CN111825772B - 具有变异衣壳蛋白的腺相关病毒及其应用 - Google Patents
具有变异衣壳蛋白的腺相关病毒及其应用 Download PDFInfo
- Publication number
- CN111825772B CN111825772B CN202010754921.2A CN202010754921A CN111825772B CN 111825772 B CN111825772 B CN 111825772B CN 202010754921 A CN202010754921 A CN 202010754921A CN 111825772 B CN111825772 B CN 111825772B
- Authority
- CN
- China
- Prior art keywords
- adeno
- associated virus
- capsid protein
- aav9
- brain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000702421 Dependoparvovirus Species 0.000 title claims abstract description 23
- 108090000565 Capsid Proteins Proteins 0.000 title abstract description 8
- 102100023321 Ceruloplasmin Human genes 0.000 title abstract description 8
- 101710197658 Capsid protein VP1 Proteins 0.000 claims abstract description 10
- 208000015181 infectious disease Diseases 0.000 claims abstract description 10
- 230000008499 blood brain barrier function Effects 0.000 claims abstract description 9
- 210000001218 blood-brain barrier Anatomy 0.000 claims abstract description 9
- 210000004958 brain cell Anatomy 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 230000014509 gene expression Effects 0.000 claims description 4
- 239000013603 viral vector Substances 0.000 claims description 4
- 210000000063 presynaptic terminal Anatomy 0.000 claims description 3
- 230000008685 targeting Effects 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 210000003050 axon Anatomy 0.000 abstract description 8
- 208000014644 Brain disease Diseases 0.000 abstract description 3
- 239000000277 virosome Substances 0.000 abstract description 2
- 210000004556 brain Anatomy 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 13
- 241000700605 Viruses Species 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 239000012634 fragment Substances 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000010253 intravenous injection Methods 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 3
- 241000711798 Rabies lyssavirus Species 0.000 description 3
- 241000700584 Simplexvirus Species 0.000 description 3
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 210000003169 central nervous system Anatomy 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 230000001537 neural effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- BOAPXDSRULBILC-UHFFFAOYSA-N 2-(5-methylthiophen-2-yl)-3-phenyl-1,2-dihydroquinazolin-4-one Chemical compound S1C(C)=CC=C1C1N(C=2C=CC=CC=2)C(=O)C2=CC=CC=C2N1 BOAPXDSRULBILC-UHFFFAOYSA-N 0.000 description 2
- 241000701157 Canine mastadenovirus A Species 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 231100000086 high toxicity Toxicity 0.000 description 2
- 230000005745 host immune response Effects 0.000 description 2
- 238000007917 intracranial administration Methods 0.000 description 2
- 210000001577 neostriatum Anatomy 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 210000002442 prefrontal cortex Anatomy 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- MCKSLROAGSDNFC-ACZMJKKPSA-N Ala-Asp-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MCKSLROAGSDNFC-ACZMJKKPSA-N 0.000 description 1
- BPAUXFVCSYQDQX-JRQIVUDYSA-N Asp-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC(=O)O)N)O BPAUXFVCSYQDQX-JRQIVUDYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 101150047856 Cav2 gene Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 241001478425 Hippocampus japonicus Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- PLOUVAYOMTYJRG-JXUBOQSCSA-N Lys-Thr-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O PLOUVAYOMTYJRG-JXUBOQSCSA-N 0.000 description 1
- 208000002678 Mucopolysaccharidoses Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000002298 density-gradient ultracentrifugation Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 1
- 229960004359 iodixanol Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000001259 mesencephalon Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 206010028093 mucopolysaccharidosis Diseases 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000002276 neurotropic effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000002509 periaqueductal gray Anatomy 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000007441 retrograde transport Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001103 thalamus Anatomy 0.000 description 1
- 230000005100 tissue tropism Effects 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 210000004515 ventral tegmental area Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0085—Brain, e.g. brain implants; Spinal cord
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Dermatology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Psychology (AREA)
- Botany (AREA)
- Inorganic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种具有变异型衣壳蛋白的腺相关病毒及其应用，所述变异型腺相关病毒AAV9‑RB，即在野生型AAV9衣壳蛋白VP1的Q588与A589之间插入氨基酸序列LADQDYTKTA。变异型腺相关病毒AAV9‑RB为首次报道的既能从轴突末端高效逆行感染，也能穿过血脑屏障靶向大脑细胞的病毒体，可以为神经科学研究和脑疾病治疗提供了更好的技术支撑，具有广泛的应用价值和前景。
Description
技术领域
本发明属于腺相关病毒技术领域，具体涉及一种具有变异衣壳蛋白的腺相关病毒及其应用。
背景技术
病毒载体，尤其是允许从轴突末端或通过血脑屏障有效地转移基因到中枢神经系统(CNS)的病毒载体，对于分析注射部位周围特定神经回路的结构和功能非常有用，而且能够通过将治疗基因传递到遥远的靶区而成为最有潜力和最有前途的治疗工具之一。与传统的逆行示踪剂相比，病毒载体可在特定神经元类群中表达基因，如光遗传的、化学遗传的和钙敏感的功能探针，被广泛应用于监测和操纵神经元活动。一些天然和工程化的嗜神经病毒表现出逆行感染能力，包括伪狂犬病毒(PRV)、单纯疱疹病毒(HSV)、狂犬病毒(RABV)、慢病毒(LV)、犬腺病毒(CAV)和腺相关病毒(AAV)等。其中，PRV具有高毒性；HSV和RV可以快速表达基因，具有很高的逆行标记效率，但对细胞也有较高的毒性，限制了长期的基因操纵和治疗。G/L双缺失的狂犬病毒降低了细胞毒性，但只有微弱的基因表达；改良RVG包装的慢病毒具有较高的逆行转运效率，但可能诱导宿主免疫应答或随机插入宿主基因组导致肿瘤发生；CAV-2的免疫原性相对较低，但基因表达水平适中且制备困难。
重组腺相关病毒(rAAVs)具有低毒、高水平的转基因表达和最小的宿主免疫应答等特性，已成为神经回路调控和基因治疗的有力工具。许多基因缺陷性脑疾病的治疗需要向整个大脑中的每个细胞补偿正确的基因，比如粘多糖症等。一些天然的和工程化的腺相关病毒可以通过轴突末端吸收或血脑屏障转运来转导大部分中枢神经系统神经元，包括AAV-TT、MNM004、AAV-PHP.eB和AAV-F等。然而，同时具有这两个重要特征的AAV还没有被报道。逆行感染AAV主要通过颅内注射来实现大部分细胞感染，没有静脉注射给药途径方便，但静脉注射给药后可能会产生抗体，不适合连续治疗，因此，开发能够多途径靶向大脑神经元细胞的腺相关病毒具有十分重要的意义。
为了实现上述目的，本发明设计了一种既能通过颅内注射从轴突末端高效逆行感染，也能通过静脉注射穿过血脑屏障等途径靶向大脑细胞的腺相关病毒病毒体，在神经科学、脑疾病模型建立和基因治疗等领域具有广泛的应用价值和市场前景。
发明内容
本发明的目的在于提供一种具有变异衣壳蛋白的腺相关病毒，该病毒既能从轴突末端高效逆行感染，也能穿过血脑屏障靶向大脑细胞。
为了实现上述目的，本发明采用以下技术方案：
具有变异衣壳蛋白的腺相关病毒(命名为AAV9-RB)，即在野生型AAV9衣壳蛋白VP1的Q588与A589之间插入氨基酸序列LADQDYTKTA(SEQ ID NO.1)。
进一步地，AAV9-RB衣壳蛋白VP1的核苷酸序列如SEQ ID NO.7所示，构建获得的Rep-Cap质粒的全序列如SEQ ID NO.8所示。
AAV9-RB在通过轴突末端逆行感染或跨血脑屏障感染大脑细胞中的应用。
在本发明的具体实施例中，将AAV9-RB的Rep-Cap质粒与pAAV-CaMKIIa-EGFP质粒通过杆状病毒-AAV表达载体系统制备相应的rAAV9-RB-CaMKIIa-EGFP，再将病毒通过尾静脉注射或脑立体定位方式注射3个月龄C57BL/6小鼠腹侧被盖区，3-4周后灌流取脑和成像，结果表明AAV9-RB既能从轴突末端高效逆行感染，也能穿过血脑屏障靶向大脑细胞。
与现有技术相比，本发明具有以下优点及有益效果：
1、变异型腺相关病毒AAV9-RB为首次报道的既能从轴突末端高效逆行感染，也能穿过血脑屏障等途径靶向大脑细胞的病毒体，为基因治疗提供更多的给药途径；
2、变异型腺相关病毒AAV9-RB能够从轴突末端高效逆行感染，解决了其亲本主要从胞体感染的缺陷。
附图说明
图1变异体AAV9-RB在VTA脑区的逆行转导。A：AAV9-RB-CaMKIIa-EGFP病毒注入VTA(腹侧被盖区)部位后能有效感染神经元，比例尺为200μm；B-E：病毒可高效逆行感染上游脑区，包括PFC(前额叶皮质)、MO(躯体运动区)、MSC(内侧隔核复合体)和MRN(中脑网状核团)等，比例尺为100μm。
图2AAV9-RB可通过静脉注射后跨中枢神经系统转导大脑神经元。将表达绿色荧光蛋白的AAV9-RB(5×1011vg/只)通过静脉注射到成年小鼠体内。A：AAV病毒表达4周后小鼠大脑中GFP信号的代表性图像，比例尺为500μm。B：A图中脑片的高倍放大图像，比例尺为200μm。Cortex：皮层；striatum：纹状体；pallidum：苍白球；hippocampus：海马；thalamus：丘脑；hypothalamus：下丘脑；periaqueductal gray：导水管周围灰质。
具体实施方式
一、AAV变异体(命名为AAV9-RB)Rep-Cap质粒的构建
AAV的血清型由AAV包装所需的Rep-Cap质粒中的Cap蛋白的序列决定，不同的血清型AAV具有不同的组织嗜性。因此，对Cap蛋白进行改造能够得到新的AAV病毒。将编码LADQDYTKTA(SEQ ID NO.1)的DNA序列(SEQ ID NO.2)插入到亲本AAV9的VP1衣壳蛋白氨基酸588和589之间，具体过程包括如下几个方面：
①以亲本AAV9质粒pAAV-RC9(购自武汉枢密脑科学技术有限公司)为模板，使用Takara Primerstar Polymerase(Takara公司)扩增Retro1片段，引物Retro1-F的序列如SEQ ID NO.3所示，引物Retro1-R的序列如SEQ ID NO.4所示；PCR的反应体系均为50μl：5×Reaction Buffer 10μl，10mM dNTPs 1μl，10μM正向引物2.5μl，10μM反向引物2.5μl，模板DNA 0.5μl，DNA Polymerase 0.5μl，ddH2O 33μl。扩增条件为：98℃3min，98℃20s，60℃20s，72℃30s，72℃10min，16℃30min，30个循环；用胶回收试剂盒(Omega公司)回收扩增出的DNA片段；
②以亲本AAV9质粒pAAV-RC9(购自武汉枢密脑科学技术有限公司)为模板，使用Takara Primerstar Polymerase(Takara公司)扩增Retro2片段，引物Retro2-F的序列如SEQ ID NO.5所示，引物Retro2-R的序列如SEQ ID NO.6所示；PCR的反应体系均为50μl：5×Reaction Buffer 10μl，10mM dNTPs 1μl，10μM正向引物2.5μl，10μM反向引物2.5μl，模板DNA：0.5μl，DNA Polymerase 0.5μl，ddH2O 33μl。扩增条件为：98℃3min，98℃20s，60℃20s，72℃30s，72℃10min，16℃30min，30个循环；用胶回收试剂盒(Omega公司)回收扩增出的DNA片段；
③以回收的Retro1和Retro2片段按质量比1:1混合液为模板，扩增Retro片段，引物为Retro1-F和Retro2-R；PCR的反应体系均为50μl：5×Reaction Buffer 10μl，10mMdNTPs 1μl，10μM正向引物2.5μl，10μM反向引物2.5μl，模板DNA 0.5μl，DNA Polymerase0.5μl，ddH2O 33μl。扩增条件为：98℃3min，98℃20s，60℃20s，72℃60s，72℃10min，16℃30min，30个循环；用胶回收试剂盒(Omega公司)回收扩增出的DNA片段；
使用BsiWI和NheI(New England Biolabs)限制性内切酶分别酶切pAAV-RC9载体和Retro片段，然后采用T4连接酶将Retro片段插入到pAAV-RC9，将连接产物转化感受态Stbl3，菌落PCR鉴定为阳性的克隆接种到5毫升LB液体培养基中进行培养并抽提质粒进行测序，测序正确的克隆命名为pAAV-RC9RB，获得质粒能够编码变异型衣壳蛋白VP1。其中，AAV9-RB衣壳蛋白VP1的多核苷酸编码序列如SEQ ID NO.7所示，构建获得的Rep-Cap质粒的全序列如SEQ ID NO.8所示。本发明所有PCR使用引物和测序均由生工生物工程(上海)股份有限公司完成。
二、AAV变异体(命名为AAV9-RB)病毒的制备与纯化
将得到的Rep-Cap质粒与pAAV-CaMKIIa-EGFP质粒(购自武汉枢密脑科学技术有限公司)通过杆状病毒-AAV表达载体系统制备相应的病毒rAAV9-RB-CaMKIIa-EGFP，并用碘克沙醇密度梯度超速离心纯化。纯化的rAAV用iQ-SYBR-Green-Supermix试剂盒(Bio-Rad)进行qPCR测定滴度，并稀释至1.0×1013vg/mL，置于-80℃冰箱备用。
三、AAV变异体(命名为AAV9-RB)病毒的活体测试
(1)将AAV9-RB-CaMKIIa-EGFP(3×1012vg/只)病毒通过脑立体定位方式注射于3个月龄C57BL/6小鼠(购自湖南斯莱克景达实验动物有限公司)的腹侧被盖区(Ventraltegmental area，VTA)区域，AAV病毒经过3周充分的表达后，于小鼠腹腔注射0.5ml 1％的戊巴比妥钠-0.9％氯化钠溶液进行麻醉，并使用磷酸缓冲盐溶液(Phosphate BufferSaline，PBS)与4％的多聚甲醛溶液(Pa raformaldehyde Solution，PFA)心脏灌流剥离脑组织。小鼠脑组织经PFA溶液固定过夜后用30％蔗糖-PBS溶液脱水，将脱水后的脑组织用组织包埋剂充分包埋并用冰冻切片机切成40μm厚度的脑片，用奥林巴斯VS120玻片扫描显微镜(奥林巴斯，日本)对其进行成像。活体检测结果如图1所示，AAV9-RB可以高效从轴突末端逆行感染上游脑区。
(2)将AAV9-RB-CaMKIIa-EGFP(5×1011vg/只)病毒通过尾静脉注射于3个月龄C57BL/6小鼠(购自湖南斯莱克景达实验动物有限公司)，4周后灌流取脑和成像。活体检测结果如图2所示，尾静脉注射AAV9-RB-CaMKIIa-EGFP的小鼠脑袋中可以检测到绿色荧光信号，表明AAV9-RB可以跨血脑屏障感染大脑细胞。
序列表
<110> 中国科学院精密测量科学与技术创新研究院
<120> 具有变异衣壳蛋白的腺相关病毒及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Leu Ala Asp Gln Asp Tyr Thr Lys Thr Ala
1 5 10
<210> 2
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctagcagacc aagactacac aaaaactgct 30
<210> 3
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
accgcatgct atgcatcagc tgctagctta cagattacga gtcagg 46
<210> 4
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctagcagacc aagactacac aaaaactgct gcacaggcgc agaccggctg 50
<210> 5
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agcagttttt gtgtagtctt ggtctgctag ttgggcactc tggtggtttg tggccac 57
<210> 6
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
acggactcag actatcagct cccgtacgtg ctcgggtcgg ctcacgag 48
<210> 7
<211> 2242
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctgggctgcc gacggttatc tacccgattg gctcgaggac aaccttagtg aaggaattcg 60
cgagtggtgg gctttgaaac ctggagcccc tcaacccaag gcaaatcaac aacatcaaga 120
caacgctcga ggtcttgtgc ttccgggtta caaatacctt ggacccggca acggactcga 180
caagggggag ccggtcaacg cagcagacgc ggcggccctc gagcacgaca aggcctacga 240
ccagcagctc aaggccggag acaacccgta cctcaagtac aaccacgccg acgccgagtt 300
ccaggagcgg ctcaaagaag atacgtcttt tgggggcaac ctcgggcgag cagtcttcca 360
ggccaaaaag aggcttcttg aacctcttgg tctggttgag gaagcggcta agacggctcc 420
tggaaagaag aggcctgtag agcagtctcc tcaggaaccg gactcctccg cgggtattgg 480
caaatcgggt gcacagcccg ctaaaaagag actcaatttc ggtcagactg gcgacacaga 540
gtcagtccca gaccctcaac caatcggaga acctcccgca gccccctcag gtgtgggatc 600
tcttacaatg gcttcaggtg gtggcgcacc agtggcagac aataacgaag gtgccgatgg 660
agtgggtagt tcctcgggaa attggcattg cgattcccaa tggctggggg acagagtcat 720
caccaccagc acccgaacct gggccctgcc cacctacaac aatcacctct acaagcaaat 780
ctccaacagc acatctggag gatcttcaaa tgacaacgcc tacttcggct acagcacccc 840
ctgggggtat tttgacttca acagattcca ctgccacttc tcaccacgtg actggcagcg 900
actcatcaac aacaactggg gattccggcc taagcgactc aacttcaagc tcttcaacat 960
tcaggtcaaa gaggttacgg acaacaatgg agtcaagacc atcgccaata accttaccag 1020
cacggtccag gtcttcacgg actcagacta tcagctcccg tacgtgctcg ggtcggctca 1080
cgagggctgc ctcccgccgt tcccagcgga cgttttcatg attcctcagt acgggtatct 1140
gacgcttaat gatggaagcc aggccgtggg tcgttcgtcc ttttactgcc tggaatattt 1200
cccgtcgcaa atgctaagaa cgggtaacaa cttccagttc agctacgagt ttgagaacgt 1260
acctttccat agcagctacg ctcacagcca aagcctggac cgactaatga atccactcat 1320
cgaccaatac ttgtactatc tctcaaagac tattaacggt tctggacaga atcaacaaac 1380
gctaaaattc agtgtggccg gacccagcaa catggctgtc cagggaagaa actacatacc 1440
tggacccagc taccgacaac aacgtgtctc aaccactgtg actcaaaaca acaacagcga 1500
atttgcttgg cctggagctt cttcttgggc tctcaatgga cgtaatagct tgatgaatcc 1560
tggacctgct atggccagcc acaaagaagg agaggaccgt ttctttcctt tgtctggatc 1620
tttaattttt ggcaaacaag gaactggaag agacaacgtg gatgcggaca aagtcatgat 1680
aaccaacgaa gaagaaatta aaactactaa cccggtagca acggagtcct atggacaagt 1740
ggccacaaac caccagagtg cccaactagc agaccaagac tacacaaaaa ctgctgcaca 1800
ggcgcagacc ggctgggttc aaaaccaagg aatacttccg ggtatggttt ggcaggacag 1860
agatgtgtac ctgcaaggac ccatttgggc caaaattcct cacacggacg gcaactttca 1920
cccttctccg ctgatgggag ggtttggaat gaagcacccg cctcctcaga tcctcatcaa 1980
aaacacacct gtacctgcgg atcctccaac ggccttcaac aaggacaagc tgaactcttt 2040
catcacccag tattctactg gccaagtcag cgtggagatc gagtgggagc tgcagaagga 2100
aaacagcaag cgctggaacc cggagatcca gtacacttcc aactattaca agtctaataa 2160
tgttgaattt gctgttaata ctgaaggtgt atatagtgaa ccccgcccca ttggcaccag 2220
atacctgact cgtaatctgt aa 2242
<210> 8
<211> 9275
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60
ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240
tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300
ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 420
aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 480
ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 540
attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 600
tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 660
gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 720
tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 780
aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 840
cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 900
agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 960
ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1020
tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1080
tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1140
caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1200
accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1260
attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1320
ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1380
taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1440
taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1500
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1560
agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1620
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1680
ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1740
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1800
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1860
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1920
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1980
tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 2040
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2100
acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2160
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 2220
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2280
ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2340
ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2400
taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2460
cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2520
tctgtgcggt atttcacacc gcagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580
agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640
caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700
aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760
cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820
gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880
gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940
ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000
ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060
cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120
ctcactacgc ggctgctcaa acctgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180
ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240
atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300
atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360
gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420
gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480
cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540
aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600
agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3660
gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720
aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780
cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc 3840
aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900
tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960
atagttctag tggttggcct acgtacccgt agtggctatg gcagggcttg ccgccccgac 4020
gttggctgcg agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga 4080
aacgcgggcg tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg 4140
ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt 4200
ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc 4260
tcccccatct cccggtaccg catgctatgc atcagctgct agcttacaga ttacgagtca 4320
ggtatctggt gccaatgggg cggggttcac tatatacacc ttcagtatta acagcaaatt 4380
caacattatt agacttgtaa tagttggaag tgtactggat ctccgggttc cagcgcttgc 4440
tgttttcctt ctgcagctcc cactcgatct ccacgctgac ttggccagta gaatactggg 4500
tgatgaaaga gttcagcttg tccttgttga aggccgttgg aggatccgca ggtacaggtg 4560
tgtttttgat gaggatctga ggaggcgggt gcttcattcc aaaccctccc atcagcggag 4620
aagggtgaaa gttgccgtcc gtgtgaggaa ttttggccca aatgggtcct tgcaggtaca 4680
catctctgtc ctgccaaacc atacccggaa gtattccttg gttttgaacc cagccggtct 4740
gcgcctgtgc agcagttttt gtgtagtctt ggtctgctag ttgggcactc tggtggtttg 4800
tggccacttg tccataggac tccgttgcta ccgggttagt agttttaatt tcttcttcgt 4860
tggttatcat gactttgtcc gcatccacgt tgtctcttcc agttccttgt ttgccaaaaa 4920
ttaaagatcc agacaaagga aagaaacggt cctctccttc tttgtggctg gccatagcag 4980
gtccaggatt catcaagcta ttacgtccat tgagagccca agaagaagct ccaggccaag 5040
caaattcgct gttgttgttt tgagtcacag tggttgagac acgttgttgt cggtagctgg 5100
gtccaggtat gtagtttctt ccctggacag ccatgttgct gggtccggcc acactgaatt 5160
ttagcgtttg ttgattctgt ccagaaccgt taatagtctt tgagagatag tacaagtatt 5220
ggtcgatgag tggattcatt agtcggtcca ggctttggct gtgagcgtag ctgctatgga 5280
aaggtacgtt ctcaaactcg tagctgaact ggaagttgtt acccgttctt agcatttgcg 5340
acgggaaata ttccaggcag taaaaggacg aacgacccac ggcctggctt ccatcattaa 5400
gcgtcagata cccgtactga ggaatcatga aaacgtccgc tgggaacggc gggaggcagc 5460
cctcgtgagc cgacccgagc acgtacggga gctgatagtc tgagtccgtg aagacctgga 5520
ccgtgctggt aaggttattg gcgatggtct tgactccatt gttgtccgta acctctttga 5580
cctgaatgtt gaagagcttg aagttgagtc gcttaggccg gaatccccag ttgttgttga 5640
tgagtcgctg ccagtcacgt ggtgagaagt ggcagtggaa tctgttgaag tcaaaatacc 5700
cccagggggt gctgtagccg aagtaggcgt tgtcatttga agatcctcca gatgtgctgt 5760
tggagatttg cttgtagagg tgattgttgt aggtgggcag ggcccaggtt cgggtgctgg 5820
tggtgatgac tctgtccccc agccattggg aatcgcaatg ccaatttccc gaggaactac 5880
ccactccatc ggcaccttcg ttattgtctg ccactggtgc gccaccacct gaagccattg 5940
taagagatcc cacacctgag ggggctgcgg gaggttctcc gattggttga gggtctggga 6000
ctgactctgt gtcgccagtc tgaccgaaat tgagtctctt tttagcgggc tgtgcacccg 6060
atttgccaat acccgcggag gagtccggtt cctgaggaga ctgctctaca ggcctcttct 6120
ttccaggagc cgtcttagcc gcttcctcaa ccagaccaag aggttcaaga agcctctttt 6180
tggcctggaa gactgctcgc ccgaggttgc ccccaaaaga cgtatcttct ttgagccgct 6240
cctggaactc ggcgtcggcg tggttgtact tgaggtacgg gttgtctccg gccttgagct 6300
gctggtcgta ggccttgtcg tgctcgaggg ccgccgcgtc tgctgcgttg accggctccc 6360
ccttgtcgag tccgttgccg ggtccaaggt atttgtaacc cggaagcaca agacctcgag 6420
cgttgtcttg atgttgttga tttgccttgg gttgaggggc tccaggtttc aaagcccacc 6480
actcgcgaat tccttcacta aggttgtcct cgagccaatc gggtagataa ccgtcggcag 6540
ccagggcggc ccgggtgatc aagtcttcgt cgagtgattg taaataaaat gtaatttaca 6600
gtatagtatt ttaattaata tacaaatgat ttgataataa ttcttattta actataatat 6660
attgtgttgg gttgaattaa aggtccgtat actccggaat attaatagat catggagata 6720
attaaaatga taaccatctc gcaaataaat aagtatttta ctgttttcgt aacagttttg 6780
taataaaaaa acctataaat attccggatt attcataccg tcccaccatc gggcgcggat 6840
ctgccgccct ggcggggttt tacgagattg tgattaaggt ccccagcgac cttgacgggc 6900
atctgcccgg catttctgac agctttgtga actgggtggc cgagaaggag tgggagttgc 6960
cgccagattc tgacttggat ctgaatctga ttgagcaggc acccctgacc gtggccgaga 7020
agctgcagcg cgactttctg acggagtggc gccgtgtgag taaggccccg gaggcccttt 7080
tctttgtgca atttgagaag ggagagagct acttccactt acacgtgctc gtggaaacca 7140
ccggggtgaa atccttagtt ttgggacgtt tcctgagtca gattcgcgaa aaactgattc 7200
agagaattta ccgcgggatc gagccgactt tgccaaactg gttcgcggtc acaaagacca 7260
gaaacggcgc cggaggcggg aacaaggtgg tggacgagtg ctacatcccc aattacttgc 7320
tccccaaaac ccagcctgag ctccagtggg cgtggactaa tttagaacag tatttaagcg 7380
cctgtttgaa tctcacggag cgtaaacggt tggtggcgca gcatctgacg cacgtgtcgc 7440
agacgcagga gcagaacaaa gagaatcaga atcccaattc tgacgcgccg gtgatcagat 7500
caaaaacttc agccaggtac atggagctgg tcgggtggct cgtggacaag gggattacct 7560
cggagaagca gtggatccag gaggaccagg cctcatacat ctccttcaat gcggcctcca 7620
actcgcggtc ccaaatcaag gctgccttgg acaatgcggg aaagattatg agcctgacta 7680
aaaccgcccc cgactacctg gtgggccagc agcccgtgga ggacatttcc agcaatcgga 7740
tttataaaat tttggaacta aacgggtacg atccccaata tgcggcttcc gtctttctgg 7800
gatgggccac gaaaaagttc ggcaagagga acaccatctg gctgtttggg cctgcaacta 7860
ccgggaagac caacatcgcg gaggccatag cccacactgt gcccttctac gggtgcgtaa 7920
actggaccaa tgagaacttt cccttcaacg actgtgtcga caagatggtg atctggtggg 7980
aggaggggaa gatgaccgcc aaggtcgtgg agtcggccaa agccattctc ggaggaagca 8040
aggtgcgcgt ggaccagaaa tgcaagtcct cggcccagat agacccgact cccgtgatcg 8100
tcacctccaa caccaacatg tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc 8160
agcagccgtt gcaagaccgg atgttcaaat ttgaactcac ccgccgtctg gatcatgact 8220
ttgggaaggt caccaagcag gaagtcaaag actttttccg gtgggcaaag gatcacgtgg 8280
ttgaggtgga gcatgaattc tacgtcaaaa agggtggagc caagaaaaga cccgccccca 8340
gtgacgcaga tataagtgag cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt 8400
cagacgcgga agcttcgatc aactacgcag acaggtacca aaacaaatgt tctcgtcacg 8460
tgggcatgaa tctgatgctg tttccctgca gacaatgcga gagaatgaat cagaattcaa 8520
atatctgctt cactcacgga cagaaagact gtttagagtg ctttcccgtg tcagaatctc 8580
aacccgtttc tgtcgtcaaa aaggcgtatc agaaactgtg ctacattcat catatcatgg 8640
gaaaggtgcc agacgcttgc actgcctgcg atctggtcaa tgtggatttg gatgactgca 8700
tctttgaaca ataatctaga gcctgcagtc tcgacaagct tgtcgagaag tactagagga 8760
tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc 8820
tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag 8880
cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 8940
cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatct 9000
gatcactgct tgagcctagg agatccgaac cagataagtg aaatctagtt ccaaactatt 9060
ttgtcatttt taattttcgt attagcttac gacgctacac ccagttccca tctattttgt 9120
cactcttccc taaataatcc ttaaaaactc catttccacc cctcccagtt cccaactatt 9180
ttgtccgccc acagcggggc atttttcttc ctgttatgtt tttaatcaaa catcctgcca 9240
actccatgtg acaaaccgtc atcttcggct acttt 9275
Claims (4)
1.腺相关病毒变异体的衣壳蛋白VP1，其特征在于，在野生型AAV9衣壳蛋白VP1的Q588与A589之间插入SEQ ID NO.1所示的氨基酸序列，编码所述衣壳蛋白VP1的核苷酸序列如SEQ ID NO .7所示。
2.腺相关病毒变异体，其特征在于，含有权利要求1所述的衣壳蛋白VP1。
3.根据权利要求2所述的腺相关病毒变异体，其特征在于，所述的腺相关病毒变异体携带目的基因进行表达。
4.权利要求2所述的腺相关病毒变异体在制备靶向大脑细胞的病毒载体中的应用，其特征在于，所述的病毒载体通过轴突末端逆行感染或跨血脑屏障感染大脑细胞。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010754921.2A CN111825772B (zh) | 2020-07-30 | 2020-07-30 | 具有变异衣壳蛋白的腺相关病毒及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010754921.2A CN111825772B (zh) | 2020-07-30 | 2020-07-30 | 具有变异衣壳蛋白的腺相关病毒及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111825772A CN111825772A (zh) | 2020-10-27 |
| CN111825772B true CN111825772B (zh) | 2023-10-20 |
Family
ID=72920517
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010754921.2A Active CN111825772B (zh) | 2020-07-30 | 2020-07-30 | 具有变异衣壳蛋白的腺相关病毒及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111825772B (zh) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057840B (zh) * | 2021-10-14 | 2022-09-20 | 和元生物技术（上海）股份有限公司 | 包含变异的aav9衣壳蛋白的重组腺相关病毒颗粒 |
| CN114213505B (zh) * | 2021-12-10 | 2022-09-20 | 和元生物技术（上海）股份有限公司 | 一种适用于特异感染u87-mg细胞的腺相关病毒突变体 |
| CN114195859B (zh) * | 2021-12-10 | 2022-11-18 | 和元生物技术（上海）股份有限公司 | 一种适用于特异感染u251细胞的腺相关病毒突变体 |
| CN115960177B (zh) * | 2022-10-09 | 2023-07-07 | 广州派真生物技术有限公司 | 腺相关病毒突变体及其应用 |
| CN117304256B (zh) * | 2022-12-08 | 2024-04-16 | 广州派真生物技术有限公司 | 腺相关病毒突变体及其应用 |
| CN116693633B (zh) * | 2023-02-21 | 2023-12-22 | 广州派真生物技术有限公司 | 腺相关病毒突变体及其应用 |
| CN117820442B (zh) * | 2023-06-09 | 2024-05-31 | 广州派真生物技术有限公司 | 腺相关病毒突变体及其应用 |
| CN117964716B (zh) * | 2023-07-21 | 2024-08-09 | 广州派真生物技术有限公司 | 腺相关病毒突变体及其应用 |
| CN117264912A (zh) * | 2023-09-25 | 2023-12-22 | 中国科学院深圳先进技术研究院 | 一种重组腺相关病毒颗粒、重组腺相关病毒载体系统及其应用 |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102071206A (zh) * | 2010-10-22 | 2011-05-25 | 中山大学 | 一种腺相关病毒衣壳蛋白基因、相应的蛋白及其应用 |
| WO2012057363A1 (ja) * | 2010-10-27 | 2012-05-03 | 学校法人自治医科大学 | 神経系細胞への遺伝子導入のためのアデノ随伴ウイルスビリオン |
| CN106062200A (zh) * | 2014-02-17 | 2016-10-26 | 伦敦国王学院 | 腺相关病毒载体 |
| WO2019043630A1 (en) * | 2017-08-31 | 2019-03-07 | Benitec Biopharma Limited | ADENO-ASSOCIATED VIRUS (AAV) WITH MODIFIED PHOSPHOLIPASE DOMAIN |
| CN110423778A (zh) * | 2019-07-26 | 2019-11-08 | 上海科技大学 | 一种逆行性重组2型腺相关病毒在感染螺旋神经元中的应用 |
| CN110437317A (zh) * | 2019-01-30 | 2019-11-12 | 上海科技大学 | 具有变异衣壳蛋白的腺相关病毒及其用途 |
| CN110950934A (zh) * | 2019-12-31 | 2020-04-03 | 复旦大学 | 一种腺相关病毒衣壳蛋白、载体及其构建方法与应用 |
| CN114231565A (zh) * | 2021-11-04 | 2022-03-25 | 中国科学院精密测量科学与技术创新研究院 | 一种可用于活体检测细胞类型特异神经连接的rAAV及其应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MA44546B1 (fr) * | 2016-06-15 | 2021-03-31 | Univ California | Virus adéno-associés variants et procédés d'utilisation |
-
2020
- 2020-07-30 CN CN202010754921.2A patent/CN111825772B/zh active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102071206A (zh) * | 2010-10-22 | 2011-05-25 | 中山大学 | 一种腺相关病毒衣壳蛋白基因、相应的蛋白及其应用 |
| WO2012057363A1 (ja) * | 2010-10-27 | 2012-05-03 | 学校法人自治医科大学 | 神経系細胞への遺伝子導入のためのアデノ随伴ウイルスビリオン |
| CN106062200A (zh) * | 2014-02-17 | 2016-10-26 | 伦敦国王学院 | 腺相关病毒载体 |
| WO2019043630A1 (en) * | 2017-08-31 | 2019-03-07 | Benitec Biopharma Limited | ADENO-ASSOCIATED VIRUS (AAV) WITH MODIFIED PHOSPHOLIPASE DOMAIN |
| CN110437317A (zh) * | 2019-01-30 | 2019-11-12 | 上海科技大学 | 具有变异衣壳蛋白的腺相关病毒及其用途 |
| CN110423778A (zh) * | 2019-07-26 | 2019-11-08 | 上海科技大学 | 一种逆行性重组2型腺相关病毒在感染螺旋神经元中的应用 |
| CN110950934A (zh) * | 2019-12-31 | 2020-04-03 | 复旦大学 | 一种腺相关病毒衣壳蛋白、载体及其构建方法与应用 |
| CN114231565A (zh) * | 2021-11-04 | 2022-03-25 | 中国科学院精密测量科学与技术创新研究院 | 一种可用于活体检测细胞类型特异神经连接的rAAV及其应用 |
Non-Patent Citations (3)
| Title |
|---|
| Kunzhang Lin等.AAV9-Retro mediates efficient transduction with axon terminal absorption and blood–brain barrier transportation.Mol Brain.2020,第13卷(第138期),第1-12页. * |
| 李嘉敏.AAV神经环路示踪病毒的改造和应用.中国优秀硕士学位论文全文数据库基础科学辑.2020,(第7期),第A006-62. * |
| 腺相关病毒外壳修饰与肿瘤靶向治疗;徐增辉;周秀梅;石文芳;钱其军;;科学通报(21);第1-5页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111825772A (zh) | 2020-10-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111825772B (zh) | 具有变异衣壳蛋白的腺相关病毒及其应用 | |
| KR101307880B1 (ko) | 폴리진을 사용한 멀티단백질 복합체의 재조합 발현 | |
| CN112666348A (zh) | 新型冠状病毒SARS-CoV-2的检测蛋白组及应用 | |
| CN111593072B (zh) | 一种在昆虫细胞中共表达非洲猪瘟病毒四种结构蛋白的方法及其应用 | |
| CN114262381B (zh) | 表面展示非洲猪瘟病毒抗原p30蛋白的重组杆状病毒、制备方法及其应用 | |
| CN112501139B (zh) | 一株重组新城疫病毒毒株及其制备方法和应用 | |
| CN112250738B (zh) | 严重急性呼吸综合征冠状病毒2病毒样颗粒的制备、纯化和鉴定方法 | |
| CN110108884A (zh) | 一种对于犬瘟热病毒及抗体的elisa检测方法 | |
| CN104328136B (zh) | 鸡新城疫病毒毒株rClone30‑fliC的制备及其在鸡新城疫病防治中的应用 | |
| CN104357409B (zh) | 表达鸡il2重组新城疫病毒及其在疫苗中的应用 | |
| CN111748034B (zh) | 一种滑液囊支原体单克隆抗体的制备方法 | |
| CN109402109B (zh) | 一种改进的重叠延伸pcr方法 | |
| CN105925610B (zh) | 昆虫杆状病毒表达载体、构建方法及其应用 | |
| CN107354172B (zh) | 重组表达载体及其构建方法和应用 | |
| CN103352042B (zh) | 编码重组新城疫病毒的cDNA、由该cDNA拯救的病毒及其在治疗恶性肿瘤中的应用 | |
| CN113234746B (zh) | 一种农药诱导蛋白互作和诱导基因表达的方法 | |
| CN114957406B (zh) | 一种犬细小病毒的三价抗原和三价病毒样颗粒以及应用 | |
| CN116640801A (zh) | 一种covid-19病毒基因工程预防性疫苗及制备方法 | |
| KR100902634B1 (ko) | 재조합 ｈｍｇｂ1 펩티드를 포함하는 핵산 전달 복합체 | |
| RU2774333C1 (ru) | Рекомбинантная плазмида pET-GST-3CL-GPG, обеспечивающая синтез протеазы 3CL SARS-CoV-2 в клетках E.coli в растворимой форме | |
| CN114231564B (zh) | 一种rAAV载体核心质粒及其应用 | |
| RU2792132C1 (ru) | Рекомбинантная плазмида pET-GST-3CL, обеспечивающая синтез протеазы 3CL SARS-CoV-2 в клетках E.coli в растворимой форме | |
| CN109321601B (zh) | Aqp5重组过表达载体及其构建方法和用途 | |
| CN102260710A (zh) | 一种适合三种宿主系统的表达载体 | |
| CN116135882A (zh) | 一株羊驼源纳米抗体m112及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |