CN114292876A - Recombinant adeno-associated virus of specific target astrocyte and application thereof - Google Patents

Abstract

The invention discloses a recombinant adeno-associated virus of a targeted astrocyte and application thereof, wherein the recombinant adeno-associated virus has a pAAV2/11 skeleton and is obtained by cotransfecting a packaging cell line by adeno-associated virus core plasmids, recombinant adeno-associated virus packaging plasmids and adenovirus element helper plasmids, the recombinant adeno-associated virus core plasmids comprise inverted terminal repetitive sequences ITR at two ends, a GfaABC1D promoter, an exogenous gene, a transcription regulating element WPRE and a transcription termination sequence SV40polyA, and the recombinant adeno-associated virus packaging plasmids comprise a Rep gene of type 2 adeno-associated virus and a Cap gene of type 11 adeno-associated virus. The invention realizes the specific targeting of astrocytes by taking pAAV2/11 as a gene transfer vector for the first time, provides better tools and technical support for neuroscience research, disease model establishment, gene therapy and the like, and has wide application value and market prospect.

Description

Recombinant adeno-associated virus of specific target astrocyte and application thereof

Technical Field

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

Background

The brain network is composed of a variety of nerve cells, which mainly include two major classes of neurons and glial cells (neuroglia), each playing a different role in the brain. The number of glial cells is several tens of times greater than that of neurons, and can be generally classified into microglia, astrocytes, oligodendrocytes, and the like. With the development of neuroscience, different types of nerve cells are found to have different network structure connection and functions, and the variation of the nerve cells can cause different brain diseases and behavior disorders. There is increasing evidence that astrocytes are associated with various brain diseases (Nature.2014; 509(7499): 189-. Therefore, there is a need to develop a tool for specifically targeting astrocytes, which is used to analyze the structure and function of astrocytes and provide theoretical basis and technical support for the treatment of brain diseases.

Currently, researchers rely mainly on transgenic animals for the analysis of astrocytes. However, transgenic animals, such as mice, rats, dogs, pigs, monkeys, etc., have long breeding cycles, high feeding costs, and are not flexible to carry the target gene for neuronal cell manipulation and disease treatment. Therefore, it is of great importance to continue to develop methods capable of specifically targeting astrocytes.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a recombinant adeno-associated virus specifically targeting astrocytes and application thereof.

The invention provides a recombinant adeno-associated virus core plasmid, which comprises a repetitive sequence ITR with two inverted ends, a GfaABC1D promoter, a foreign gene, a transcription regulatory element WPRE and a transcription termination sequence SV40 polyA.

Further, the exogenous gene includes a reporter gene;

preferably, the reporter gene is EGFP.

Further, the nucleotide sequence of the recombinant adeno-associated virus core plasmid is shown as SEQ ID NO: 3, respectively.

The second aspect of the invention provides a recombinant adeno-associated virus vector system, which comprises the adeno-associated virus core plasmid, recombinant adeno-associated virus packaging plasmid and adenovirus element helper plasmid.

Further, the recombinant adeno-associated virus packaging plasmid comprises a Rep gene of adeno-associated virus type 2 and a Cap gene of adeno-associated virus type 11;

preferably, the recombinant adeno-associated virus packaging plasmid takes pAAV-RC2/1 as a framework;

preferably, the adenovirus element Helper plasmid is pAd-Helper.

Further, the nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is shown as SEQ ID NO: 4, respectively.

The third aspect of the invention provides a recombinant adeno-associated virus, which is obtained by cotransfecting a packaging cell line with the adeno-associated virus core plasmid, the recombinant adeno-associated virus packaging plasmid and the adenovirus element helper plasmid.

Further, the packaging cell line is selected from HEK-293 cells, HEK-293T cells or HEK-293FT cells;

preferably, the molecular number of the adeno-associated virus core plasmid, the recombinant adeno-associated virus packaging plasmid and the adenovirus element helper plasmid is 1:1: 1.

Further, the recombinant adeno-associated virus packaging plasmid comprises a Rep gene of adeno-associated virus type 2 and a Cap gene of adeno-associated virus type 11;

preferably, the recombinant adeno-associated virus packaging plasmid takes pAAV-RC2/1 as a framework;

preferably, the nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is as shown in SEQ ID NO: 4 is shown in the specification;

preferably, the adenovirus element Helper plasmid is pAd-Helper.

The fourth aspect of the present invention provides the use of the recombinant adeno-associated virus core plasmid, the recombinant adeno-associated virus vector system or the recombinant adeno-associated virus in specific targeting of astrocytes.

The invention has the beneficial effects that;

the invention realizes the specific targeting of astrocytes by taking pAAV2/11 as a gene transfer vector for the first time, provides better tools and technical support for neuroscience research, disease model establishment, gene therapy and the like, and has wide application value and market prospect.

Drawings

FIG. 1 is a map of recombinant adeno-associated virus core plasmid expression vector.

FIG. 2 is a graph of the signals of recombinant adeno-associated virus infecting the dorsal hippocampal region. Wherein "DAPI" represents a signal to stain the nucleus, blue fluorescence; "GFAP" represents a signal after staining an astrocyte marker GFAP, and is red fluorescence; the EGFP represents a cell signal marked by rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA, is green fluorescence and is marked together with red fluorescence.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Example 1: construction of recombinant adeno-associated virus core plasmid

BglII and NheI (NewEngland Biolabs) restriction endonuclease was used to digest vector pZacc 2.1-GfaABC1D-rM 3D-mChery-SV 40polyA (purchased from Addgene, accession number: 92285), a gel recovery kit (Omega) was used to recover the GfaABC1D promoter fragment, T4 ligase (purchased from TaKaRa) was used to ligate the adeno-associated viral vector AV-hSyn-EGFP-WPRE-SV40polyA (Blindac Biotech, Inc.) which was also double digested with BglII and NheI, the ligation product was used to transform E.coli Stbl3 competent cells, which were placed in a 35 ℃ incubator overnight, a single clone was selected for colony PCR identification, and the primers were GfaABC1D-F (SEQ ID NO.1) and GfaABC1D-R (SEQ ID NO.2), and performing amplification culture and plasmid extraction on the positive colonies, performing double enzyme digestion verification and sequencing on the plasmids by BglII and NheI, and naming the correctly sequenced plasmid as pAAV-GFaABC1D-EGFP-WPRE-SV40 polyA. The map of the constructed recombinant adeno-associated virus core plasmid pAAV-GFaABC1D-EGFP-WPRE-SV40polyA expression vector is shown in figure 1, and the gene sequence is shown in SEQ ID NO. 3. All PCR-based primer synthesis and sequencing used in the present invention were performed by Biotechnology engineering (Shanghai) Inc.

Example 2: preparation of recombinant adeno-associated virus

Packaging the recombinant adeno-associated virus by using a recombinant adeno-associated virus packaging plasmid pAAV-RC2/11 expression vector, and mixing a recombinant adeno-associated virus core plasmid pAAV-GFaABC1D-EGFP-WPRE-SV40polyA, a pAAV-RC2/11 serotype AAV capsid plasmid and an adenovirus element Helper plasmid pAd-Helper according to the plasmid molecular number of 1:1:1 co-transfecting HEK-293T cells, collecting supernatant and cell sediment after transfecting for 72 hours, concentrating and purifying by iodixanol gradient centrifugation, and finally detecting the titer of recombinant adeno-associated virus by SYBRGreenqPCR (systematic amplified polymorphic nucleic acid) method to finally obtain rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA virus with the titer of 6.0 multiplied by 10¹²VG/mL。

The recombinant adeno-associated virus packaging plasmid pAAV-RC2/11 has the nucleotide sequence shown in SEQ ID NO: 4.

Example 3: in vivo use of recombinant adeno-associated virus

Prepared rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA (100 nL/single) virus is injected into the dorsal hippocampal region of 8-10 week-old C57BL/6 mice (purchased from Hunan Sleka Jingida laboratory animals Co., Ltd.) in a brain stereotactic manner, the brain is perfused and taken after 3 weeks, the mouse brain tissue is fixed by a PFA solution treated by DEPC for 4 hours and then dehydrated by a 30% sucrose-PBS solution treated by DEPC for 48 hours, and the dehydrated brain tissue is fully embedded by a tissue embedding medium and cut into brain slices with the thickness of 40 mu m by a freezing microtome. Brain slices containing dorsal hippocampal regions were immunohistochemically stained with GFAP antibody, after which they were mounted and imaged using a slide scanner microscope. In vivo detection results show that a green fluorescent signal marked by rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA and a red signal of an astrocyte marker GFAP are labeled together, and the rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA recombinant adeno-associated virus can specifically target astrocytes. FIG. 2 shows signals of rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA recombinant adeno-associated virus infecting the dorsal hippocampus.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

The nucleotide sequence of the invention is as follows:

SEQ ID NO：1

aacatatcctggtgtggagt

SEQ ID NO：2

gctagcctatagtgagtcgta

SEQ ID NO：3

cgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattaggctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtagccatgctctaggaagatctaacatatcctggtgtggagtaggggacgctgctctgacagaggctcgggggcctgagctggctctgtgagctggggaggaggcagacagccaggccttgtctgcaagcagacctggcagcattgggctggccgccccccagggcctcctcttcatgcccagtgaatgactcaccttggcacagacacaatgttcggggtgggcacagtgcctgcttcccgccgcaccccagcccccctcaaatgccttccgagaagcccattgagcagggggcttgcattgcaccccagcctgacagcctggcatcttgggataaaagcagcacagccccctaggggctgcccttgctgtgtggcgccaccggcggtggagaacaaggctctattcagcctgtgcccaggaaaggggatcaggggatgcccaggcatggacagtgggtggcagggggggagaggagggctgtctgcttcccagaagtccaaggacacaaatgggtgaggggagagctctccccatagctgggctgcggcccaaccccaccccctcaggctatgccagggggtgttgccaggggcacccgggcatcgccagtctagcccactccttcataaagccctcgcatcccaggagcgagcagagccagagcaggttggagaggagacgcatcacctccgctgctcgcaagctttattgcggtagtttatcacagttaaattgctaacgcagtcagtgcttctgacacaacagtctcgaacttaagctgcagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagttcaattacagctcttaaggctagagtacttaatacgactcactataggctagcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctatgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgcggccgcttcgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggagatgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtaaaatcgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaata

SEQ ID NO：4

gctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccccctcgaggtcgacggtatcgggggagctcgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccgtggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaagggagagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaactgattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggctgctgacggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacctgaaacctggagccccgaagcccaaggccaaccagcagaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcggtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaagaagagggtactcgaacctctgggcctggttgaagaaggtgctaaaacggctcctggaaagaagagaccgttagagtcaccacaagagcccgactcctcctcgggcatcggcaaaaaaggcaaacaaccagccagaaagaggctcaactttgaagaggacactggagccggagacggaccccctgaaggatcagataccagcgccatgtcttcagacattgaaatgcgtgcagcaccgggcggaaatgctgtcgatgcgggacaaggttccgatggagtgggtaatgcctcgggtgattggcattgcgattccacctggtctgagggcaaggtcacaacaacctcgaccagaacctgggtcttgcccacctacaacaaccacttgtacctgcgtctcggaacaacatcaagcagcaacacctacaacggattctccaccccctggggatattttgacttcaacagattccactgtcacttctcaccacgtgactggcaaagactcatcaacaacaactggggactacgaccaaaagccatgcgcgttaaaatcttcaatatccaagttaaggaggtcacaacgtcgaacggcgagactacggtcgctaataaccttaccagcacggttcagatatttgcggactcgtcgtatgagctcccgtacgtgatggacgctggacaagaggggagcctgcctcctttccccaatgacgtgttcatggtgcctcaatatggctactgtggcatcgtgactggcgagaatcagaaccaaacggacagaaacgctttctactgcctggagtattttccttcgcaaatgttgagaactggcaacaactttgaaatggcttacaactttgagaaggtgccgttccactcaatgtatgctcacagccagagcctggacagactgatgaatcccctcctggaccagtacctgtggcacttacagtcgactacctctggagagactctgaatcaaggcaatgcagcaaccacatttggaaaaatcaggagtggagactttgccttttacagaaagaactggctgcctgggccttgtgttaaacagcagagattctcaaaaactgccagtcaaaattacaagattcctgccagcgggggcaacgctctgttaaagtatgacacccactataccttaaacaaccgctggagcaacatcgcgcccggacctccaatggccacagccggaccttcggatggggacttcagtaacgcccagcttatattccctggaccatctgttaccggaaatacaacaacttcagccaacaatctgttgtttacatcagaagaagaaattgctgccaccaacccaagagacacggacatgtttggccagattgctgacaataatcagaatgctacaactgctcccataaccggcaacgtgactgctatgggagtgctgcctggcatggtgtggcaaaacagagacatttactaccaagggccaatttgggccaagatcccacacgcggacggacattttcatccttcaccgctgattggtgggtttggactgaaacacccgcctccccagatattcatcaagaacactcccgtacctgccaatcctgcgacaaccttcactgcagccagagtggactctttcatcacacaatacagcaccggccaggtcgctgttcagattgaatgggaaattgaaaaggaacgctccaaacgctggaatcctgaagtgcagtttacttcaaactatgggaaccagtcttctatgttgtgggctcctgatacaactgggaagtatacagagccgcgggttattggctctcgttatttgactaatcatttgtaattacgtgttaatcaataaaccggttgattcgtttcagttgaactttggtctcctgtccttcttatcttatcggttaccatggttatagcttacacattaactgcttggttgcgcttcgcgataaaagacttacgtcatcgggttacccctagggggatccactagttctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatccatcacactggcggccgctcgactagagcggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcag

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute of Chinese academy of sciences

<120> recombinant adeno-associated virus of specific target astrocyte and application thereof

<130> CP121011174C

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

aacatatcct ggtgtggagt 20

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<400> 2

gctagcctat agtgagtcgt a 21

<210> 3

<211> 5737

<212> DNA

<213> Artificial sequence

<400> 3

cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 60

cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 120

gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga 180

taacaatttc acacaggaaa cagctatgac catgattacg ccagatttaa ttaaggcctt 240

aattaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcggcctca 300

gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 360

ttaatgatta acccgccatg ctacttatct acgtagccat gctctaggaa gatctaacat 420

atcctggtgt ggagtagggg acgctgctct gacagaggct cgggggcctg agctggctct 480

gtgagctggg gaggaggcag acagccaggc cttgtctgca agcagacctg gcagcattgg 540

gctggccgcc ccccagggcc tcctcttcat gcccagtgaa tgactcacct tggcacagac 600

acaatgttcg gggtgggcac agtgcctgct tcccgccgca ccccagcccc cctcaaatgc 660

cttccgagaa gcccattgag cagggggctt gcattgcacc ccagcctgac agcctggcat 720

cttgggataa aagcagcaca gccccctagg ggctgccctt gctgtgtggc gccaccggcg 780

gtggagaaca aggctctatt cagcctgtgc ccaggaaagg ggatcagggg atgcccaggc 840

atggacagtg ggtggcaggg ggggagagga gggctgtctg cttcccagaa gtccaaggac 900

acaaatgggt gaggggagag ctctccccat agctgggctg cggcccaacc ccaccccctc 960

aggctatgcc agggggtgtt gccaggggca cccgggcatc gccagtctag cccactcctt 1020

cataaagccc tcgcatccca ggagcgagca gagccagagc aggttggaga ggagacgcat 1080

cacctccgct gctcgcaagc tttattgcgg tagtttatca cagttaaatt gctaacgcag 1140

tcagtgcttc tgacacaaca gtctcgaact taagctgcag aagttggtcg tgaggcactg 1200

ggcaggtaag tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt 1260

cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact 1320

ttgcctttct ctccacaggt gtccactccc agttcaatta cagctcttaa ggctagagta 1380

cttaatacga ctcactatag gctagcgcca ccatggtgag caagggcgag gagctgttca 1440

ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 1500

tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 1560

ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 1620

agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 1680

ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 1740

gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 1800

acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 1860

acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 1920

acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 1980

gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca 2040

aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 2100

tcactctcgg catggacgag ctgtacaagt aagtcgacaa tcaacctctg gattacaaaa 2160

tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 2220

ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2280

tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2340

gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2400

gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 2460

ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 2520

tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc 2580

tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 2640

gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc 2700

ggatctccct ttgggccgcc tccccgcgcg gccgcttcga gcagacatga taagatacat 2760

tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 2820

ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa 2880

caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa 2940

gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat ggctacgtag 3000

ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca 3060

ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 3120

cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaatt 3180

cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 3240

gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 3300

gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat 3360

taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 3420

cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 3480

aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 3540

ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 3600

ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 3660

caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 3720

cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 3780

taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 3840

atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 3900

tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 3960

cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 4020

agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 4080

taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 4140

tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 4200

catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 4260

ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 4320

ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 4380

catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 4440

aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 4500

aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 4560

taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 4620

atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 4680

gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 4740

tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 4800

ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 4860

gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 4920

agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 4980

aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 5040

agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 5100

tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 5160

atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 5220

taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 5280

gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 5340

gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 5400

aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 5460

tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 5520

gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 5580

cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 5640

ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 5700

cgagtcagtg agcgaggaag cggaagagcg cccaata 5737

<210> 4

<211> 7400

<212> DNA

<213> Artificial sequence

<400> 4

gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc 60

gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg 120

acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg 180

taccgggccc cccctcgagg tcgacggtat cgggggagct cgcagggtct ccattttgaa 240

gcgggaggtt tgaacgcgca gccgccatgc cggggtttta cgagattgtg attaaggtcc 300

ccagcgacct tgacgagcat ctgcccggca tttctgacag ctttgtgaac tgggtggccg 360

agaaggaatg ggagttgccg ccagattctg acatggatct gaatctgatt gagcaggcac 420

ccctgaccgt ggccgagaag ctgcagcgcg actttctgac ggaatggcgc cgtgtgagta 480

aggccccgga ggctcttttc tttgtgcaat ttgagaaggg agagagctac ttccacatgc 540

acgtgctcgt ggaaaccacc ggggtgaaat ccatggtttt gggacgtttc ctgagtcaga 600

ttcgcgaaaa actgattcag agaatttacc gcgggatcga gccgactttg ccaaactggt 660

tcgcggtcac aaagaccaga aatggcgccg gaggcgggaa caaggtggtg gatgagtgct 720

acatccccaa ttacttgctc cccaaaaccc agcctgagct ccagtgggcg tggactaata 780

tggaacagta tttaagcgcc tgtttgaatc tcacggagcg taaacggttg gtggcgcagc 840

atctgacgca cgtgtcgcag acgcaggagc agaacaaaga gaatcagaat cccaattctg 900

atgcgccggt gatcagatca aaaacttcag ccaggtacat ggagctggtc gggtggctcg 960

tggacaaggg gattacctcg gagaagcagt ggatccagga ggaccaggcc tcatacatct 1020

ccttcaatgc ggcctccaac tcgcggtccc aaatcaaggc tgccttggac aatgcgggaa 1080

agattatgag cctgactaaa accgcccccg actacctggt gggccagcag cccgtggagg 1140

acatttccag caatcggatt tataaaattt tggaactaaa cgggtacgat ccccaatatg 1200

cggcttccgt ctttctggga tgggccacga aaaagttcgg caagaggaac accatctggc 1260

tgtttgggcc tgcaactacc gggaagacca acatcgcgga ggccatagcc cacactgtgc 1320

ccttctacgg gtgcgtaaac tggaccaatg agaactttcc cttcaacgac tgtgtcgaca 1380

agatggtgat ctggtgggag gaggggaaga tgaccgccaa ggtcgtggag tcggccaaag 1440

ccattctcgg aggaagcaag gtgcgcgtgg accagaaatg caagtcctcg gcccagatag 1500

acccgactcc cgtgatcgtc acctccaaca ccaacatgtg cgccgtgatt gacgggaact 1560

caacgacctt cgaacaccag cagccgttgc aagaccggat gttcaaattt gaactcaccc 1620

gccgtctgga tcatgacttt gggaaggtca ccaagcagga agtcaaagac tttttccggt 1680

gggcaaagga tcacgtggtt gaggtggagc atgaattcta cgtcaaaaag ggtggagcca 1740

agaaaagacc cgcccccagt gacgcagata taagtgagcc caaacgggtg cgcgagtcag 1800

ttgcgcagcc atcgacgtca gacgcggaag cttcgatcaa ctacgcagac aggtaccaaa 1860

acaaatgttc tcgtcacgtg ggcatgaatc tgatgctgtt tccctgcaga caatgcgaga 1920

gaatgaatca gaattcaaat atctgcttca ctcacggaca gaaagactgt ttagagtgct 1980

ttcccgtgtc agaatctcaa cccgtttctg tcgtcaaaaa ggcgtatcag aaactgtgct 2040

acattcatca tatcatggga aaggtgccag acgcttgcac tgcctgcgat ctggtcaatg 2100

tggatttgga tgactgcatc tttgaacaat aaatgattta aatcaggtat ggctgctgac 2160

ggttatcttc cagattggct cgaggacaac ctctctgagg gcattcgcga gtggtgggac 2220

ctgaaacctg gagccccgaa gcccaaggcc aaccagcaga agcaggacga cggccggggt 2280

ctggtgcttc ctggctacaa gtacctcgga cccttcaacg gactcgacaa gggggagccc 2340

gtcaacgcgg cggacgcagc ggccctcgag cacgacaagg cctacgacca gcagctcaaa 2400

gcgggtgaca atccgtacct gcggtataac cacgccgacg ccgagtttca ggagcgtctg 2460

caagaagata cgtcttttgg gggcaacctc gggcgagcag tcttccaggc caagaagagg 2520

gtactcgaac ctctgggcct ggttgaagaa ggtgctaaaa cggctcctgg aaagaagaga 2580

ccgttagagt caccacaaga gcccgactcc tcctcgggca tcggcaaaaa aggcaaacaa 2640

ccagccagaa agaggctcaa ctttgaagag gacactggag ccggagacgg accccctgaa 2700

ggatcagata ccagcgccat gtcttcagac attgaaatgc gtgcagcacc gggcggaaat 2760

gctgtcgatg cgggacaagg ttccgatgga gtgggtaatg cctcgggtga ttggcattgc 2820

gattccacct ggtctgaggg caaggtcaca acaacctcga ccagaacctg ggtcttgccc 2880

acctacaaca accacttgta cctgcgtctc ggaacaacat caagcagcaa cacctacaac 2940

ggattctcca ccccctgggg atattttgac ttcaacagat tccactgtca cttctcacca 3000

cgtgactggc aaagactcat caacaacaac tggggactac gaccaaaagc catgcgcgtt 3060

aaaatcttca atatccaagt taaggaggtc acaacgtcga acggcgagac tacggtcgct 3120

aataacctta ccagcacggt tcagatattt gcggactcgt cgtatgagct cccgtacgtg 3180

atggacgctg gacaagaggg gagcctgcct cctttcccca atgacgtgtt catggtgcct 3240

caatatggct actgtggcat cgtgactggc gagaatcaga accaaacgga cagaaacgct 3300

ttctactgcc tggagtattt tccttcgcaa atgttgagaa ctggcaacaa ctttgaaatg 3360

gcttacaact ttgagaaggt gccgttccac tcaatgtatg ctcacagcca gagcctggac 3420

agactgatga atcccctcct ggaccagtac ctgtggcact tacagtcgac tacctctgga 3480

gagactctga atcaaggcaa tgcagcaacc acatttggaa aaatcaggag tggagacttt 3540

gccttttaca gaaagaactg gctgcctggg ccttgtgtta aacagcagag attctcaaaa 3600

actgccagtc aaaattacaa gattcctgcc agcgggggca acgctctgtt aaagtatgac 3660

acccactata ccttaaacaa ccgctggagc aacatcgcgc ccggacctcc aatggccaca 3720

gccggacctt cggatgggga cttcagtaac gcccagctta tattccctgg accatctgtt 3780

accggaaata caacaacttc agccaacaat ctgttgttta catcagaaga agaaattgct 3840

gccaccaacc caagagacac ggacatgttt ggccagattg ctgacaataa tcagaatgct 3900

acaactgctc ccataaccgg caacgtgact gctatgggag tgctgcctgg catggtgtgg 3960

caaaacagag acatttacta ccaagggcca atttgggcca agatcccaca cgcggacgga 4020

cattttcatc cttcaccgct gattggtggg tttggactga aacacccgcc tccccagata 4080

ttcatcaaga acactcccgt acctgccaat cctgcgacaa ccttcactgc agccagagtg 4140

gactctttca tcacacaata cagcaccggc caggtcgctg ttcagattga atgggaaatt 4200

gaaaaggaac gctccaaacg ctggaatcct gaagtgcagt ttacttcaaa ctatgggaac 4260

cagtcttcta tgttgtgggc tcctgataca actgggaagt atacagagcc gcgggttatt 4320

ggctctcgtt atttgactaa tcatttgtaa ttacgtgtta atcaataaac cggttgattc 4380

gtttcagttg aactttggtc tcctgtcctt cttatcttat cggttaccat ggttatagct 4440

tacacattaa ctgcttggtt gcgcttcgcg ataaaagact tacgtcatcg ggttacccct 4500

agggggatcc actagttcta gaggtcctgt attagaggtc acgtgagtgt tttgcgacat 4560

tttgcgacac catgtggtca cgctgggtat ttaagcccga gtgagcacgc agggtctcca 4620

ttttgaagcg ggaggtttga acgcgcagcc gccaagccga attctgcaga tatccatcac 4680

actggcggcc gctcgactag agcggccgcc accgcggtgg agctccagct tttgttccct 4740

ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 4800

ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 4860

gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 4920

gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4980

tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 5040

gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 5100

ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 5160

ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 5220

acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 5280

tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 5340

ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 5400

ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 5460

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 5520

actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 5580

gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 5640

tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 5700

caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 5760

atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 5820

acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5880

ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5940

ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 6000

tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 6060

tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 6120

gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 6180

tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 6240

tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 6300

ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 6360

tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 6420

ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 6480

gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 6540

ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 6600

cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 6660

ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 6720

ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 6780

gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 6840

ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6900

gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc 6960

gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc 7020

ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag 7080

agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc 7140

gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa 7200

gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg 7260

aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt 7320

gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc 7380

gcgtcccatt cgccattcag 7400

Claims (10)

1. The recombinant adeno-associated virus core plasmid is characterized by comprising a repetitive sequence ITR at the two ends in opposite directions, a GfaABC1D promoter, a foreign gene, a transcription regulatory element WPRE and a transcription termination sequence SV40 polyA.

2. The recombinant adeno-associated virus core plasmid of claim 1 wherein the foreign gene comprises a reporter gene;

preferably, the reporter gene is EGFP.

3. The recombinant adeno-associated virus core plasmid according to claim 1 wherein the nucleotide sequence of the recombinant adeno-associated virus core plasmid is as set forth in SEQ ID NO: 3, respectively.

4. A recombinant adeno-associated virus vector system, comprising the adeno-associated virus core plasmid, the recombinant adeno-associated virus packaging plasmid, and the adenovirus element helper plasmid of claim 1.

5. The recombinant adeno-associated virus vector system according to claim 4 wherein the recombinant adeno-associated virus packaging plasmid comprises the Rep gene of adeno-associated virus type 2 and the Cap gene of adeno-associated virus type 11;

preferably, the recombinant adeno-associated virus packaging plasmid takes pAAV-RC2/1 as a framework;

preferably, the adenovirus element Helper plasmid is pAd-Helper.

6. The recombinant adeno-associated virus packaging plasmid of claim 5 wherein the nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is as set forth in SEQ ID NO: 4, respectively.

7. A recombinant adeno-associated virus obtained by cotransfecting the packaging cell line with the adeno-associated virus core plasmid, the recombinant adeno-associated virus packaging plasmid and the adenovirus element helper plasmid of claim 1.

8. The recombinant adeno-associated virus according to claim 7 wherein the packaging cell line is selected from a HEK-293 cell, a HEK-293T cell or a HEK-293FT cell;

preferably, the molecular number of the adeno-associated virus core plasmid, the recombinant adeno-associated virus packaging plasmid and the adenovirus element helper plasmid of claim 1 is 1:1: 1.

9. The recombinant adeno-associated virus according to claim 7 wherein the recombinant adeno-associated virus packaging plasmid comprises a Rep gene of adeno-associated virus type 2 and a Cap gene of adeno-associated virus type 11;

preferably, the recombinant adeno-associated virus packaging plasmid takes pAAV-RC2/1 as a framework;

preferably, the nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is as shown in SEQ ID NO: 4 is shown in the specification;

preferably, the adenovirus element Helper plasmid is pAd-Helper.

10. Use of the recombinant adeno-associated virus core plasmid according to any one of claims 1 to 3, the recombinant adeno-associated virus vector system according to any one of claims 4 to 6, or the recombinant adeno-associated virus according to any one of claims 7 to 9 for specifically targeting astrocytes.

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