CN115354048B - Construction method and application of Alzheimer disease animal model and Abeta 42 recombinant expression vector - Google Patents
Construction method and application of Alzheimer disease animal model and Abeta 42 recombinant expression vector Download PDFInfo
- Publication number
- CN115354048B CN115354048B CN202210640718.1A CN202210640718A CN115354048B CN 115354048 B CN115354048 B CN 115354048B CN 202210640718 A CN202210640718 A CN 202210640718A CN 115354048 B CN115354048 B CN 115354048B
- Authority
- CN
- China
- Prior art keywords
- abeta
- bri
- expression vector
- recombinant expression
- aav
- 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
- 239000013604 expression vector Substances 0.000 title claims abstract description 48
- 238000003259 recombinant expression Methods 0.000 title claims abstract description 38
- 108010064539 amyloid beta-protein (1-42) Proteins 0.000 title abstract description 66
- 238000010276 construction Methods 0.000 title abstract description 11
- 238000011565 Alzheimer-disease animal model Methods 0.000 title abstract description 6
- 208000024827 Alzheimer disease Diseases 0.000 claims abstract description 45
- 241000282414 Homo sapiens Species 0.000 claims abstract description 28
- 239000013607 AAV vector Substances 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 238000010171 animal model Methods 0.000 claims description 16
- 241000282693 Cercopithecidae Species 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 238000002347 injection Methods 0.000 abstract description 29
- 239000007924 injection Substances 0.000 abstract description 29
- 238000003776 cleavage reaction Methods 0.000 abstract description 20
- 230000007017 scission Effects 0.000 abstract description 20
- 208000037259 Amyloid Plaque Diseases 0.000 abstract description 18
- 230000001575 pathological effect Effects 0.000 abstract description 18
- 230000014509 gene expression Effects 0.000 abstract description 13
- 230000002776 aggregation Effects 0.000 abstract description 12
- 238000004220 aggregation Methods 0.000 abstract description 12
- 102000004961 Furin Human genes 0.000 abstract description 10
- 108090001126 Furin Proteins 0.000 abstract description 10
- 238000001727 in vivo Methods 0.000 abstract description 6
- 231100000331 toxic Toxicity 0.000 abstract description 6
- 230000002588 toxic effect Effects 0.000 abstract description 6
- 230000007774 longterm Effects 0.000 abstract description 5
- 238000007877 drug screening Methods 0.000 abstract description 4
- 230000002025 microglial effect Effects 0.000 abstract description 4
- 210000004556 brain Anatomy 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 18
- 239000013598 vector Substances 0.000 description 17
- 238000000034 method Methods 0.000 description 16
- 241000700605 Viruses Species 0.000 description 13
- 210000001320 hippocampus Anatomy 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 13
- 241001465754 Metazoa Species 0.000 description 11
- 238000011532 immunohistochemical staining Methods 0.000 description 8
- 210000002442 prefrontal cortex Anatomy 0.000 description 8
- 102100037935 Polyubiquitin-C Human genes 0.000 description 7
- 238000009825 accumulation Methods 0.000 description 6
- 210000005013 brain tissue Anatomy 0.000 description 6
- 239000013603 viral vector Substances 0.000 description 6
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 5
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 5
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 5
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 210000001061 forehead Anatomy 0.000 description 5
- 230000007170 pathology Effects 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 108010043324 Amyloid Precursor Protein Secretases Proteins 0.000 description 4
- 102000002659 Amyloid Precursor Protein Secretases Human genes 0.000 description 4
- 210000001642 activated microglia Anatomy 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 210000003140 lateral ventricle Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000010173 Alzheimer-disease mouse model Methods 0.000 description 3
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 3
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 3
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 241000282567 Macaca fascicularis Species 0.000 description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 210000000274 microglia Anatomy 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 2
- 101000831205 Danio rerio Dynein axonemal assembly factor 11 Proteins 0.000 description 2
- 102100024282 Dynein axonemal assembly factor 11 Human genes 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241001559542 Hippocampus hippocampus Species 0.000 description 2
- 101000831210 Homo sapiens Dynein axonemal assembly factor 11 Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 102000044159 Ubiquitin Human genes 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 108010056354 Ubiquitin C Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 101150063416 add gene Proteins 0.000 description 2
- 230000006934 amyloid beta 42 aggregation Effects 0.000 description 2
- 230000006933 amyloid-beta aggregation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 108091005703 transmembrane proteins Proteins 0.000 description 2
- 102000035160 transmembrane proteins Human genes 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical group NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 108010050254 Presenilins Proteins 0.000 description 1
- 102000015499 Presenilins Human genes 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003941 amyloidogenesis Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004900 c-terminal fragment Anatomy 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 210000001652 frontal lobe Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000007326 intracellular aggregation Effects 0.000 description 1
- -1 kozak sequences Proteins 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000011809 primate model Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/106—Primate
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0312—Animal model for Alzheimer's disease
-
- 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
Abstract
The invention relates to a construction method of an Alzheimer disease animal model and an Abeta 42 recombinant expression vector, wherein the recombinant expression vector comprises an AAV vector, the AAV vector comprises a BRI-Abeta 42 sequence, and the BRI-Abeta 42 sequence is used for encoding Abeta 42. The Abeta 42 recombinant expression vector disclosed by the invention can release Abeta 42 in vivo through cleavage of furin, and the released Abeta 42 is aggregated to form amyloid plaques in the same way due to the strong toxic effect and the characteristic of easy aggregation; the construction method of the Alzheimer disease animal model has short modeling time, can realize long-term expression of Abeta 42 and aggregation in a non-human primate body through one injection, so that activated microglial cells are increased, and the pathological characteristics of Alzheimer disease patients can be well simulated in the non-human primate body, thereby being used for large-scale drug screening for treating Alzheimer disease.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a construction method and application of an Alzheimer disease animal model and an Abeta 42 recombinant expression vector.
Background
Alzheimer's Disease (AD) is a neurodegenerative disease, which is common in the elderly. Alzheimer's disease patients are clinically manifested as reduced cognitive function, mental and motor skills, and gradually reduced life; its pathological features are mainly represented by extracellular deposition to form amyloid plaques, neurofibrillary tangles produced by intracellular aggregation, and abnormal accumulation of these plaques and tangles leads to loss of neurons in the brain and activation proliferation of glial cells. When the amyloid precursor protein or presenilin is mutated, the secretory enzymatic hydrolysis pathway of the amyloid precursor protein is altered, resulting in overproduction and accumulation of aβ42, aβ42 being a monomeric peptide necessary for amyloid deposition formation, and thus abnormal accumulation of aβ42 is considered to be the origin of the cascade that initiates AD.
There is no effective means or drug for treating alzheimer's disease, among which the main reason is the lack of suitable animal models for research and drug development, because abnormal accumulation of aβ42 is a major influencing factor driving alzheimer's disease, and thus it is important to construct an animal model that rapidly and stably overexpresses aβ42 for a long period in the brain.
The current animal model of Alzheimer disease is mainly a transgenic AD mouse model and mainly comprises PDAPP, tg2576, APP23, tgCRND8 and PSEN1 M146V Or PSEN1 M146L 、PSAPP、APP Dutch BRI-Abeta 40 and BRI-Abeta 42, JNPL3, tau P301S 、Tau V337M 、Tau R406W rTg4510, htau, TAPP, 3×TgAD, etc.
Because rodents are far from human beings in weight, brain structure, genome similarity, physiological biochemistry and other aspects, the currently reported transgenic AD mouse model can not well simulate pathology and behavior characteristics of AD patients and is not suitable for research, development and screening of medicines.
In recent years, more and more studies have been conducted to study neurodegenerative diseases using non-human primate models. Continuous injection of aβ oligomers into the ventricles of the cynomolgus brain has been studied, also causing alzheimer-like pathology. However, although alzheimer-like pathology is caused by continuous injection of aβ oligomers into the lateral ventricles of the cynomolgus brain, this method of constructing an AD animal model by multiple injections of aβ oligomers into the lateral ventricles takes a long time, causes an acute response, and requires multiple administrations, which does not simulate aβ aggregation well in vivo.
Thus, how to rapidly and stably overexpress aβ42 in the brain of animals over a long period to mimic the pathological features of AD patients is a difficulty in constructing animal models of AD disease to study AD disease.
Disclosure of Invention
In order to solve the above problems, a first object of the present invention is to provide a method for constructing an animal model of alzheimer's disease, comprising the steps of:
the Abeta 42 recombinant expression vector is injected into the hippocampus and prefrontal cortex of the brain of the non-human primate by a stereotactic injection method, and the Abeta 42 recombinant expression vector is an AAV vector capable of expressing Abeta 42.
In one implementation of the invention, the non-human primate is a monkey.
In one implementation of the invention, one month after injection, the hippocampus and prefrontal cortex have amyloid plaques formed by aggregation of aβ42.
In one implementation of the invention, aβ42 is protein tagged.
In one implementation of the invention, the protein tag includes at least one of a FLAG tag, an HA tag, and a c-Myc tag.
In one implementation of the invention, the viral titer of the Abeta 42 recombinant expression vector is greater than 10 during injection 12 vg/mL。
A second object of the present invention is to provide an Abeta 42 recombinant expression vector comprising an AAV vector comprising a BRI-Abeta 42 sequence, wherein the BRI-Abeta 42 sequence is used for encoding Abeta 42.
In one implementation of the invention, the BRI-Abeta 42 sequence is linked to a protein tag sequence, which is used to encode a protein tag.
In one implementation of the invention, the protein tag includes at least one of a FLAG tag, an HA tag, and a c-Myc tag.
The third object of the present invention is to provide a transformant containing the above-mentioned aβ42 recombinant expression vector.
It is a fourth object of the present invention to provide the use of the above recombinant expression vector or the above transformant for constructing an animal model of Alzheimer's disease.
In one implementation of the invention, the animal is a non-human primate.
In one implementation of the invention, the animal is a monkey.
The fifth object of the present invention is to provide the use of the above-mentioned Alzheimer's disease animal model in screening a drug for treating Alzheimer's disease.
According to the method for constructing the Alzheimer's disease animal model, disclosed by the invention, the long-term expression of the Abeta 42 in the non-human primate body can be realized through one injection, the expressed Abeta 42 is aggregated to form amyloid plaques due to the strong toxic effect and the characteristic of easy aggregation, the activated microglial cell number is increased, the pathological characteristics of the Alzheimer's disease patient can be well simulated in the non-human primate body, and the method can be used for large-scale drug screening for treating the Alzheimer's disease.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows two plasmid expression vectors constructed in example 1 of the present invention;
FIG. 2 is a schematic diagram showing the comparison between the sequence of AAV-UBC-BRI-Abeta 42 plasmid expression vector constructed in example 1 of the present invention and Abeta 42 gene sequence;
FIG. 3 is a graphical representation of the results of immunohistochemical staining assays in example 2 of the present invention;
FIG. 4 is a schematic diagram of Western blotting verification and identification result in example 2 of the present invention;
FIG. 5 is a schematic representation of the results of immunohistochemical staining of FLAG tags of brain tissue injected with the viral vector of interest and wild-type control brain tissue in example 3 of the present invention;
FIG. 6 is a schematic diagram showing the results of immunohistochemical staining of amyloid plaques in brain tissue injected with the viral vector of interest and wild-type control brain tissue in example 3 of the present invention;
FIG. 7 is a schematic representation of microglial immunohistochemical staining results of brain tissue injected with the viral vector of interest and wild-type control brain tissue in example 3 of the present invention.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Accordingly, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention will be disclosed in or be apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
As mentioned above, the transgenic AD mouse model reported so far cannot well simulate the pathological and behavioral characteristics of AD patients, and is not beneficial to research, development and screening of drugs.
Although Alzheimer's disease-like pathology is caused by continuous injection of Abeta oligomers into the lateral ventricles of the cynomolgus brain, this method of constructing an AD animal model by multiple injection of Abeta oligomers into the lateral ventricles takes a long time, causes an acute response, and requires multiple administrations, which does not mimic Abeta aggregation well in vivo.
In order to solve at least one of the above problems, a first aspect of the present invention provides an aβ42 recombinant expression vector comprising an AAV vector comprising a BRI-aβ42 sequence, the BRI-aβ42 sequence being used to encode aβ42.
The Abeta 42 recombinant expression vector is a recombinant AVV vector, wherein Adeno-associated virus (AAV) is a tiny, non-enveloped, tiny single-stranded DNA virus with icosahedron, and the recombinant AAV vector is a gene vector modified on the basis of nonpathogenic wild AAV, and can be used for effectively transfecting nerve cells.
As used herein, the terms "recombinant AAV vector," "recombinant AAV virus," "recombinant AAV virion," "recombinant AAV viral particle," or "AAV gene expression vector" are used interchangeably herein to mean that the genomic DNA enclosed by the AAV viral capsid contains a heterologous polynucleotide.
The vector is prepared by replacing a sequence encoding a target gene with a sequence encoding GFP by an AAV-UBC-GFP vector, and expressing the carried genetic material element in a host cell by infection, transformation, transduction or transfection into the host cell. The vector may contain a number of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, introns, kozak sequences, genes of interest, polyA sequences, selection elements, and reporter genes. In addition, the vector may also comprise an origin of replication.
BRI in the vector is a transmembrane protein with a furin cleavage site, and Aβ42 is released by cleavage of furin. In animal experiments, BRI-Abeta 42 is cleaved to release Abeta 42 by the action of furin in animals.
The Abeta 42 recombinant expression vector disclosed by the invention can release Abeta 42 in vivo through cleavage of furin, and the released Abeta 42 is aggregated to form amyloid plaques in the same way due to the strong toxic effect and the characteristic of easy aggregation; the administration times are few, the long-term expression of Abeta 42 in the non-human primate body can be realized through one injection, more microglial cells can be observed to be activated, the pathological characteristics of the Alzheimer disease patient can be well simulated in the non-human primate body, and the method is used for large-scale drug screening for treating the Alzheimer disease.
In some embodiments, the BRI-aβ42 sequence is linked to a protein tag sequence that encodes a protein tag for distinguishing between endogenous aβ42.
In some embodiments, the protein tag comprises at least one of a FLAG tag, an HA tag, and a c-Myc tag. For example, the Abeta 42 recombinant expression vector contains a BRI-Abeta 42-flag sequence or a BRI-Abeta 42-ha sequence. It should be noted that, for GFP tags with larger molecular weight, the present invention replaces GFP in the vector with the gene encoding the target protein, which can prevent the great difference between the aβ42 with GFP and the aβ42 generated by the actual patient, and can not well simulate the aβ42 in the patient, and affect the cleavage, release and aggregation of the aβ42 expressed by the recombinant expression vector.
The FLAG tag is a polypeptide consisting of 8 amino acid residues: N-DYKDDDDK-C (1012 Da) which functions as a tag, the flag sequence of the coding tag is: GATTACAAGGACGACGATGACAAG. In the protein expression and localization research, a target gene to be researched and a FLAG gene sequence can be connected through a genetic engineering technology means, the target gene can be connected at the C end or the N end of the target protein, the integrated gene is transferred into cells, the subsequent detection is mainly realized through the specific combination of an immunodeterminant formed by a FLAG peptide chain and a monoclonal antibody thereof, and the detection means include immunofluorescence (immunofluorescence), immunoblotting (Western Blotting) and the like.
In some embodiments, the Abeta 42 recombinant expression vector comprises a promoter UBC and the Abeta 42 recombinant expression vector is AAV-UBC-BRI-BRI-Abeta 42-flag.
In AAV-UBC-BRI-Abeta 42-flag expression vectors, high expression of BRI-Abeta 42-flag is controlled by UBC transcripts. Ubiquitin (Ub) is a protein that is abundant and highly conserved in cells, and ubiquitin C promoter can be used to express high levels of protein in a wide range of species and tissue types, and the use of UBC promoter to initiate transcription of BRI-aβ42-flag allows for high expression of BRI-aβ42-flag in multiple brain regions and in multiple cell types.
In some embodiments, the cleavage site comprises the cleavage sites of the restriction enzymes BamHI and EcoRV.
In some embodiments, the recombinant expression vector AAV-UBC-AAV-UBC-BRI-Abeta 42-flag of the invention is modified by an AAV-UBC-GFP virus expression vector, the GFP sequence of the AAV-UBC-GFP plasmid is deleted by enzyme cutting sites BamHI and EcoRV, and the BRI-Abeta 42-flag sequence with BamHI and EcoRV double enzyme cutting is connected, so that the AAV-UBC-BRI-Abeta 42-flag recombinant expression vector is constructed and obtained for expressing Abeta 42.
The cloning site on GFP is adopted to insert the sequence for encoding Abeta 42 in the vector, the expressed Abeta 42 has no GFP, and the GFP can be prevented from influencing the cleavage, release and aggregation of Abeta 42.
Specifically, the restriction enzyme BamHI and EcoR V restriction sites on GFP are adopted to delete the GFP sequence in the AAV-UBC-GFP virus expression vector, and the BamHI and EcoR V restriction sites are further adopted to connect the BRI-Abeta 42-flag sequence, so that the cloning site on GFP is inserted into the Abeta 42 coding sequence, and the expressed Abeta 42 is free of GFP, thereby avoiding the GFP from influencing the cutting, release and aggregation of Abeta 42.
In a second aspect, the present invention provides a transformant containing the above-described aβ42 recombinant expression vector.
The transformant is used for packaging the Abeta 42 recombinant expression vector, and carrying out positioning infection on a non-human primate through packaged viruses, so that the Abeta 42 is expressed in the non-human primate for a long time, amyloid plaques can be formed by aggregation, the flag tag can distinguish endogenous Abeta 42, the aggregation of Abeta 42 can also increase the number of activated microglia, and the pathological characteristics of Alzheimer disease patients can be well simulated in the non-human primate.
In some embodiments, the transformant comprises a host cell and a recombinant expression vector transformed into the host cell.
In some embodiments, the host cell is an escherichia coli cell, a HEK293 cell, a Hela cell, or a Vero cell.
The third aspect of the present invention provides a method for preparing the aβ42 recombinant expression vector, comprising:
the GFP sequence was deleted by the BamHI and EcoRV cleavage sites on the AAV-UBC-GFP plasmid, and the BRI-Abeta 42-flag sequence carrying both BamHI and EcoRV cleavage sequences was ligated to obtain the AAV-UBC-BRI-Abeta 42 vector.
AAV-UBC-GFP viral expression vectors are common expression vectors available from Addgene Inc. Wherein in this expression vector ubiquitin (Ub) is a protein which is abundant and highly conserved in cells, ubiquitin C promoter is useful for high level expression in a wide range of species and tissue types, UBC promoter is selected to initiate transcription of BRI-aβ42-flag, enabling as high expression of BRI-aβ42-flag in multiple brain regions as possible as well as in multiple cell types.
The invention adopts cloning site on GFP to insert coding sequence of Abeta 42, which can avoid GFP from affecting Abeta 42 cutting, releasing and gathering.
In AD patients aβ42 is produced by 2 stages of cleavage of amyloid precursor protein. First, the extracellular domain of APP is cleaved by β -secretase and the extracellular domain fragment is secreted. Gamma-secretase then cleaves the C-terminal fragment of APP remaining in the cell membrane and produces aβ42. Aβ42 produced by β -and γ -secretase cleavage in patients further aggregates extracellular and forms amyloid plaques.
In the AAV-UBC-BRI-Abeta 42 vector constructed by the invention, BRI is also a transmembrane protein, and has a furin cleavage site, and Abeta 42 can be released by cleavage of furin. In animal experiments, BRI-Abeta 42 is cleaved to release Abeta 42 by the action of furin in animals.
The Abeta 42 recombinant expression vector obtained by replacing GFP sequence with BRI-Abeta 42-flag sequence, specifically, the AAV-UBC-BRI-Abeta 42 vector can release Abeta 42 in vivo through cleavage of furin, and the released Abeta 42 is aggregated to form amyloid plaques in the same way due to strong toxic effect and aggregation easily, thereby playing the role of simulating the accumulation of amyloid plaques in AD patients.
In some embodiments, the BRI-Abeta 42 sequence is:
CGCGGATCCGCCACCATGGACTACAAAGACGATGACGACAAGATGGTGAAGGTGACGTTCAACTCCGCTCTGGCCCAGAAGGAGGCCAAGAAGGACGAGCCCAAGAGCGGCGAGGAGGCGCTCATCATCCCCCCCGACGCCGTCGCGGTGGACTGCAAGGACCCAGATGATGTGGTACCAGTTGGCCAAAGAAGAGCCTGGTGTTGGTGCATGTGCTTTGGACTAGCATTTATGCTTGCAGGTGTTATTCTAGGAGGAGCATACTTGTACAAATATTTTGCACTTCAACCAGATGACGTGTACTACTGTGGAATAAAGTACATCAAAGATGATGTCATCTTAAATGAGCCCTCTGCAGATGCCCCAGCTGCTCTCTACCAGACAATTGAAGAAAATATTAAAATCTTTGAAGAAGAAGAAGTTGAATTTATCAGTGTGCCTGTCCCAGAGTTTGCAGATAGTGATCCTGCCAACATTGTTCATGACTTTAACAAGAAACTTACAGCCTATTTAGATCTTAACCTGGATAAGTGCTATGTGATCCCTCTGAACACTTCCATTGTTATGCCACCCAGAAACCTACTGGAGTTACTTATTAACATCAAGGCTGGAACCTATTTGCCTCAGTCCTATCTGATTCATGAGCACATGGTTATTACTGATCGCATTGAAAACATTGATCACCTGGGTTTCTTTATTTATCGACTGTGTCATGACAAGGAAACTTACAAACTGCAACGCAGAGAAACTATTAAAGGTATTCAGAAACGTGATGCAGAATTCCGACATGACTCAGGATATGAAGTTCATCATCAAAAATTGGTGTTCTTTGCAGAAGATGTGGGTTCAAACAAAGGTGCAATCATTGGACTCATGGTGGGCGGTGTTGTCATAGCGTGAGATATCGC。
in a third aspect, the present invention provides the use of the recombinant expression vector described above or the transformant described above in the construction of an animal model of Alzheimer's disease. Specifically, the transformant containing the Abeta 42 recombinant expression vector can stably express Abeta 42 in a non-human primate animal for a long time, and Abeta 42 is aggregated to form amyloid plaques due to the strong toxic effect and the characteristic of easy aggregation.
In some embodiments, the animal comprises a non-human primate. The non-human primate is more similar to human in various aspects such as brain structure, brain volume, brain weight and the like, and can simulate the pathological characteristics of Alzheimer's disease patients more than traditional rodents and other small animals.
In some embodiments, the animal comprises a monkey, more particularly a cynomolgus monkey.
In a fifth aspect, the invention provides a method for constructing an animal model of alzheimer's disease, wherein the BRI-aβ42 recombinant expression vector is injected into the brain hippocampus and prefrontal cortex of the brain of a non-human primate by a stereotactic injection method, and is an AAV vector capable of expressing aβ42.
It should be noted that the hippocampus and the prefrontal cortex are brain regions where AD is early and is susceptible to lesions and injuries, and the hippocampus is located deep in the brain, and the accurate position thereof is difficult to be determined.
In addition, the shape of the sea horse is slender, the area of the forehead leaf cortex is wider, the virus diffusion coverage area is limited, and the AAV-UBC-BRI-Abeta 42 recombinant expression vector is injected into the brain sea horse area and the forehead leaf cortex in a multi-point injection mode, so that Abeta 42 is over-expressed in an injection area, and the over-expressed Abeta 42 is aggregated in the injection area, so that the pathological characteristics of Alzheimer disease patients are formed.
The method for expressing the Abeta 42 by the non-human primate is simple, convenient and quick, and can cause pathological features of Alzheimer's disease patients after injecting the Abeta 42 for one month, and can cause pathological features similar to Alzheimer's disease patients near an injection site by only carrying out one-time stereotactic injection on the tested animal, wherein the pathological features comprise amyloid plaques formed by Abeta 42 aggregation and more microglia cells are activated.
In some embodiments, the non-human primate is a monkey.
In some embodiments, the non-human primate is a cynomolgus monkey.
In some embodiments, one month after injection, the hippocampus and prefrontal cortex have amyloid plaques formed by aβ42 aggregation.
In some embodiments, aβ42 carries a protein tag that serves to localize aβ42 or aggregated aβ42 and to distinguish between endogenous aβ42.
Specifically, the aβ42 and flag tags expressed by the aβ42 recombinant expression vector can be detected by immunohistochemical staining, and whether aβ42 aggregates to form amyloid plaques is observed.
In some embodiments, the protein tag comprises at least one of a FLAG tag, an HA tag, and a c-Myc tag.
In some embodiments, the viral titer of the aβ42 recombinant expression vector at the time of injection is 10 13 vg/mL to realize the over-expression of the Abeta 42 recombinant expression vector in the injection area, so that Abeta 42 is aggregated to form amyloid plaques, and more microglia are activated, thereby forming pathological characteristics of Alzheimer disease patients, and having the effect of simulating the accumulation of amyloid plaques and the increase of the activated microglia in AD patients.
Embodiments of the present invention will be described in detail below with reference to examples.
EXAMPLE 1 construction of recombinant expression vectors
The AAV-UBC-GFP Plasmid used in this example was purchased from Addgene under the accession number Plasmid #62518, and the restriction endonucleases BamHI and EcoR V were purchased from New England Biolabs, U.S.A..
The construction of the viral vector expressing the target plasmid is shown in figure 1, and the specific construction flow method is as follows:
1. the GFP sequence was deleted by the BamHI and EcoRV cleavage sites on the AAV-UBC-GFP plasmid, and the BRI-Abeta 42-flag sequence carrying both BamHI and EcoRV cleavage sequences was ligated to obtain the AAV-UBC-BRI-Abeta 42 vector.
2. The GFP sequence was deleted by the BamHI and EcoRV cleavage sites on the AAV-UBC-GFP plasmid, and the BRI-flag sequence carrying both BamHI and EcoRV cleavage sequences was ligated to obtain an AAV-UBC-BRI vector.
The constructed AAV plasmid vector is verified to be successfully constructed by sequencing results, the specific results are shown in figure 2, and figure 2 is a schematic diagram showing the comparison of the sequencing results of the AAV-UBC-BRI-Abeta 42 plasmid expression vector and Abeta 42 gene fragment sequences.
Example 2 validation of constructed plasmids and packaging of viruses at 293T level
AAV-UBC-BRI-Abeta 42 and AAV-UBC-BRI plasmids are respectively transfected to 293T for 48 hours, and then cells are harvested for respectively carrying out immunohistochemical staining and western blotting verification, and the identification results are shown in figures 3 and 4.
AAV-UBC-BRI-Aβ42 and AAV-UBC-BRI plasmids that demonstrate effectiveness in expression in 293T are virus packaged.
Example 3 construction of animal model and detection of pathology
The virus packaged in example 2 was injected into the hippocampus and forehead She Buwei of monkey brain by stereotactic injection. The specific injection scheme is as follows: stereotactic of the monkey brain hippocampus and forehead She Buwei was performed by magnetic resonance scanning image (MRI), the parameters were based on MRI actual measurements. Subsequently, 20ul of AAV-UBC-BRI-Abeta 42 was injected into the right hippocampus and prefrontal cortex of the monkey, and 20ul of AAV-UBC-BRI (AAV-UBC-BRI-Abeta 42 virus titre 10) was injected into the left hippocampus and prefrontal cortex 13 vg/mL, AAV-UBC-BRI virus titre of 10 13 vg/mL)。
After 1 month of virus injection, the injected monkeys were anesthetized and perfused to remove hippocampus and prefrontal cortex for immunohistochemical staining. As the constructed AAV-UBC-BRI-Abeta 42-FLAG vector carries a FLAG tag, immunohistochemical staining of FLAG antibody shows that there is a large area of cell-expressing viral vector near the injection site, and the results are shown in FIG. 5. In the virus injection zone, the formation of typical amyloid plaques was observed, the detection results are shown in fig. 6, and an increase in the number of activated microglia was observed, the detection results are shown in fig. 7.
As can be seen from fig. 4 and 5, the present invention adopts a stereotactic injection method to inject a viral vector carrying BRI-aβ42-flag into the brain hippocampus and frontal lobe layer of a test cynomolgus monkey, and after 1 month of injection, the pathological examination of the test monkey revealed that the expression level of aβ42 in the vicinity of the injected region was significantly increased, and the number of activated microglia was increased.
The AAV-UBC-BRI-Abeta 42-flag vector disclosed in the embodiment can release Abeta 42 in vivo through cleavage of furin, and the released Abeta 42 can be aggregated to form amyloid plaques in the same way due to the strong toxic effect and aggregation-prone property, and can distinguish endogenous Abeta 42; the method has the advantages that the administration frequency is low, the pathological characteristics of the Alzheimer's disease patient can be simulated in a short period after the administration, the long-term expression of Abeta 42 and the formation of aggregates in the non-human primate body can be realized through one injection, more microglia cells are activated, the pathological characteristics of the Alzheimer's disease patient can be well simulated in the non-human primate body, and the method is used for large-scale drug screening for treating the Alzheimer's disease.
The method for constructing the Alzheimer disease animal model adopts a three-dimensional positioning multipoint injection mode to rapidly inject the AAV-UBC-BRI-Abeta 42-flag vector into the hippocampus and the forehead leaf layer of the non-human primate, is simple and rapid, has less administration times, and can realize rapid and stable long-term overexpression of Abeta 42 in the brain of the non-human primate by one administration; the period of occurrence of pathological features is short, and the pathological features of Alzheimer disease patients can be simulated only by about one month after administration; furthermore, the Abeta 42 expressed by the Alzheimer's disease animal model constructed by the invention is controlled by a UBC promoter, can widely express Abeta 42 in different cell types and HAs at least one tag of FLAG tag, HA tag and c-Myc tag, and can be different from endogenous Abeta 42.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> and university of south China
<120> construction method and use of Alzheimer's disease animal model and Abeta 42 recombinant expression vector
<140> 2022106407181
<141> 2022-06-08
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 908
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
cgcggatccg ccaccatgga ctacaaagac gatgacgaca agatggtgaa ggtgacgttc 60
aactccgctc tggcccagaa ggaggccaag aaggacgagc ccaagagcgg cgaggaggcg 120
ctcatcatcc cccccgacgc cgtcgcggtg gactgcaagg acccagatga tgtggtacca 180
gttggccaaa gaagagcctg gtgttggtgc atgtgctttg gactagcatt tatgcttgca 240
ggtgttattc taggaggagc atacttgtac aaatattttg cacttcaacc agatgacgtg 300
tactactgtg gaataaagta catcaaagat gatgtcatct taaatgagcc ctctgcagat 360
gccccagctg ctctctacca gacaattgaa gaaaatatta aaatctttga agaagaagaa 420
gttgaattta tcagtgtgcc tgtcccagag tttgcagata gtgatcctgc caacattgtt 480
catgacttta acaagaaact tacagcctat ttagatctta acctggataa gtgctatgtg 540
atccctctga acacttccat tgttatgcca cccagaaacc tactggagtt acttattaac 600
atcaaggctg gaacctattt gcctcagtcc tatctgattc atgagcacat ggttattact 660
gatcgcattg aaaacattga tcacctgggt ttctttattt atcgactgtg tcatgacaag 720
gaaacttaca aactgcaacg cagagaaact attaaaggta ttcagaaacg tgatgcagaa 780
ttccgacatg actcaggata tgaagttcat catcaaaaat tggtgttctt tgcagaagat 840
gtgggttcaa acaaaggtgc aatcattgga ctcatggtgg gcggtgttgt catagcgtga 900
gatatcgc 908
Claims (1)
1. An aβ42 recombinant expression vector useful for constructing an animal model of alzheimer's disease from a non-human primate, said aβ42 recombinant expression vector comprising an AAV vector comprising a BRI-aβ42 sequence;
the BRI-Abeta 42 sequence is as follows:
CGCGGATCCGCCACCATGGACTACAAAGACGATGACGACAAGATGGTGAAGGTGACGTTCAACTCCGCTCTGGCCCAGAAGGAGGCCAAGAAGGACGAGCCCAAGAGCGGCGAGGAGGCGCTCATCATCCCCCCCGACGCCGTCGCGGTGGACTGCAAGGACCCAGATGATGTGGTACCAGTTGGCCAAAGAAGAGCCTGGTGTTGGTGCATGTGCTTTGGACTAGCATTTATGCTTGCAGGTGTTATTCTAGGAGGAGCATACTTGTACAAATATTTTGCACTTCAACCAGATGACGTGTACTACTGTGGAATAAAGTACATCAAAGATGATGTCATCTTAAATGAGCCCTCTGCAGATGCCCCAGCTGCTCTCTACCAGACAATTGAAGAAAATATTAAAATCTTTGAAGAAGAAGAAGTTGAATTTATCAGTGTGCCTGTCCCAGAGTTTGCAGATAGTGATCCTGCCAACATTGTTCATGACTTTAACAAGAAACTTACAGCCTATTTAGATCTTAACCTGGATAAGTGCTATGTGATCCCTCTGAACACTTCCATTGTTATGCCACCCAGAAACCTACTGGAGTTACTTATTAACATCAAGGCTGGAACCTATTTGCCTCAGTCCTATCTGATTCATGAGCACATGGTTATTACTGATCGCATTGAAAACATTGATCACCTGGGTTTCTTTATTTATCGACTGTGTCATGACAAGGAAACTTACAAACTGCAACGCAGAGAAACTATTAAAGGTATTCAGAAACGTGATGCAGAATTCCGACATGACTCAGGATATGAAGTTCATCATCAAAAATTGGTGTTCTTTGCAGAAGATGTGGGTTCAAACAAAGGTGCAATCATTGGACTCATGGTGGGCGGTGTTGTCATAGCGTGAGATATCGC；
the non-human primate is a monkey;
the AAV vector contains a nucleotide sequence encoding a protein tag;
the A beta 42 protein overexpressed by the A beta 42 recombinant expression vector is provided with a protein tag;
the protein tag is a FLAG tag.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210640718.1A CN115354048B (en) | 2022-06-08 | 2022-06-08 | Construction method and application of Alzheimer disease animal model and Abeta 42 recombinant expression vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210640718.1A CN115354048B (en) | 2022-06-08 | 2022-06-08 | Construction method and application of Alzheimer disease animal model and Abeta 42 recombinant expression vector |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115354048A CN115354048A (en) | 2022-11-18 |
| CN115354048B true CN115354048B (en) | 2024-02-02 |
Family
ID=84030698
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210640718.1A Active CN115354048B (en) | 2022-06-08 | 2022-06-08 | Construction method and application of Alzheimer disease animal model and Abeta 42 recombinant expression vector |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115354048B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115997722A (en) * | 2022-12-26 | 2023-04-25 | 海南大学 | Abeta 42-hIAPP co-oligomer-induced AD non-human primate model method |
| CN116716351B (en) * | 2023-03-30 | 2024-02-23 | 湖北天勤生物技术研究院有限公司 | Composition for constructing cynomolgus monkey Alzheimer's disease model, application and construction method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107261144A (en) * | 2009-11-03 | 2017-10-20 | 法奈科斯公司 | Treat the new treatment of Alzheimer disease |
| CN108699139A (en) * | 2015-07-16 | 2018-10-23 | 前体生物药物股份公司 | Humanized antibody |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003002754A2 (en) * | 2001-06-29 | 2003-01-09 | Mayo Foundation For Medical Education & Research | Bri constructs and methods of using |
| WO2015067668A1 (en) * | 2013-11-05 | 2015-05-14 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New alzheimer's disease animal model |
-
2022
- 2022-06-08 CN CN202210640718.1A patent/CN115354048B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107261144A (en) * | 2009-11-03 | 2017-10-20 | 法奈科斯公司 | Treat the new treatment of Alzheimer disease |
| CN108699139A (en) * | 2015-07-16 | 2018-10-23 | 前体生物药物股份公司 | Humanized antibody |
Non-Patent Citations (6)
| Title |
|---|
| Patricia A Lawlor 等.Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Abeta levels.《Molecular Neurodegeneration》.2007,第2卷参见摘要，第9页左栏2-5段. * |
| Summers KL 等.NCBI Reference Sequence: NM_001204303.1.《NCBI-BLAST》.2019,参见核苷酸和氨基酸序列信息. * |
| Tuhina Prasad 等.Amyloid β peptides overexpression in retinal pigment epithelial cells via AAV-mediated gene transfer mimics AMD-like pathology in mice.《Scientific Reports》.2017,第7卷(第1期),参见摘要. * |
| Vidal，R. 等.GenBank: AF152462.1.《NCBI-BLAST》.1999,参见核苷酸和氨基酸序列信息. * |
| 卢静 主编.《实验动物专业技术人员等级培训教材（中级）》.中国协和医科大学出版社,2016,参见第151-152页. * |
| 罕园园 等.阿尔兹海默症转基因小鼠模型研究进展及评价.《中国实验动物学报》.2013,第21卷(第06期),参见第3页左栏倒数第2段. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115354048A (en) | 2022-11-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115354048B (en) | Construction method and application of Alzheimer disease animal model and Abeta 42 recombinant expression vector | |
| Klein et al. | Dopaminergic cell loss induced by human A30P α-synuclein gene transfer to the rat substantia nigra | |
| Kim et al. | Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo | |
| JP6836999B2 (en) | Adeno-associated virus mutants and how to use them | |
| KR102604159B1 (en) | Tissue-selective transgene expression | |
| KR20200107949A (en) | Engineered DNA binding protein | |
| CN107466325A (en) | Multi-carrier system and its application | |
| CN109154002B (en) | AAV vectors for the treatment of dominant retinitis pigmentosa | |
| US20220125948A1 (en) | Compositions and methods for the diagnosis and treatment of retinopathies | |
| US20120231014A1 (en) | Neural Regeneration | |
| CN110249051A (en) | Enhance the method and composition that functional myelin generates | |
| US20230295287A1 (en) | Anti-vegf antibody constructs and related methods for treating vestibular schwannoma associated symptoms | |
| Lawlor et al. | Aβ infusion and related models of Alzheimer dementia | |
| KR20230002788A (en) | Artificial expression constructs for selectively modulating gene expression in neocortical layer 5 glutamatergic neurons | |
| EP1418806A2 (en) | Human disease modeling using somatic gene transfer | |
| JP2022521432A (en) | DNA-binding domain transactivator and its use | |
| US20230295654A1 (en) | Methods and compositions for treatment of fragile x syndrome | |
| JP2007319073A (en) | Screening of utrophin gene expression enhancer | |
| Feng et al. | AAV-GRN partially corrects motor deficits and ALS/FTLD-related pathology in Tmem106b−/− Grn−/− mice | |
| Prokofeva et al. | Structure and Function of Neuronal Circuits Linking Ventrolateral Preoptic Nucleus and Lateral Hypothalamic Area | |
| KR20220034833A (en) | Treatment of diseases derived from overt mutant genes | |
| CN112639107A (en) | Methods of treating hearing loss and/or vision loss associated with CLRN1 | |
| US11895993B2 (en) | Method of producing a novel animal model for tauopathies | |
| KR102108107B1 (en) | Producing method of dominant intermediate Charcot-Marie-Tooth disease type C animal model and uses thereof | |
| TW202342525A (en) | Anti-vegf antibody constructs and related methods for treating vestibular schwannoma associated symptoms |
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 |