the PCR product size of the positive transformant was 163 bp.

7. Sequencing

Inoculating the identified positive clone transformant into a proper amount of LB liquid culture medium containing corresponding antibiotics, culturing for 12-16h at 37 ℃, taking a proper amount of bacterial liquid for sequencing, performing sequencing by Shanghai Baiqu biomedical science and technology Limited, and comparing and analyzing a sequencing result with a target gene sequence. The alignment results are as follows:

CCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGTGTCTAGAGTCTGTAAGCTGAAGACCTGGCAGTGCTGAGCTGGTCAGCCCCCAGGACCTCCTTTTGTGCCCACGAGTGACTCACCTTGGCATAGACATAATGGTCAGGGGTGGGCACGCAGCCTGCTTCCCGCTGTGCTCCAGGCCTCCTTCGATGCTTTCCGAGAAGTCTATTGAGCTGGGAGCTTGTACTGCACCCGGGGCTGACATCCTGGCATCCTGGGATAAAAGCAGCCCACGGGGCTGCCCTTGCCATATGCCTCACTGGCGGCAGAGAACAAGGCTCTATTCAGCGAGTACCCTGGAGTAGACACCAGAAGCCCAAGCATGGGCAGAGGAAGGCAGGGGTTGGGGGGAGCAGAGCTGTCTGTGTTCCAGAAGCCCAAGGACACAGATGGCTAAGGCGCCTGGGAGAGGGACCTGAGTGGAAGAGATAGATGGGCCTGAAGTCTCAAGCAGCAACAGCCTCCTCCCCGCCATTGGTGAGGGTGGGGTTTGGTTTCCCGGACCTACATATCCCTCAGAGGCCTGGTGTGTAGGAATTTAAAGGGGGTAAATCTCCTGAGAGAATGAGGGGTACCCAGGAAGACGGGGTGTTACAGAAAGAAAGACTCCAGCATGCACAGCCAACTCATTCAAAACTACTCTGTCAGGGGCTGCCAGGGGCCAGGCTCGGGGTGGGGGGTGGGGGGCAACGAGAAGCTGGATCAGGGAGAAATGGCCCACTAGGCTGGATAAGAGGCCACAGAGGGGCTCAGGAATGAAGCCTGCTGTCTTACCCTATTAGGATCTGCGTGCATACCTTCTGCCGTGCACTCTAAACACACAGCCAGAGGCTCAAGTTGACCCTGGAGTCACAGAGAGGGCTCCAACCTTAGCCCTCCACTCCTGAACTCCAGGAATGAGAAGATAGAGTTGGAGAGATTCAGGGGAGAGGACTCTGTTGAGAATGGGGGTCACAGGAAACTGTAATATAGGTTGATCCCGGAGGAAGGGAATAGGTTCTTCAAGTTCCTAGCATCTCACAGGCCCCCAGAGAAGGACAGAGTTGGGGTGGTCCTGGCTTACAGGCTCTAAGAACTGGAAGCTGATTACCCCACCGAGCTGTGCACTCTCTGTCTCTGTCTCTGTGTGTGCGCTCGTGCACACTTATCACACAAATGTTCATGTGTGTGCACATACATGTGTTGAGACCAGAGGTCAACCTCAGGCACTGTTGCCTTGGTTTTCTGAGAGAGCATTTCTCTCTGGATCTGGAACTCGCCAATTAGTGAGAGCCAGGAAGTCTGCTGATTTTCACTGCCCAGCACTGGAGTTTACAAGTATGCACTGTCAACCCAGGCCTTTTGTATTCATTCTGCAGCTAGAACTTGGGTGGGTCTTCATGCTTGACAGGCAAGCAATTTATGGACTAAGCTGTTCCCTCGGCCCTCTCTTGACCCATTTACCAGAAAGGGGGTTCCTTGATCAATGGCGAAGCCAGGCTGGTGTTCCCAAGAAAGCCTTGACTCTGGGTACAGTGACCTCAGTGGGGTGAGAGGAGTTCTCCCCCTAGCTGGGCTGGGGCCCAGCTCCACCCCCTCAGGCTATTCAATGGGGGTGCTTCCAGGAAGTCAGGGGCAGATTTAGTCCAACCCGTTCCTCCATAAAGGCCCTGACATCCCAGGAGCCAGCAGAGGCAGGGCAGGGTCGACTCCGGACGCCACCATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCAAGCGGAACTACATCCTGGGCCTGGCCATCGGCATCACCAGCGTGGGCTACGGCATCATCGACTACGAGACACGGGACGTGATCGATGCCGGCGTGCGGCTGTTCAAAGAGGCCAACGTGGAAAACAACGAGGGCAGGCGGAGCAAGAGAGGCGCCAGAAGGCTGAAGCGGCGGAGGCGGCATAGAATCCAGAGAGTGAAGAAGCTGCTGTTCGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAACCCCTACGAGGCCAGAGTGAAGGGCCTGAGCCAGAAGCTGAGCGAGGAAGAGTTCTCTGCCGCCCTGCTGCACCTGGCCAAGAGAAGAGGCGTGCACAACGTGAACGAGGTGGAAGAGGACACCGGCAACGAGCTGTCCACCAAAGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAATACGTGGCCGAACTGCAGCTGGAACGGCTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAAGACCAGCGACTACGTGAAAGAAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGACCAGAGCTTCATCGACACCTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGACCTGGCGAGGGCAGCCCCTTCGGCTGGAAGGACATCAAAGAATGGTACGAGATGCTGATGGGCCACTGCACCTACTTCCCCGAGGAACTGCGGAGCGTGAAGTACGCCTACAACGCCGACCTGTACAACGCCCTGAACGACCTGAACAATCTCGTGATCACCAGGGACGAGAACGAGAAGCTGGAATATTACGAGAAGTTCCAGATCATCGAGAACGTGTTCAAGCAGAAGAAGAAGCCCACCCTGAAGCAGATCGCCAAAGAAATCCTCGTGAACGAAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCAAGCCCGAGTTCACCAACCTGAAGGTGTACCACGACATCAAGGACATTACCGCCCGGAAAGAGATTATTGAGAACGCCGAGCTGCTGGATCAGATTGCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACATCCAGGAAGAACTGACCAATCTGAACTCCGAGCTGACCCAGGAAGAGATCGAGCAGATCTCTAATCTGAAGGGCTATACCGGCACCCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGCTGTGGCACACCAACGACAACCAGATCGCTATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGTGGACCTGTCCCAGCAGAAAGAGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTCGTGAAGAGAAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAACGACATCATTATCGAGCTGGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAACGAGATGCAGAAGCGGAACCGGCAGACCAACGAGCGGATCGAGGAAATCATCCGGACCACCGGCAAAGAGAACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGACATGCAGGAAGGCAAGTGCCTGTACAGCCTGGAAGCCATCCCTCTGGAAGATCTGCTGAACAACCCCTTCAACTATGAGGTGGACCACATCATCCCCAGAAGCGTGTCCTTCGACAACAGCTTCAACAACAAGGTGCTCGTGAAGCAGGAAGAAGCCAGCAAGAAGGGCAACCGGACCCCATTCCAGTACCTGAGCAGCAGCGACAGCAAGATCAGCTACGAAACCTTCAAGAAGCACATCCTGAATCTGGCCAAGGGCAAGGGCAGAATCAGCAAGACCAAGAAAGAGTATCTGCTGGAAGAACGGGACATCAACAGGTTCTCCGTGCAGAAAGACTTCATCAACCGGAACCTGGTGGATACCAGATACGCCACCAGAGGCCTGATGAACCTGCTGCGGAGCTACTTCAGAGTGAACAACCTGGACGTGAAAGTGAAGTCCATCAATGGCGGCTTCACCAGCTTTCTGCGGCGGAAGTGGAAGTTTAAGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCGAGGACGCCCTGATCATTGCCAACGCCGATTTCATCTTCAAAGAGTGGAAGAAACTGGACAAGGCCAAAAAAGTGATGGAAAACCAGATGTTCGAGGAAAAGCAGGCCGAGAGCATGCCCGAGATCGAAACCGAGCAGGAGTACAAAGAGATCTTCATCACCCCCCACCAGATCAAGCACATTAAGGACTTCAAGGACTACAAGTACAGCCACCGGGTGGACAAGAAGCCTAATAGAGAGCTGATTAACGACACCCTGTACTCCACCCGGAAGGACGACAAGGGCAACACCCTGATCGTGAACAATCTGAACGGCCTGTACGACAAGGACAATGACAAGCTGAAAAAGCTGATCAACAAGAGCCCCGAAAAGCTGCTGATGTACCACCACGACCCCCAGACCTACCAGAAACTGAAGCTGATTATGGAACAGTACGGCGACGAGAAGAATCCCCTGTACAAGTACTACGAGGAAACCGGGAACTACCTGACCAAGTACTCCAAAAAGGACAACGGCCCCGTGATCAAGAAGATTAAGTATTACGGCAACAAACTGAACGCCCATCTGGACATCACCGACGACTACCCCAACAGCAGAAACAAGGTCGTGAAGCTGTCCCTGAAGCCCTACAGATTCGACGTGTACCTGGACAATGGCGTGTACAAGTTCGTGACCGTGAAGAATCTGGATGTGATCAAAAAAGAAAACTACTACGAAGTGAATAGCAAGTGCTATGAGGAAGCTAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGTTTATCGCCTCCTTCTACAACAACGATCTGATCAAGATCAACGGCGAGCTGTATAGAGTGATCGGCGTGAACAACGACCTGCTGAACCGGATCGAAGTGAACATGATCGACATCACCTACCGCGAGTACCTGGAAAACATGAACGACAAGAGGCCCCCCAGGATCATTAAGACAATCGCCTCCAAGACCCAGAGCATTAAGAAGTACAGCACAGACATTCTGGGCAACCTGTATGAAGTGAAATCTAAGAAGCACCCTCAGATCATCAAAAAGGGCAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCCGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCAAGAGACTACAAGGATGACGATGACAAGGATTACAAAGACGACGATGATAAGGACTATAAGGATGATGACGACAAATAACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACT。

the alignment results show that: the sequencing result is completely consistent with the target sequence.

8. Plasmid extraction

Transferring the bacterial liquid with correct sequencing into 10ml LB liquid culture medium containing corresponding antibiotic, culturing overnight at 37 ℃,

and (4) carrying out plasmid extraction by using a small-extraction medium-amount kit of Tiangen endotoxin-free plasmids, and allowing the extracted qualified plasmids to enter a downstream process. The detailed operation steps are as follows:

1. collecting overnight cultured bacteria liquid in a marked 5ml centrifuge tube, centrifuging at 12000rpm for 2min, and collecting bacteria;

2. discarding the supernatant, adding 250 μ l of cell resuspension, and fully oscillating to make the bacterial mass suspend uniformly;

3. adding 250 μ l cell lysate, adding 10 μ l proteinase K, reversing the mixture from top to bottom for 5-6 times, and mixing gently;

standing for 1-2min to make thallus cracking and clarifying;

4. adding 350 μ l of neutralizing solution, turning upside down, mixing to completely separate out protein, and standing in ice bath for 5 min;

5.10000rpm for 10min, discarding protein, collecting supernatant in another clean sterile 1.5ml EP tube;

6.12000rpm for 5min while preparing the labeled recovery column, transferring the supernatant to the recovery column,

centrifuging at 12000rpm for 1min, and discarding the lower layer waste liquid;

7. adding 600 μ l of pre-prepared rinsing liquid, centrifuging at 12000rpm for 1min, discarding the lower layer waste liquid, and repeating

Once, performing idle separation at 12000rpm for 2min, and further removing residual rinsing liquid;

8. transferring the recovery column to a new 1.5ml EP tube in a super clean bench, standing for 10-20min, and naturally drying;

9. adding 95 μ l of nucleic-Free Water into the recovery column, standing for 2min, centrifuging at 12000rpm for 2min, collecting sample, numbering, performing agarose gel electrophoresis (the same as the agarose gel electrophoresis method), measuring concentration, and performing quality inspection;

wherein, the concentration determination method comprises the following steps:

1, starting a UV-240 ultraviolet spectrophotometer and preheating for 10 min;

2. washing the cuvette with distilled water, blotting the cuvette with absorbent paper, adding TE buffer solution, putting the cuvette on a cell S frame of a sample chamber, and closing a cover plate;

3. setting a slit and then zeroing;

4. diluting the standard sample and the sample to be tested (5ul DNA to 1000 ul);

5. putting the cuvette filled with the standard sample or the sample to be detected on an S frame of the sample chamber, and closing the cover plate;

6. setting ultraviolet wavelength, and respectively measuring OD values at 230nm, 260nm and 280 nm;

7. calculating the concentration of the sample: the concentration of the DNA sample is (ug/ul: OD 260X dilution times X50/1000);

8. the concentration of the DNA sample was calculated to be 0.87 ug/ul.

The quality inspection method comprises the following steps: the DNA purity is judged by comparing the ratio of OD260/OD280, and when the OD260/OD280 is less than 1.8, the protein content is high; when OD260/OD280 is greater than 2.0, the RNA content is higher; when OD260/OD280 was between 1.8 and 2.0, it means that the DNA was relatively pure.

Through detection, the ratio of OD260/OD280 of the sample is 1.87, and the purity is good. (II) construction of U6-sagRNA (HUWE1) × 3 vector

1. Experimental Material

1.1 Primary reagents

1.2 Main instruments and Equipment

1.3 Gene of interest and information on tool vector

The target gene is as follows: U6-sagRNA (HUWE1) × 3

Carrier name: CV182

The element sequence is as follows: MCS (modulation and coding scheme)

Cloning site: XbaI/PmlI

The map of the constructed vector is shown in FIG. 5.

2. Vector cleavage

2.1 prepare 50 μ l enzyme system according to Table 9, and add various reagents in sequence according to the list order, gently blow and beat by a pipette, mix well, centrifuge briefly, and react for 3h at 37 ℃. And (4) carrying out agarose gel electrophoresis on the vector enzyme digestion product, wherein the agarose gel electrophoresis method is the same as the above method, and recovering the target band.

TABLE 9 enzyme digestion System

Reagent	Volume (μ l)
		ddH2O	42
10×CutSmart Buffer2	5
		Purified plasmid DNA (1. mu.g/. mu.L)	2
AgeI(10U/μl)	1
		Total	50

2.2 enzyme cutting result:

the agarose gel electrophoresis of the cleaved products is shown in FIG. 6.

3. Obtaining a target Gene fragment

3.1PCR amplification primers are shown in Table 10.

TABLE 10PCR amplification primers

ID	seq
		SEQ ID NO.5	GCCGCACGCGTGTGTCTAGAGAGGGC
SEQ ID NO.6	GGCCGCTCGGTCCGCACGTGAAAAATCTCG

3.2PCR amplification of Gene fragments of interest

A reaction system shown in Table 11 was prepared, gently blown and mixed, centrifuged for a short time, and placed in a PCR apparatus for reaction.

TABLE 11 PCR reaction System

Reagent	Volume (μ L)
		ddH2O	32.5
5×PS Buffer	10
		dNTP Mix(2.5mM each)	4
Upstream amplification primer (10. mu.M)	1
		Downstream amplification primer (10. mu.M)	1
Stencil (10 ng/. mu.L)	1
		PrimeSTAR HS DNA polymerase	0.5
Total	50

TABLE 12 PCR reaction conditions

3.3 PCR amplification results

The PCR amplification products were subjected to agarose gel electrophoresis in the same manner as described above, and the results are shown in FIG. 7.

As can be seen from FIG. 7, the size of the PCR amplification product was 1127 bp.

Exchanging the PCR product with the vector

A reaction system shown in Table 13 was prepared in an ice-water bath. And lightly blowing and beating the mixture by using a pipettor, and centrifuging the mixture for a short time to avoid generating bubbles. The reaction was carried out at 37 ℃ for 30min, followed by cooling in an ice-water bath for 5min and then immediately followed by conversion.

Table 13 reaction system:

5. transformation of

6. Colony PCR identification

6.1 primers

TABLE 14 PCR identification primers for colonies

ID	seq
		SEQ ID NO.7	GATTTTTGCTAGCGTTTAAACG
SEQ ID NO.8	AAACTTTGGGGCTCCACCCAC

6.2 PCR identification

A reaction system shown in Table 15 was prepared, mixed by shaking, and centrifuged for a short time. Picking single colony in a super clean bench by using a sterile gun head to a 20 mu L identification system, blowing, uniformly mixing, and placing in a PCR instrument for reaction.

Table 15 identification of reaction systems

TABLE 16 PCR identification reaction conditions

6.3 identification results

The PCR-identified product was subjected to agarose gel electrophoresis in the same manner as described above, and the results are shown in FIG. 8.

As can be seen from FIG. 8, the size of the PCR amplification product was 296 bp.

TGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGTGTCTAGAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTTCCGCGGCTTCCACCGTGCCGTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTGCTAGCGTTTAAACGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTGCGGTGGGTGGAGCCCCAAAGTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTGGGCCCTCTAGACTCGAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTATGGTTGCATTTACTGGGCCGTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTCACGTGCGGACCGAGCGGCCGCAGGAACCC。

the above comparison results show that: the sequencing result is completely consistent with the target sequence.

8. Plasmid extraction

Transferring the correctly sequenced bacterium liquid into 10ml LB liquid culture medium containing corresponding antibiotics, culturing overnight at 37 ℃, performing plasmid extraction by using a small-medium-amount plasmid extraction kit without endotoxin from Tiangen, and introducing the qualified plasmids into a downstream process. The detailed operation steps are as follows:

3. adding 250 mul cell lysis solution, adding 10 mul proteinase K, reversing the solution for 5-6 times, and mixing gently;

standing for 1-2min to make thallus cracking and clarifying;

centrifuging at 6.12000rpm for 5min while preparing labeled recovery column, transferring supernatant to the recovery column, centrifuging at 12000rpm for 1min, and discarding lower layer waste liquid;

7. adding 600 μ l of pre-prepared rinsing liquid, centrifuging at 12000rpm for 1min, discarding the lower layer waste liquid, repeating once, and allowing to idle at 12000rpm for 2min to further remove the residual rinsing liquid;

9. adding 95 μ l of nucleic-Free Water into the recovery column, standing for 2min, centrifuging at 12000rpm for 2min, collecting sample, numbering, performing agarose gel electrophoresis (same as the agarose gel electrophoresis method), determining concentration, and performing quality control;

wherein, the concentration determination method comprises the following steps:

1, starting a UV-240 ultraviolet spectrophotometer and preheating for 10 min;

3. setting a slit and then zeroing;

7. calculating the concentration of the sample: the concentration of the DNA sample is (ug/ul: OD 260X dilution X50/1000)

8. The concentration of the DNA sample was calculated to be 0.57 ug/ul.

The ratio of OD260/OD280 of the sample is 1.91 through detection, and the purity is good.

(III) construction of U6-sagRNA (NC) vector

1. Experimental Material

1.1 Primary reagents

1.2 Main instruments and Equipment

1.3 Gene of interest and information on tool vector

The target gene is as follows: U6-sagRNA (NC)

Carrier name: CV182

The element sequence is as follows: MCS (modulation and coding scheme)

Cloning site: XbaI/PmlI

The structure of the constructed U6-sagRNA (NC) vector is shown in FIG. 9;

2. vector cleavage

2.1A 50. mu.l digestion system was prepared according to Table 17. Adding various reagents in sequence according to the list sequence, gently blowing and uniformly mixing by using a pipette, centrifuging for a short time, and reacting for 3h at 37 ℃. And (4) carrying out agarose gel electrophoresis on the vector enzyme digestion product, wherein the agarose gel electrophoresis method is the same as the above method, and recovering the target band.

TABLE 17 enzyme digestion System

2.2 enzyme cutting result:

the results of agarose gel electrophoresis of the cleaved products are shown in FIG. 10.

3. Obtaining a target Gene fragment

3.1PCR amplification primers are shown in Table 18.

TABLE 18 PCR amplification primers

ID	seq
		SEQ ID NO.9	GCCGCACGCGTGTGTCTAGAGAGGGCCTATTTCCCATGATTCC
SEQ ID NO.10	GGCCGCTCGGTCCGCACGTGAAAAATCTCGCCAACAAGTTGACGAG

3.2PCR amplification of Gene fragments of interest

A reaction system shown in Table 19 was prepared, gently blown and mixed, centrifuged for a short time, and placed in a PCR apparatus for reaction.

TABLE 19 PCR reaction System

TABLE 20 PCR reaction conditions

3.3 PCR amplification results

The PCR amplification products were subjected to agarose gel electrophoresis in the same manner as described above, and the results of the agarose gel electrophoresis of the PCR amplification products are shown in FIG. 11.

As can be seen from FIG. 11, the PCR amplification product was 360bp in size.

Exchanging the PCR product with the vector

The following reaction system was prepared in an ice-water bath. And lightly blowing and beating the mixture by using a pipettor, and centrifuging the mixture for a short time to avoid generating bubbles. The reaction was carried out at 37 ℃ for 30min, followed by cooling in an ice-water bath for 5min and then immediately followed by conversion.

Reaction system:

5. transformation of

Add 10. mu.L of the exchange reaction product to 100. mu.L of competent cells, flick the tube wall and mix well, and leave on ice for 30 min. Heat shock at 42 deg.C for 90s, and incubating in ice water bath for 2 min. Adding 500. mu.L LB medium, and shaking-culturing at 37 deg.C for 1 h. Taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and placing the flat plate in a constant-temperature incubator

Culturing for 12-16 h.

6. Colony PCR identification

6.1 colony PCR identifying primers are shown in Table 20.

TABLE 20 colony PCR identification primers

ID	seq
		SEQ ID NO.11	GCCGCACGCGTGTGTCTAGAGAGGGCCTATTTCCCATGATTCC
SEQ ID NO.12	ATTCTGTTTCCAGAGTAC

6.2 PCR identification

A reaction system shown in Table 21 was prepared, mixed by shaking, and centrifuged for a short time. Picking single colony in a super clean bench by using a sterile gun head to a 20 mu L identification system, blowing, uniformly mixing, and placing in a PCR instrument for reaction.

Table 21 identification of reaction systems

Reagent	Volume (μ L)
		ddH2O	9.2
2×Taq Plus Master Mix	10
		Upstream primer (10. mu.M)	0.4
Downstream primer (10. mu.M)	0.4
		Single colony	-
Total	20

TABLE 22PCR reaction conditions

6.3 identification results

The PCR-identified product was subjected to agarose gel electrophoresis in the same manner as described above, and the result of agarose gel electrophoresis of the PCR-identified product is shown in FIG. 12.

From FIG. 12, the sizes of PCR products of positive transformants are: 313 bp.

7. Sequencing

GAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGTGTC TAGAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGACCACGGCAGGTCTCAGTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCT。

8. Plasmid extraction

1. collecting overnight cultured bacteria liquid in a marked 5ml centrifuge tube at 12000rpm, and separating for 2min to collect bacteria;

standing for 1-2min to make thallus cracking and clarifying;

6.12000rpm for 5min, preparing the marked recovery column, transferring the supernatant to the recovery column,

centrifuging at 12000rpm for 1min, and discarding the lower layer waste liquid;

7. adding 60 μ l of pre-prepared rinsing liquid, centrifuging at 12000rpm for 1min, discarding the lower layer waste liquid, and repeating

9. adding 95 μ l of nucleic-Free Water to the recovery column, standing for 2min, and centrifuging at 12000rpm for 2min

And min, collecting samples, numbering, performing electrophoresis, measuring concentration, and performing quality inspection.

Performing agarose gel electrophoresis (the same as the agarose gel electrophoresis method), measuring concentration, and performing quality inspection;

wherein, the concentration determination method comprises the following steps:

1, starting a UV-240 ultraviolet spectrophotometer and preheating for 10 min;

3. setting a slit and then zeroing;

4. standard and test samples were diluted (5ul DNA to 1000ul)

7. calculating the concentration of the sample: the concentration of the DNA sample was (ug/ul: OD 260X dilution X50/1000).

8. The concentration of the DNA sample was calculated to be 1.2 ug/ul.

The quality inspection method comprises the following steps: the DNA purity is judged by comparing the ratio of OD260/OD280, and when the OD260/OD280 is less than 1.8, the protein content is high; when OD260/OD280 is greater than 2.0, the RNA content is higher; when OD260/OD280 was between 1.8 and 2.0, it indicated that the DNA was purer.

Through detection, the ratio of OD260/OD280 of the sample is 1.85, and the purity is good.

(IV) recombinant adeno-associated virus packaging

The AAV Helper-Free System (AAV Helper-Free System) is used to produce recombinant adeno-associated virus without Helper virus. As no live Helper virus is required anymore, the AAV Helper-Free System of this example consists of a viral vector, a pAAV-RC vector and a pHelper vector:

1, respectively constructing hGFAP-promoter-dsacas9-VP64-3flag-polyA, U6-sagRNA (HUWE1)3 and U6-sagRNA (NC) vectors according to the (I), (II) and (III);

the pAAV-RC vector comprises rep and cap genes encoding AAV replication proteins and viral capsid proteins; stabilizing the expression levels of rep and cap genes is a key step in obtaining the desired high titer viral product; pAAV-RC uses two different promoters to control the expression of Rep and Cap, respectively, to obtain optimal expression levels and ratios of the respective gene products. This example used the AAV9 serotype, thus the pAAV-RC9 vector was used. A vector map of pAAV-RC9 is shown in FIG. 13.

The pHelper plasmid contains a collection of adenoviral genes VA, E2A and E4 necessary for AAV-293 Shencell production of high titer viruses. The map of the pHelper plasmid vector is shown in FIG. 13.

The production and packaging method of the recombinant adeno-associated virus particles comprises the following specific steps:

1. preparation of AAV-293 cells

3X 10 addition to 10ml DMEM growth Medium per 100-mm group of Fine culture plates⁶AAV-293 cells were used for transfection 48 hours later.

Transfection of AAV-293 cells

1. Two days prior to transfection, host cells passaged should reach 70-80% confluence;

2. the constructed co-transfected plasmid hGFAP-promoter-dsacas9-VP64-3flag-polyA, U6-sagRNA (HUWE1)3, U6-sagRNA (NC) vector, and commercial pHelper and pAAV-RC9 plasmids (purchased from Yuzhou Biotechnology, Guangzhou, Ltd.) were removed from the freezer at-20 ℃ and the concentration of the plasmids was adjusted to 1mg/ml with pH7.5TE buffer;

3. calculating the required transfection system and plasmid dosage according to the number of packaging trays, and sucking if packaging traysThree plasmids 10ul each (10ug each) were taken into a 1.5ml EP centrifuge tube and 1ml of 0.3M CaCl was added₂Lightly mixing;

4. aspirate 1ml of 2X HBS solution into another 15ml conical bottom tube. To this was added dropwise 1.03ml of a DNA/CaCl2 mixture (previous step). Repeatedly blowing, beating and mixing uniformly by turning over the ring;

5. immediately mixing the DNA/CaCl₂the/HBS solution was added dropwise to the cell culture dish. Gently shaking the cell culture plate while adding the solution to ensure that the solution is uniformly distributed in the culture medium as much as possible;

6. the cell culture plate is returned to the incubator at 37 ℃ and placed for 6 hours;

7. after transfection, the medium in the dish was replaced with 10ml of fresh medium;

8. the plates were returned to the incubator and incubated for an additional 66-72 hours.

Toxic materials recovering

1. Preparing a dry ice ethanol bath (only pouring ethanol into a foam box filled with dry ice, or using liquid nitrogen to replace the dry ice ethanol bath) and a water bath at 37 ℃;

2. the toxigenic cells were collected into a 15ml centrifuge tube along with the culture medium. When collecting cells, the culture tray is inclined at a certain angle to scrape the cells into the culture medium;

3.200 Xg, centrifugate for 3 minutes, centrifugate and separate Shencyst and supernatant, deposit the supernatant separately, Shenthin resuspend with 1ml PBS; 4. the cell suspension was repeatedly transferred in a dry ice ethanol bath and a water bath at 37 ℃ and freeze-thawed four times. After each melting, the mixture was shaken slightly. Cell debris was removed by centrifugation at 5.10,000 Xg and the centrifuged supernatant was transferred to a fresh centrifuge tube.

Concentration of AAV viruses

1. 40% PEG-8000 was added to the supernatant until the concentration was 8%, and after standing on ice for 2 hours (mixing back and forth every 15 minutes), centrifuged at 2,500 Xg for 30 minutes. Removing the supernatant, resuspending the precipitate with PBS, and mixing with cell lysis supernatant; 2.3,000 Xg for 30 minutes, and the supernatant was transferred to another clean tube. At this point, no cell debris should be visible in the supernatant. If partial debris still exists, centrifuging again;

3. residual plasmid DNA (final concentration of 50U/ml) was removed by digestion with Benzonase nuclease. Close the tube cover and temporalis several times to mix well. Incubation at 37 ℃ for 30 min;

4. filtering with a 0.45 μm filter head to obtain filtrate.

Purification of AAV

1. Adding solid CsCl into the virus concentrated solution until the density is 1.41g/ml (the refractive index is 1.372), adding 6.5g CsCl into about 10ml virus solution, and shaking to dissolve the CsCl, wherein the dissolved CsCl absorbs heat and cools;

2. adding the sample into an ultracentrifuge tube, and filling the residual space of the centrifuge tube with a pre-prepared 1.41g/ml CsCl solution;

3. centrifugation was carried out at 175,000 Xg for 24 hours to form a density gradient. Samples of different densities were collected in sequential steps and sampled for titre determination. Collecting the fraction enriched in AAV particles;

4. the above process is repeated once.

Determination of viral titre

1. Preparing sample and standard substance, diluting the standard substance plasmid and sample to be tested to

original concentration

10^-5，10^-6，10^-7，10^-8；

2. The reaction manifold volume was calculated from the number of reactions (X) (two duplicate wells were made for each gradient, 1 more was prepared for each 10 reactions):

TABLE 23 reaction System

Reaction components	Each reaction (μ l)	X number of reactions (μ l)
			2×SYBR-Green Mixture	10	10X
Upstream and downstream primers	0.5 each	Each 0.5X
			Water (W)	4	4X
Total volume	15	15X

Remarking: an upstream primer: CTGACGGAATGGCGCCGTCTTTCGAAGGCCCCGGAGGCCCTT, respectively;

a downstream primer: AAGGGCCTCCGGGGCCTTCGAAAGACGGCGCCATTCCGTCAG

3. Adding 15 mul of reaction solution into each reaction hole, and then adding 5 mul of template;

4. the sample was loaded on the machine, annealing temperature was set at 60 ℃, Ct values were obtained according to standard procedures and the copy number in AAV samples was calculated, and the results are shown in table 24.

TABLE 24 test results

Name of virus	rAAV-dsaCas9-HUWE1	rAAV-HUWE1-saGRNA	rAAV-U6-NC-saGRNA
				Number of copies	1.17E+13Copies/mL	1.12E+13Copies/mL	1.14E+13Copies/mL

(V) experiments on glioblastoma cell transplantation in situ model treatment (the flow is shown in FIG. 15), which specifically include:

firstly, constructing a glioblastoma in-situ transplanted tumor model, which comprises the following specific steps: (1) LN229 cells were collected in the logarithmic growth phase and adjusted to a cell density of 2X 10⁵The ratio of the total amount of the acid to the total amount of the acid; (2) 4-6 weeks old female NOD/SCID mice (purchased from the center of laboratory animals in southwestern hospital affiliated to army and military medical university) are anesthetized with pentobarbital sodium anesthetic, flattened and placed on a wood board, 5uL of cell suspension is injected into the cranium of the mice by a sterilized micro-injection needle, and the needle insertion site is selected at the position 5mm on the right side after the intersection of the anterior midline of the brain of the mice and the connection line of the outer canthus, and the needle insertion is performed for 5 mm.

The rAAV9-dCas9-HUWE1 is used for treating tumor-bearing mice, and the method comprises the following specific steps: (1) recombinant adeno-associated virus therapy experiments were performed on day 10 of orthotopic transplantation tumor establishment. The experiment is divided into two groups, namely a tail vein injection group and an in-situ injection group; each group is divided into an experimental group, a carrier control group and a pbs control group; (2) mixing 10ul rAAV-dsaCas9-HUWE1 virus and 10ul rAAV-HUWE1-sagRNA virus, and injecting into mouse intracranial by using a micro-injector, wherein the operation method is the same as the above; similarly, 10ul rAAV-dsaCas9-HUWE1 virus and 10ul rAAV-U6-NC-sagRNA are mixed and then separately injected into the mouse intracranial as a carrier control group and a pbs control group; (3) 10ul rAAV-dsaCas9 virus and 10ul rAAV-HUWE1-sagRNA virus were mixed, and the mixture was adjusted to 100ul volume with PBS and injected into the mouse tail vein. Similarly, 10ul rAAV-dsaCas9-HUWE1 virus and 10ul rAAV-U6-NC-sagRNA are mixed and then are subjected to volume metering by PBS to 100ul, and 100ul PBS alone is used as a carrier control group and is also injected into the tail vein of the mouse as well as a PBS control group;

selecting irradiation time from the 10 th day of intracranial transplantation tumor construction according to mouse states, injecting 200uL Luciferin working solution into the abdominal cavity of each mouse every time, and observing respective tumor forming conditions of the experimental group and the control group of mice by using an IVIS living animal imaging instrument; the results are shown in FIG. 16;

the method comprises the following steps of carrying out protein immunohistochemical staining on a Flag tag of an orthotopic transplantation tumor of a tumor-bearing mouse: (1) dewaxing: baking the sample for 30min under a baking lamp, and performing dewaxing treatment according to xylene I (15min) → xylene II (15 min);

(2) hydration: passing anhydrous alcohol I (10min) → anhydrous alcohol II (10min) → 95% alcohol (5min) → 85% alcohol (5min) → 75% alcohol (5min) through the cylinder in this order, rinsing the slide glass with ultrapure water for 3min under tap water, and washing the slide glass with ultrapure water 3 times for 5min each time; (3) antigen retrieval: adding EDTA repair liquid (working liquid) into a pressure cooker on an induction cooker, and putting the glass slide until the glass slide is completely submerged; heating for 2.5min in the repairing solution after pressurizing and steaming, placing the slide in a repairing box, covering the cover, and placing in a ventilation place to restore the slide to room temperature; (4) blocking endogenous peroxidase: selecting a freshly prepared 3% H2O2 blocking solution, adding the blocking solution into a repair box to enable the blocking solution to submerge a slide, sealing the box at 37 ℃ for 30min, washing the box with PBS for 4 times, lasting for 10min each time, and adding 30uLTween-20 during the last washing; (5) and (3) sealing: wiping off water around the slices, and dripping 30uL of goat instant sealing serum for sealing at 37 ℃ for 40 min; (6) incubating the primary antibody: spin-drying the serum on the slide by force, dripping Flag primary antibody working solution (obtained by diluting primary antigen solution with PBS (phosphate buffer solution) according to the proportion of 1: 250) on the surface of the slice, and placing the slice in a wet box for incubation at 4 ℃ overnight; (7) incubation of secondary antibody: spin-dry the primary antibody on the slide with force, wash with PBS 4 times, last 10min each time, add 30uLTween-20 while washing for the last time; dripping 25uL drops of DAB general secondary antibody on the surface of each section, and incubating for 30min at 37 ℃; (8) color development: according to the following steps of 1: uniformly mixing DAB developing A, B solution according to the proportion of 50, dripping DAB working solution to the glass slide with 40uL of each slide, and rinsing with tap water after the development is finished; (9) nuclear staining: placing the slide in hematoxylin solution for 20sec, washing with tap water, adding into hydrochloric acid alcohol, differentiating for 2sec, washing with tap water for 10min, and performing reverse blue reaction; (10) dewatering and sealing: sequentially dehydrating and transparentizing the slide glass by the sequence of 75% alcohol (3min) → 85% alcohol (3min) → 95% alcohol (3min) → 100% alcohol (5min) → xylene I (20min) → xylene II (20min), placing the slide glass in a ventilation place overnight, and then sealing the slide glass by neutral resin; the results are shown in FIG. 17;

the method for detecting the life cycle of the tumor-bearing mouse comprises the following specific steps: recording time after mice of the experimental group and the control group die naturally from the construction day of the transplanted tumor (namely, day 0), wherein the difference value of the death time and the tumor construction time is the survival time of the mice, and drawing a survival curve after all the mice die; the results are shown in FIG. 18;

HUWE1 and Ki67 proteins of tumor-bearing mice orthotopic transplantation tumor are subjected to immunohistochemical staining, and the specific steps are as follows:

(1) dewaxing: baking the sample for 30min under a baking lamp, and performing dewaxing treatment according to xylene I (15min) → xylene II (15 min);

(2) hydration: passing anhydrous alcohol I (10min) → anhydrous alcohol II (10min) → 95% alcohol (5min) → 85% alcohol (5min) → 75% alcohol (5min) through the cylinder in this order, rinsing the slide glass with ultrapure water for 3min under tap water, and washing the slide glass with ultrapure water 3 times for 5min each time; (3) antigen retrieval: adding EDTA repair liquid (working liquid) into a pressure cooker on an induction cooker, and putting the glass slide until the glass slide is completely submerged; heating for 2.5min in the repairing solution after pressurizing and steaming, placing the slide in a repairing box, covering the cover, and placing in a ventilation place to restore the slide to room temperature; (4) blocking endogenous peroxidase: selecting a freshly prepared 3% H2O2 blocking solution, adding the blocking solution into a repair box to enable the blocking solution to submerge a slide, sealing the box at 37 ℃ for 30min, washing the box with PBS for 4 times, lasting for 10min each time, and adding 30uLTween-20 during the last washing; (5) and (3) sealing: wiping off water around the slices, and dripping 30uL of goat instant sealing serum for sealing at 37 ℃ for 40 min; (6) incubating the primary antibody: spin-drying the serum on the slide, dripping HUWE1 and Ki67 primary antibody working solution (obtained by diluting primary antigen solution with PBS solution according to the proportion of 1: 250) on the surface of the slide, and placing the slide in a wet box for incubation at 4 ℃ overnight; (7) incubation of secondary antibody: spin-dry the primary antibody on the slide with force, wash with PBS 4 times, last 10min each time, add 30uLTween-20 while washing for the last time; dripping 25uL drops of DAB general secondary antibody on the surface of each section, and incubating for 30min at 37 ℃; (8) color development: according to the following steps of 1: uniformly mixing DAB developing A, B solution according to the proportion of 50, dripping DAB working solution to the glass slide with 40uL of each slide, and rinsing with tap water after the development is finished; (9) nuclear staining: placing the slide in hematoxylin solution for 20sec, washing with tap water, adding into hydrochloric acid alcohol, differentiating for 2sec, washing with tap water for 10min, and performing reverse blue reaction; (10) dewatering and sealing: sequentially dehydrating and transparentizing the slide glass by the sequence of 75% alcohol (3min) → 85% alcohol (3min) → 95% alcohol (3min) → 100% alcohol (5min) → xylene I (20min) → xylene II (20min), placing the slide glass in a ventilation place overnight, and then sealing the slide glass by neutral resin; the results are shown in FIG. 19.

As shown in fig. 4, in both the in situ injection and tail vein injection of rAAV9 virus significantly inhibited glioblastoma in situ graft tumor growth compared to the control virus and pbs injection.

As can be seen from fig. 5, both tail vein injection and in situ injection of rAAV9 can achieve expression of rAAV9 in glioblastoma, and the results indicate that rAAV9 successfully breaches the blood brain barrier and enters the brain parenchyma of mice, and is expressed in the injected human glial cells; flag immunohistochemistry for PBS control group was negative.

As can be seen from FIG. 6, in-situ injection and tail vein injection of rAAV9 virus can significantly prolong the survival time of tumor-bearing mice compared with the control virus and pbs injection groups, and the result shows that rAAV9-dsaCas9 mediates HUWE1 overexpression and can significantly inhibit the growth of glioblastoma.

As shown in fig. 7, both in situ and tail vein injections of rAAV9 achieved overexpression of HUWE1 in glioblastoma, with in situ injections of rAAV9 significantly increased the expression level of HUWE1 compared to tail vein injections of rAAV 9. Compared with the rAAV-control group and the PBS control group, the ratio of Ki67 of the rAAV9-HUWE1 treatment group is obviously reduced, which indicates that the proliferation of glioma cells is obviously inhibited.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> first subsidiary hospital of China civil liberation army, military and medical university

<120> recombinant adeno-associated virus expression vector

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gccgcacgcg tgtgtctaga gtctgtaagc tgaagacctg gcagtgc 47

<210> 2

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggccgctcgg tccgcacgtg gaacaaacga cccaacaccc gtgcg 45

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gagagaatga ggggtaccca g 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtgggccatt tctccctgat c 21

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gccgcacgcg tgtgtctaga gagggc 26

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggccgctcgg tccgcacgtg aaaaatctcg 30

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gatttttgct agcgtttaaa cg 22

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aaactttggg gctccaccca c 21

<210> 9

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gccgcacgcg tgtgtctaga gagggcctat ttcccatgat tcc 43

<210> 10

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggccgctcgg tccgcacgtg aaaaatctcg ccaacaagtt gacgag 46

<210> 11

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gccgcacgcg tgtgtctaga gagggcctat ttcccatgat tcc 43

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

attctgtttc cagagtac 18

Claims

1. A recombinant adeno-associated virus expression vector, which comprises hGFAP-promoter-dsacas9-VP64-3flag-polyA virus vector, U6-sagRNA (HUWE1) × 3 virus vector, U6-sagRNA (NC) virus vector, pAAV-RC vector and pHelper plasmid.

2. The method for preparing the recombinant adeno-associated virus expression vector according to claim 1, comprising the steps of:

and transfecting the pHelper plasmid and the pAAV-RC plasmid with an hGFAP-promoter-dsacas9-VP64-3flag-polyA viral vector, a U6-sagRNA (HUWE1)3 viral vector and a U6-sagRNA (NC) viral vector to AAV-293 cells respectively, and completing assembly after 2-3 days of transfection to obtain the recombinant adeno-associated virus expression vector.

3. The method of claim 2, wherein the hGFAP-promoter-dsacas9-VP64-3flag-polyA viral vector is constructed by: and (3) carrying out gel electrophoresis on the enzyme digestion product by using the XbaI/PmlI enzyme digestion CV182 vector, recovering a target band, carrying out PCR amplification, exchanging the PCR amplification product with the enzyme digestion vector, and placing the exchange reaction product in a competent cell for conversion to obtain the recombinant plasmid.

4. The method for preparing the primer of claim 3, wherein the nucleotide sequence of the upstream amplification primer amplified by PCR is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream amplification primer is shown as SEQ ID NO. 2.

5. The method of claim 2, wherein the U6-saGRNA (HUWE1)3 viral vector is constructed by: and (3) carrying out gel electrophoresis on the enzyme digestion product by using the XbaI/PmlI enzyme digestion CV182 vector, recovering a target band, carrying out PCR amplification, exchanging the PCR amplification product with the enzyme digestion vector, and placing the exchange reaction product in a competent cell for conversion to obtain the recombinant plasmid.

6. The method for preparing the primer of claim 5, wherein the nucleotide sequence of the upstream amplification primer amplified by PCR is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream amplification primer is shown as SEQ ID NO. 4.

7. Use of the recombinant adeno-associated virus expression vector according to any one of claims 1 to 6 in the preparation of a medicament for treating glioblastoma.