WO2004050876A1 - Vaccin genique concernant un adenovirus recombinant convenant a la therapie et la prophylaxie de la maladie d'alzheimer, et utilisation correspondante - Google Patents
Vaccin genique concernant un adenovirus recombinant convenant a la therapie et la prophylaxie de la maladie d'alzheimer, et utilisation correspondante Download PDFInfo
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- WO2004050876A1 WO2004050876A1 PCT/CN2003/001019 CN0301019W WO2004050876A1 WO 2004050876 A1 WO2004050876 A1 WO 2004050876A1 CN 0301019 W CN0301019 W CN 0301019W WO 2004050876 A1 WO2004050876 A1 WO 2004050876A1
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Classifications
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- 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
- 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
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- 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
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Definitions
- Recombinant adeno-associated virus gene vaccine for treating and preventing Alzheimer's disease with stem and use thereof
- the present invention relates to a recombinant adeno-associated virus gene vaccine for use in the treatment and prevention of Alzheimer's disease (AD) and its use. More specifically, the recombinant adeno-associated virus gene vaccine of the present invention comprises a recombinant DNA molecule containing a core sequence encoding a fusion protein of a cholera toxin B subunit and an A ⁇ peptide fragment. The invention also relates to a pharmaceutical composition comprising said recombinant DNA molecule or fusion protein. Background technique
- AD Alzheimer's disease
- presenile dementia is a common degenerative disease of the central nervous system. Its main clinical manifestations are progressive memory loss and cognitive impairment.
- the neuropathological features of AD are excessive extracellular ⁇ -amyloid precipitation, increased neurofibrillary tangles, decreased synaptic density, and decreased neuronal cells. Among them, the deposition of extracellular P amyloid, which forms scattered senile plaques and amyloid angiopathy, is considered to be a key link in the pathogenesis of the disease.
- Amyloid ⁇ protein is a ⁇ -amyloid precursor protein (APP) that is cleaved by ⁇ -secretase (BACE) and ⁇ -secretase and contains 39 to 43 amino acid residues. Enzymatic fragment. Under normal circumstances, most of APP is cleaved by ⁇ -secretase and ⁇ -secretase, does not form A ⁇ peptide, and cannot form extracellular ⁇ -amyloid precipitate. The occurrence of AD is related to several mutants of the AP protein.
- the APP V717I Goate et al, Nature 349; 704), APP V717G (Hai an et al, Nature 353; 844), APP V717F (MurreIl, Science 254; 97), APP lysine 595 - methionine Yue 596 changed into a double mutation of aspartic acid 595 -leucine 596 (Mullan et al, Nature Genet., 1; 345).
- the APP gene was sequenced and localized on chromosome 21 (Kang et al, Nature 325; 733). Expression of the APP gene produces several 695, 751, and 770 JJ3 ⁇ 4-containing isomers of A ⁇ . (Kang et al, Nature 325; 733, Kitaguchi et al, Nature 331; 530). Although there is evidence that APP has a role in mediating adhesion and growth of neurons (Schubert et al, Neuron 3; 689) and a role in G protein-linked signaling pathways (Nishimoto et al, Nature 362; 75), The exact function of APP in the nervous system is unclear.
- ⁇ -amyloid plays a key role in the occurrence of AD. All factors related to the disease either promote the deposition of ⁇ -amyloid or strengthen the pathological changes caused by ⁇ -amyloid. Therefore, the main target site currently developed by researchers for the treatment of AD drugs is to reduce the amount of beta amyloid deposits in the central nervous system (CNS). This type of research is mainly divided into two major areas, namely reducing the production of beta amyloid and increasing its clearance.
- patent application WO 98/44955 discloses a method for preventing or preventing Alzheimer's disease using a recombinant antibody specific to ⁇ -amyloid terminus, and this patent application protects a recombinant antibody specific to amyloid terminus and encoding Their DNA and how to use it.
- WO 99/27944 discloses the use of A ⁇ 42 peptide to produce anti-P amyloid antibody. The antibody can enter the brain tissue through the blood-brain barrier to bind to P amyloid protein, and clear P starch by phagocytosis of microglia. Like Protein deposition.
- the patent application protects the preparation of A & 42 peptide vaccine and its use method. Although the patent application mentions the potential of the AP 42 DNA fragment as a gene vaccine, it does not disclose how the gene vaccine is used in therapy.
- a ⁇ 42 peptide has a small molecule and low immunogenicity. In order to obtain an anti-Amyloid antibody that can clear senile plaques, repeated injections of A ⁇ 42 peptide are required. Adopting long-term subcutaneous immunization, the operation is tedious, and it also brings greater pain to AD patients. Secondly, the use of passive immunotherapy to directly inject anti-beta amyloid antibody into AD patients also has obvious shortcomings, that is, the half-life of the antibody in the body is very short. Not suitable for the treatment of chronic diseases like AD.
- a new genetic recombinant vaccine which contains a recombinant DNA sequence encoding a cholera toxin B subunit and an A ⁇ peptide fragment or a derivative thereof, and also contains a recombinant DNA molecule operatively linked to the recombinant DNA molecule Promoter capable of expressing fusion proteins (cholera toxin B subunit and A ⁇ peptide fragment) in eukaryotic cells.
- a leader sequence is also provided at the 5 end of the recombinant DNA molecule, which encodes the N-terminal signal peptide.
- the nucleotide sequence of the fusion protein encoding the cholera toxin beta subunit and the A ⁇ peptide fragment is shown in Figure 4 and the sequence listing (SEQ ID NO. 1).
- Another aspect of the present invention is to provide a recombinant DNA molecule containing a gene encoding a fusion protein of a cholera toxin B subunit and an A ⁇ peptide fragment.
- the fusion protein can induce the body to produce anti-beta amyloid antibodies.
- Yet another aspect of the present invention is to provide a recombinant viral vector comprising the recombinant DNA molecule of the present invention, the recombinant viral vector encoding A fusion protein is used to introduce the recombinant DNA molecule into a body cell.
- Suitable viral vectors include genovirus, adenovirus, herpes virus, retrovirus, vaccinia virus, fowlpox virus, Sindbis virus or fowlpox virus.
- the pharmaceutical composition contains a fusion protein called CB- ⁇ (sequence listing SEQ IDNO. 2).
- the protein CB- ⁇ is composed of a cholera toxin B subunit "head-A ⁇ peptide fragment.
- the protein CB- ⁇ may be an expression product of a recombinant DNA molecule in a prokaryotic cell, or a eukaryotic cell such as Yeast expression products.
- the fusion protein of the present invention is not limited to being formed from the cholera toxin B subunit and the A ⁇ peptide.
- the fusion protein can also be phycoglobin ⁇ first 3 peptides; in some pharmaceutical compositions, the fusion protein can also be a modified bacterial toxin-linker-A ⁇ peptide; in some pharmaceutical compositions, the fusion The protein may also be a 3 ⁇ 48 8 "head 0 peptide of HBV; in some pharmaceutical compositions, the fusion protein may also be a lipid or liposome- ⁇ peptide.
- the present invention also provides a pharmaceutical composition containing the recombinant DNA molecule of the present invention encoding a fusion protein (cholera toxin B subunit-linker-A ⁇ peptide) and a suitable gene transport vector.
- the composition is suitable for a variety of routes of administration for preventing or inhibiting the further development of Alzheimer's disease.
- the pharmaceutical composition can be administered orally, nasally, intradermally, subcutaneously, intramuscularly, topically or intravenously.
- Figure 1 shows the mechanism of beta amyloid production and the pathway of beta amyloid precursor protein (APP) hydrolysis.
- P-amyloid is a peptide fragment produced by ⁇ -amyloid precursor protein (APP) cleavage by ⁇ -secretase (BACE) and ⁇ -secretase.
- Figure 1 also shows the degradation process of APP after cleavage by ⁇ -secretase and ⁇ -secretase.
- Figure 2 shows a column of beta amyloid-producing regions in AP. Arrows indicate ⁇ -, Cleavage sites for ⁇ -, or ⁇ -secretase. Beta amyloid is a peptide fragment containing 39 to 43 amino acid residues.
- Figure 3 shows the technical route for the construction of genetically recombinant vaccines, including the cloning, ligation, sequencing, construction of plasmid plants, packaging and harvesting of CB and ⁇ 42 DNA.
- Figure 3 shows the technical route for the construction of genetically recombinant vaccines, including the cloning, ligation, sequencing, construction of plasmid plants, packaging and harvesting of CB and ⁇ 42 DNA.
- Embodiment 1 shows the technical route for the construction of genetically recombinant vaccines, including the cloning, ligation, sequencing, construction of plasmid plants, packaging and harvesting of CB and ⁇ 42 DNA.
- Figure 4 shows the DNA sequence encoding the fusion protein in the recombinant DNA molecule, which contains a total of 510 nucleotides.
- Figure 5 shows a 3 ⁇ 4 ⁇ column of the gene recombinant DNA molecule expression product-fusion protein, consisting of 170 amino acid residues.
- Figure 6 shows the titers of anti-beta amyloid antibodies in peripheral blood after immunizing PDAPP transgenic mice and non-transgenic mice with a genetically modified immune vaccine.
- A PDAPP transgenic mice in the treatment group
- B PDAPP transgenic mice in the prevention group
- C Non-transgenic mice in the treatment group
- D Non-transgenic mice in the prevention group.
- FIG. 7 shows the neutralizing effect of serum anti-A ⁇ IgG antibodies.
- rAAV / CB-A ⁇ 42 immune serum was diluted 1:10, the neurotoxic effect of A ⁇ 42 was partially neutralized.
- FIG. 8 shows the mean latency (MeaniSEM) of PDAPP transgenic mice in the treatment group searching for hidden platforms in the water maze experiment for 1 to 6 days.
- A Non-treatment group and AAV / GFP group.
- B rAAV / A ⁇ 42 group.
- C rAAV / CB-A ⁇ 42 group.
- im indicates intramuscular injection, in indicates nasal feeding, and oral indicates intragastric administration (the following figures are the same).
- Figure 9 shows the average incubation period (Mean soil SEM) of PDAPP transgenic mice in the prevention group searching for hidden platforms in the water maze experiment for 1 to 6 days.
- Figure 10 shows the percentage of search time in the TQ quadrant of the PDAPP transgenic mice in each group in the treatment group (A) and the prevention group (B). * P ⁇ 0.05, ** P ⁇ 0.01, compared with non-treatment group or AAV-GFP group.
- FIG. 11 shows the number of times the mice of each group of the treatment group (A) and the prevention group (B) passed the platform position in 30 seconds in the exploration experiment.
- Figure 12 shows the mean latency (Mean SEM) of PD APP transgenic mice in the treatment group in a visible platform water maze test.
- Figure 13 shows the mean latency (MeaniSEM) of PDAPP transgenic mice in the prevention group in the visible platform water maze test.
- FIG. 14 Immunohistochemical detection of A ⁇ amyloid in the brain tissue of PDAPP transgenic mice in the treatment group (cerebral cortex).
- FIG. 15 Immunohistochemical detection of ⁇ amyloid in brain tissue of PDAPP transgenic mice in the treatment group (hippocampal region).
- FIG. 16 Immunohistochemical detection of A ⁇ amyloid in the brain tissue of PDAPP transgenic mice in the prevention group (cerebral cortex).
- FIG. 1 Immunohistochemical detection of ⁇ amyloid in brain tissue of PDAPP transgenic mice in the prevention group (Hippocampus region).
- FIG. 1 Congo red histochemical detection (brain cortex) of brain tissue from PDAPP transgenic mice in the treatment group.
- FIG. 19 Histochemical detection of Congo red brain tissue in hippocampal regions of PDAPP transgenic mice in the treatment group.
- FIG. 20 Congo red histochemical detection (brain cortex) of the brain tissue of PDAPP transgenic mice in the prevention group.
- FIG. 21 Congo red histochemical detection of brain tissue in PDAPP transgenic mice in the prevention group (Hippocampus region).
- Figure 22 GFAP (Glial fibrillary acidic) in brain tissue of PDAPP transgenic mice in the treatment group protein glial fibrillary acidic protein) immunohistochemical detection.
- FIG. 23 Immunohistochemical detection of GFAP in brain tissue of PDAPP transgenic mice in the prevention group.
- the genetically recombinant vaccine of the present invention can be used for the prevention and treatment of Alzheimer's disease (a disease characterized by ⁇ amyloid deposition).
- the recombinant DNA molecule of the present invention can express a polypeptide such as the cholera toxin B subunit ⁇ head-A ⁇ peptide, which refers to a nucleotide sequence that contains transcriptional and translational regulatory information, and this sequence is effectively related to encoding this polypeptide The nucleotide sequences are linked.
- Effective linking refers to the linking that regulates the DNA sequence and the way the DNA sequence is linked to cause gene expression.
- the regulatory regions required for gene expression include a promoter region and a DNA sequence that, when transcribed into RNA, will become the starting signal for protein synthesis. Such regions will typically include those 5, -noncoding sequences involved in transcription and translation.
- the promoter used is, for example, a CMV promoter, which is suitable for a variety of cells and can promote the expression of a gene of interest in a variety of cells.
- Vectors used for gene transfer include viral vectors (such as adeno-associated virus vectors), lipids / liposomes, and ligands for cell surface receptors.
- the recombinant DNA molecule needs to be combined with a gene transfer vector.
- the gene transfer vector in some viral vectors and the recombinant DNA molecule encoding a fusion protein are integrated into the DNA of the viral vector or packaged into virus particles; some lipids or lipids
- the plastid gene transfer vector can encapsulate the recombinant DNA molecule therein; some ligands of cell surface receptors are used as gene transfer vectors to combine with the recombinant DNA molecule in a coupling or other manner. Therefore, the term "combination" includes integration or packaging, compounding, coupling, and the like.
- Adeno-associated virus was originally contaminated in tissue culture It was later found that a non-pathogenic co-infection factor was isolated during the onset of adenovirus infection in children (Blacklow et al, 1986), which was called the eye related virus or adenovirus-associated virus.
- AAV is a single-stranded DNA virus of the genus Parvovirus, having a 4.7 kb genome.
- AAV is a replication-deficient virus that requires a co-infection of a helper virus (usually an adenovirus or herpes virus) to replicate efficiently and complete its life cycle.
- helper virus usually an adenovirus or herpes virus
- AAV becomes a latent form and undergoes stable integration at a high frequency, usually at a specific site on chromosome 19 (Kotin et al, 1992).
- the AAV genome has been sequenced, and the only sequence necessary for AAV integration was found to be an inverted terminal repeat (ITR) of 145 nucleotides.
- ITR inverted terminal repeat
- the clone capacity of AAV is about 4.7Kb (Muzyczka, 1992). Because the virus has its own superior characteristics (non-pathogenic, extensive host cells, efficient integration ability, heat resistance, acid resistance, and long-term efficient expression), it is particularly suitable for use as a gene transfer vector.
- AAV can infect cells and integrate onto the cell chromosome, express the fusion protein (cholera toxin B subunit head-A ⁇ peptide), and secrete it outside the cell for antigen presentation
- fusion protein cholera toxin B subunit head-A ⁇ peptide
- present antigens to T and B lymphocytes induce immune responses, and produce anti-beta amyloid antibodies.
- the antibody can enter the extracellular environment of neurons in brain tissue and combine with soluble or aggregated beta amyloid to form a beta amyloid-antibody complex.
- the antibodies regulate the beta amyloid-antibody complex through the opsonization of Fc receptors on the surface of small neuroglia shield cells to clear the deposition of amyloid beta, or the beta amyloid-antibody complex passes through The excretion of sagittal sinus arachnoid villi is cleared from the central nervous system, thereby avoiding the deposition of beta amyloid in brain tissue and the neurotoxic effects induced by it.
- the genetic recombination vaccine and the pharmaceutical composition of the present invention are not only suitable for patients who have apparently developed Alzheimer's disease pathological features, but also suitable for the general population to prevent the occurrence of Alzheimer's disease, so that the general population can obtain a response to this epidemic.
- Sexual and destructive disease immunity especially patients with Down's syndrome or people with familial Alzheimer's disease-related gene mutations who are prone to develop Alzheimer's disease.
- the best route of administration is subcutaneous, intradermal, intramuscular, oral, nasal or intravenous.
- a recombinant DNA molecule containing a fusion protein gene combined with a gene transfer vector can be used in the production or preparation of a pharmaceutical composition containing such a required effective amount of the recombinant DNA molecule.
- a suitable dose of virus particles in a pharmaceutical composition for treating or preventing Alzheimer's disease is about 5 ⁇ 10 4 to 1 ⁇ 10 12 virus particles.
- a ligand of a cell surface receptor is used as a gene transfer carrier, the amount of DNA molecules bound by the ligand used is about 0.5 to 100 ⁇ g.
- the amount of DNA molecules used is about 1 to 500 g.
- PDAPP transgenic mice refers to C57 mice transfected with APP V7171 gene mutants. The mice begin to have behavioral changes at 3 to 5 months and appear in brain tissues at 11 to 13 months. Similar to the pathological changes in patients with Alzheimer's disease, there is obvious deposition of amyloid and senile plaque.
- non-transgenic mice refers to C57 normal mice that are not transfected with the APP V71 "gene mutant.
- APP V717I , APP V717G, and APP V717F refer to mutations in the 717 site of the APP protein encoded by the human APP gene from the original valine to isoleucine, glycine, and phenylalanine, respectively.
- ⁇ peptide refers to AP peptide fragments containing acid residues 1-39, 1-40, 1-41, 1-42, and 1-43, mainly including AP 39 , ⁇ 40 , ⁇ 41 , 0 42 and 0 43 .
- "Alpha] [beta peptide” include ⁇ 39, ⁇ ⁇ 40, ⁇ 41, ⁇ derivatives ⁇ ⁇ 43 and ⁇ 42 a.
- a ⁇ 41 , A ⁇ 40 and A ⁇ 39 differ from A ⁇ 42 in that they lack the C-terminal Ala, Ala-Ile and Ala-Ile-Val, respectively.
- ⁇ 43 differs from ⁇ 42 in that it adds a Thr to the C-terminus.
- amyloid ⁇ protein is equivalent in meaning to the term “A ⁇ peptide” and includes A ⁇ 39 , A ⁇ 40 , A ⁇ 41 , A ⁇ 42 and A ⁇ 43 peptides.
- immune response refers to the generation of a response in a patient being treated against amyloid beta antibodies and / or ⁇ specific T cells or their secreted products.
- adjuvant refers to a substance that can enhance the body's immune response to an antigen when used in combination with an antigen, but does not produce an immune response to the antigen when used alone.
- adjuvants can enhance the immune response through several mechanisms, including aggregation of lymphocytes, activation of B cells and / or T cells, activation of macrophages Cell.
- the term "patient” includes humans and other mammalian subjects who receive prophylactic or therapeutic treatments.
- the term "fusion protein” is referred to herein as containing a new fusion protein shield called CB-Ap.
- the new protein (CB- ⁇ ) consists of the cholera toxin B subunit head- ⁇ peptide fragment.
- the linker amino acid sequence is glycine-proline-glycine-proline.
- recombinant DNA molecule refers to a nucleotide sequence containing derivatives encoding the cholera toxin beta subunit and A ⁇ peptide fragments, and derivatives thereof.
- derivative refers to a sequence containing a nucleotide that encodes a polypeptide that substantially retains the biological function or activity of human amyloid. Or refers to a polypeptide that substantially retains the biological function or activity of human beta amyloid.
- promote sequence refers to the part of a DNA molecule that can specifically bind to RNA polymerase, that is, the part where transcription starts.
- gene transfer vector refers to any technology and / or substance suitable for introducing DNA molecules into the body.
- the AAV gene recombinant vaccine of the present invention After the AAV gene recombinant vaccine of the present invention is administered to a patient, it ectopically expresses a fusion protein molecule in the patient's body, and induces the body to produce an antibody against beta amyloid.
- the antibody is capable of neuron extracellular environment in brain tissue, and Soluble or aggregated beta amyloid binds to form a beta amyloid-antibody complex.
- the antibodies promote the phagocytosis of ⁇ -amyloid-antibody complex by the opsonization of Fc receptors on the surface of microglia to clear the deposition of P amyloid, or the ⁇ -amyloid-antibody complex passes through The excretion of sagittal sinus arachnoid villi is cleared from the central nervous system, thereby avoiding the deposition of beta amyloid in brain tissue and the neurotoxic effects induced by it.
- the amyloid proteins involved in the pathological process of Alzheimer's disease namely A ⁇ 39 , A ⁇ 4 ⁇ A ⁇ 41 , AP 42 and A ⁇ 43 are the main clearance targets. Therefore, the genetic recombinant vaccine of the present invention can be used to prevent or treat Alzheimer's disease, or to inhibit the further development of Alzheimer's disease.
- beta amyloid eggs can be prevented or eliminated
- the invention also avoids the problems caused by long-term repeated use of pharmaceutical preparations that need to cross the blood-brain barrier.
- experiments show that the structural design of the fusion protein of the present invention and the selection of the linker allow the two polypeptides linked by the linker to maintain their respective activities in the body, thereby pre-paying or treating the Alzheimer's gene recombinant vaccine and the fusion protein of the present invention. Symptoms provide a guarantee.
- the content of the present invention can be more easily understood by referring to the following examples, which are only for further explanation and are not meant to limit the scope of the present invention.
- Upstream primer 5, -GGTCCTGGTCCTGATGCAGAATTCCGACATG AC-3, (SEQIDNO. 3),
- Downstream primer 5, -GGAAGATCTTTACTACGCTATGACAACACCGCCC-3, (SEQIDNO. 4).
- CB cholera toxin B subunit
- the universal AAV shield particle vector pSNAV (Chinese patent Application number: 99119038.6, Title of invention: Construction and application of a series of general-purpose adenovirus-associated virus vectors, Publication No .: CN 1252450A), after double digestion with Kpn I and Bgl ll, large fragments were recovered to obtain pSNAV vectors containing sticky ends Fragment.
- the digested pSNAV vector fragment and the CB-A P 42 DNA fragment were ligated into the pSNAV-CB-A 0 42 plasmid. Then, the ligation product was transformed, and a small amount of purified plasmid DNA was extracted.
- pSNAV-A ⁇ 42 plasmid was constructed, and pSNAV-A ⁇ 42 plasmid and pSNAV-GFP plasmid were used as controls for pSNAV-CB-A ⁇ 42 plasmid.
- the pSNAV-GFP plasmid was previously constructed by Wu Xiaobing et al. (Chinese patent application number: 99119038.6, invention name: Construction and use of a series of general-purpose adenovirus-associated virus vectors).
- the pSNAV-A ⁇ 42 plasmid contains only A ⁇ 42 DNA fragments and is preceded by an AAP leader sequence, but does not contain the CB gene DNA fragment.
- the green fluorescent protein (GFP) gene was inserted into the pSNAV-GFP plasmid.
- the pSNAV-CB-A P 42 plasmid obtained in the above Example 1 was packaged into a recombinant AAV virus (rAAV / CB-A ⁇ 42 virus) containing a CB-A ⁇ 42 expression cassette, that is, a genetic recombinant vaccine of the present invention.
- rAAV / CB-A ⁇ 42 virus a recombinant AAV virus
- CB-A ⁇ 42 expression cassette that is, a genetic recombinant vaccine of the present invention.
- It is the method of the patent applied by Wu Xiaobing et al. Choinese patent application number: 98120033.8, invention name: Production and use of fully functional helper virus for recombinant adeno-associated virus production; Chinese patent application number: 99119039.4, invention name: available Production method and use of recombinant adenovirus-associated virus produced in Da Mo; Chinese Patent Application No. 99123723.4, Title of Invention: A method and
- Step 1 Establishment of rAAV / CB-A ⁇ 42 virus vector cell line BHK cells were transfected with the pSNAV-CB-A P 42 plasmid, and the rAAV / CB-A ⁇ 42 virus vector cell line BHK / pSNAV-CB-A ⁇ 42 was obtained by selective culture.
- BHK cells were cultured in RPMI1640 medium 37 containing 10% fetal bovine serum.
- the pSNAV-CB-A ⁇ 42 plasmid was transfected into BHK cells with lipofectamine (GIBCO BRL), digested 24 hours later, and passaged at 1: 2 to 5.
- Apparently resistant cell clones formed after 10 days. Cell clones were singled out for further culture and cryopreserved.
- the cloned and cultured virus vector cells obtained are infected with a full-function helper virus HSVl-rc (Chinese patent application number: 98120033.8, invention name: Production and use of full-function helper virus for recombinant adeno-associated virus production)
- HSVl-rc Full-function helper virus
- the cells and their culture fluids were harvested after the cells became virulent.
- the results of SDS-PAGE electrophoresis showed three characteristic electrophoretic bands of AAV virus.
- Southern hybridization results showed that the rAAV virus contained the CB-A ⁇ 42 nucleic acid sequence.
- the titer of rAAV / CB-A ⁇ 42 virus was determined.
- a cell line with high rAAV virus titer was selected as the AAV-GFP virus plant cell line.
- the cloned cultured virus vector cells obtained above were further cultured to a large number of roller bottle culture scales with RPMI1640 medium containing 10% fetal bovine serum. After the cells reached a certain number, the full-function helper virus HSVl-rc was used to expand the culture Cell.
- the cells and their culture fluids are harvested, and the recombinant rAAV / CB-A ⁇ 42 virus is isolated and purified using the following steps: 1) chloroform disrupts the cells, inactivates HSV helper viruses, and denatures a large number of cellular proteins; 2) Treat cell lysate with DNasel and RNase to degrade nucleic acid; 3) Add NaCI to separate rAAV from cell debris and centrifuge to remove cell debris; 4) Precipitate rAAV with PEG / NaCl; 5) Extract with chloroform to remove impurities and residues 6) dialysis and desalting; 7) further purification of rAAV by density gradient centrifugation or affinity chromatography.
- the titer of rAAV / CB-A ⁇ 42 virus was determined by the method of Roche's digoxin DNA labeling and detection kit (Cat. No. 1093657), and serially dried plasmid pSNAV-CB-A P 42 plasmid As a standard control, southern hybridization was performed, and the hybridization signal of the sample was compared and quantified with the standard control. The results showed that the rAAV virus contained CB-A ⁇ 42 Nucleic acid sequence. rAAV virus titer of 2 X 10 12 - 13 viral particles / ml.
- the electrophoretic purity of rAAV / CB-A p 42 virus was determined by SDS-PAGE method, with a sample volume of 5 g, and the gel was stained with Coomassie blue after electrophoresis. See the three main bands of AAV-2 coat protein VP1, VP2, and VP3 After scanning with a scanner, calculate the percentage of total VP1, VP2, and VP3 proteins to the total protein. The results showed three characteristic electrophoretic bands of AAV virus.
- the packaged virus was orally, nasally, and intramuscularly injected to immunize 1- and 11-month-old PDAPP transgenic mice (PDAPP V7171 transgenic mice, see literature: Qin Chuan, Chang Yang, etc.).
- peripheral blood was collected from mice, and serum was isolated.
- ELISA was used to detect the titer of anti- ⁇ antibody in serum.
- rAAV / CB-A P 42 immunized mice were able to produce high titers of anti-AP l g G antibodies in either the treatment group or the prevention group, whether in transgenic mice or non-transgenic mice.
- the titer of anti-AP lgG antibody was the highest in the serum (the antibody titer was between 5,000-15,000, which is equivalent to 10 times the antibody titer produced by the corresponding mice immunized with rAAV / AP 42).
- Antibody (antibody titer below 1: 64). It was also found that the titers of antibody production were different between the three pathways of rAAV / CB-A P 42 immunized mice. At two months, the antibody titer was significantly higher than that of the gavage group and the nasal feeding group, and there was no significant difference between the gavage group and the nasal feeding group. The rAAV / CB-A P 42 immunized mice had a lower anti-A ⁇ I g G antibody titer in the peripheral blood of the treatment group than the prevention group (P ⁇ 0.05).
- Example 4 Example 4
- the SH-S ⁇ 5 ⁇ human neuroblastoma cells purchased from Chinese Peking Union Medical University cells) per well in 100 ⁇ containing 10 4 cells were seeded in 96-well plates, culture medium serum-free medium DMEM / F12 (available (Gibicol, USA).
- a ⁇ 42 peptide (Sigma Company, USA) was diluted to 0.12 ⁇ M, and incubated at 37 ° C for 1 week to produce fibril filaments. Then, mix 10: 1 and 50: 1 with the immune serum, and incubate for 24h at 7. Meanwhile, non-immune serum was used as a control. Each sample was duplicated. This reaction mixture was added to culture wells containing SH-SY5Y human neuroblastoma cells, 37. C.
- rAAV-GFP intramuscular group and non-treated groups were statistically analyzed with rAAV / A ⁇ 42 immunized intramuscularly in groups rAAV / CB-A ⁇ 42 intramuscularly Immunohistochemistry showed rAAV / CB-A ⁇ 42
- the average latency of the intramuscularly immunized PDAPP transgenic mice group was significantly shorter than that of the rAAV / GFP group (p ⁇ 0.01) and the untreated group at 5-6 days.
- the rAAV / CB-A ⁇ 42 gene recombinant vaccine was used in one-month-old mice. Behavioral indicators of mice were observed after 12 months. Since the ⁇ ⁇ / 42 gene recombinant vaccine was found in pre-experiments to produce anti-A ⁇ antibody titers in PDAPP transgenic mice was very low, and the vaccine did not significantly improve the behavior of transgenic mice in the treatment group, so we The rAAV / A ⁇ 42 group was not added to the prevention group.
- the percentage of search time in the TQ quadrant of rAAV / CB-A ⁇ 42 intramuscularly immunized mice was 37.5%, compared with 27.2% in the rAAV / GFP group and 25.9% in the untreated group. There were significant differences between the AAV-GFP group and the untreated group and the rAAV / CB-A ⁇ 42 intramuscular injection group (p ⁇ 0.05). rAAV-A P 42 42 intramuscularly immunized mice had a search time percentage in the TQ quadrant and the average number of times they passed through the platform were 28.9%, respectively.
- the r AAV / A ⁇ 42 intramuscular injection immunization group averaged 0.66 ⁇ 0.25 in the 30-second search time, and the rAAV / GFP group There was no significant difference between the treatment group and the non-treatment group (both P> 0.05), and it was also significantly less than the rAAV / CB-A ⁇ 42 intramuscular injection group (PO.01). There was no significant difference in the percentage of search time in the TQ quadrant and the number of times to pass through the platform position among non-transgenic mice in each group.
- the average latency of the rAAV / CB-A ⁇ 42 intramuscular injection group on the fourth day was 15.1 ⁇ l.ls, while the rAAV / GFP group, the untreated group, and the rAAV / A ⁇ 42 intramuscular injection group were respectively It was 19.4 ⁇ 3.1s, 21 ⁇ 3.6s, and 19.9 ⁇ 3.3s.
- rAAV / CB-A ⁇ 42 was injected into the immunized group and AAV-GFP group, untreated group and rAAV / A ⁇ 42 muscle. There were significant differences between the injection groups (all P values were less than 0.05).
- a ⁇ 42 levels were significantly lower than those in the treatment group.
- rAAV / CB-Ap 42 showed no significant differences among the three immune pathways, the contents of A ⁇ 40 and A ⁇ 42 in the brain tissue of PDAPP transgenic mice immunized with rAAV / CB-Ap 42 were significantly lower than those in the other groups.
- rAAV / CB-Ap 42 was 90.1 ⁇ 13.2 ng / g in the brain tissue of PDAPP transgenic mice in the immunized PDAPP transgenic mice, and the content of A ⁇ 42 was 40.2 ⁇ 10.3 ng / g, which were significantly lower than those in non- Treatment group (A ⁇ 40 was 130.6 ⁇ 17.9 ng / g, A 42 was 51.9 ⁇ 10.2 ng / g) and rAAV / GFP group (127.8 ⁇ 18.6 ng /, A ⁇ 42 was 55.3 ⁇ 13.5 ng / g) (all p values were Less than 0.05).
- Example 7 Immunohistochemical staining of amyloid P in brain tissue of PDAPP mice
- the CB-A P 42 and A ⁇ 42 DNA fragments were amplified by a polymerization reaction (PCR) (the specific steps are the same as those in Example 1), and they were respectively loaded into pET30a (Novagen) and pET42a (Novagen) prokaryotic cells. Expression vector. Then, it was transformed into an E. coli BL21 (DE3) -expressing strain and induced by IPTG to obtain a fusion protein. The fusion protein was purified through a His Trap purification column, and then the CB-A ⁇ 42 protein and the A ⁇ 42 peptide fragment were respectively cut with thrombin Xa and purified, lyophilized, and stored at -20.
- PCR polymerization reaction
- the lyophilized CB-A ⁇ 42 protein and AP 42 peptide obtained in Example 10 were separately dissolved in phosphate buffered saline (PBS), and Balb / c mice were immunized subcutaneously without adjuvant, and crusted Shows that after one month of immunization, the CB-A ⁇ 42 protein immunization group can produce high titers of anti-P amyloid
- the antibody (antibody titer is 1: 10,000 ⁇ 20,000), while the A ⁇ 42 peptide immunization group produced only very low anti-beta amyloid antibodies (antibody titer: 1: 200 ⁇ 500). This shows that CB-AP 42 protein can also be used as a drug for the prevention and treatment of Alzheimer's disease.
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