CN100450551C - Recombinated adeno-associated virus genes vaccine for prevention and cure of Alzheimer disease and use thereof - Google Patents
Recombinated adeno-associated virus genes vaccine for prevention and cure of Alzheimer disease and use thereof Download PDFInfo
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Abstract
The present invention relates to a recombinant adeno-associated virus gene vaccine for treating and preventing Alzheimer's disease, and an application thereof. More specifically, the recombinant adeno-associated virus gene vaccine of the present invention comprises recombinant DNA molecules which comprise a nucleotide sequence for encoding cholera toxin B subunits and fusion protein of A beta peptide fragments. The present invention also relates to a medicine composition which comprises the recombinant DNA molecules or the fusion protein.
Description
Technical field
The present invention relates to a kind of be used for Alzheimer (Alzheimer ' s disease, recombinant adeno-associated virus gene vaccine of treatment AD) and prevention and uses thereof.More specifically, recombinant adeno-associated virus gene vaccine of the present invention comprises a kind of recombinant DNA molecules, and this recombinant DNA molecules contains the nucleotide sequence of the fusion rotein of a kind of choleratoxin B subunit of coding and A β fragments of peptides.The invention still further relates to the pharmaceutical composition that comprises described recombinant DNA molecules or fusion rotein.
Background technology
Alzheimer (Alzheimer ' s disease, AD) claim presenile dementia (Presenile dementia) again, be a kind of carrying out property of common central nervous system degenerative disease, its main clinical manifestation is carrying out property hypomnesis and cognitive disorder.The neuropathological feature of AD is that over-drastic extracellular amyloid beta precipitation, neurofibrillary tangles increase, synaptic density reduces, neuronal cell minimizing etc.Wherein the deposition of extracellular amyloid beta forms dispersive senile plaque and amyloid angiopathy, is considered to the key link in this disease pathogenesis.Amyloid beta (amyloid β protein) is by beta-amyloyd precursor protein (β amyloid precursor protein, APP) enzymatic fragment that contains 39～43 amino acid residues through producing after beta-secretase (BACE) and the gamma-secretase cracking.Under the normal condition, APP is most of not to form A β peptide through alpha-secretase enzyme and gamma-secretase cracking, can not form extracellular amyloid beta precipitation.The generation of AD is relevant with the proteic several mutants of APP.As APP
V717I(Goate et al, Nature 349; 704), APP
V717G(Harlan et al, Nature353; 844), APP
V717F(Murrell, Science 254; 97), the lysine of APP
595-methionine
596Become aspartic acid
595-leucine
596Two sudden change (Mullan et al, NatureGenet., 1; 345).These sudden changes have changed the restriction enzyme site of APP, make most of APP through beta-secretase and gamma-secretase cracking, produce the A β peptide of 39～43 amino acid residues, form extracellular amyloid beta aggregate and precipitate.Particularly these sudden changes have increased A β
42Peptide and A β
43The generation of peptide is because A β
42Peptide and A β
43The conformation of peptide carboxyl terminal extending part more is insoluble in water, be easy to assemble to form the preferential precipitation of antiparallel βZhe Die lamella (Joachim et al, Nature 325; 733, Halverson et al, Biochemistry 29; 2639, Barrow et al, J.Mol.Bio.225; 1075).Yet, A β
42The gathering of peptide can promote A β
40Peptide aggregation, thus senile plaque formed.
App gene is checked order the sixth of the twelve Earthly Branches, and is positioned (Kang et al, Nature325 on No. 21 chromosome; 733).The expression of app gene produces the isomer of several 695,751 and 770 the amino acid whose A of containing β.(Kang?et?al，Nature?325；733、Kitaguchi?et?al，Nature331；530)。Though evidence suggests APP have the effect of mediation neuronic adhesion and growth (Schubert et al, Neuron 3; 689) and in the signal transduction path that G protein connects work (Nishimoto et al, Nature 362; 75), but the definite function of APP in nervous system also it be unclear that.
Generally believe at present, play key effect in the generation evolution that is deposited on AD of amyloid beta, the factor of all and this disease association or promote the auspicious proteic deposition of β starch, or strengthen the pathological change that causes by amyloid beta.Therefore, to be used for the treatment of the main target site of AD medicine be the quantity that reduces the auspicious proteinosis of β starch among the central nervous system (CNS) in research worker exploitation at present.This type of research mainly is divided into two big fields, promptly reduces the auspicious proteic generation of β starch and increases its removing.
For first field, from the angle of materia medica, the most direct mode that reduces the auspicious proteic generation of β starch is by direct inhibition β or gamma secretase.But, do not study clear to beta-secretase (BACE) and gamma-secretase as yet in intravital other function of people at present, and they are present on the inner membrance of the endoplasmic reticulum of cell or interior system, also be difficult to now find and pass through blood brain barrier, can suppress the specificity mortifier of their enzymatic activity again by the medicine of cell membrane and endoplasmic reticulum.
For second field, especially for the patient of developing into AD, the deposition of removing amyloid beta in the cerebral tissue is even more important.The research comparative heat in present this field.Wherein, patent application WO 98/44955 discloses the method for using the recombinant antibodies that is specific to the amyloid-beta end to prevent or prevent Alzheimer, this patent application protection be recombinant antibodies and coding their DNA and the using method thereof that is specific to the amyloid-beta end.WO 99/27944 discloses use A β
42Peptide produces the antibody of anti-beta amyloid, and this antibody can enter cerebral tissue by blood brain barrier and combine with amyloid beta, and removes the deposition of amyloid beta by the phagocytosis of microgliacyte.That this patent application is protected is A β
42The preparation of peptide vaccine and using method thereof.Though be mentioned to relevant A β in this patent application
42Dna fragmentation is the particular content how to use in treatment as the potential probability of gene vaccine but do not disclose gene vaccine.
Though it is above-mentioned peptide vaccine or antibody the passive immunity Therapeutic Method of AD have more in the past had bigger breakthrough, still perfect inadequately.At first, A β
42Peptide molecule is less, and immunogenicity is low, for obtaining removing the anti-beta amyloid antibody of senile plaque, need inject A β repeatedly
42Peptide.Adopt secular subcutaneous immunity, complex operation brings bigger misery also for simultaneously patient AD.Secondly, adopt the passive immunotherapy method to give patient's AD direct injection anti-beta amyloid antibody, also exist significant disadvantages, promptly the antibody half-life in vivo is very short.Be not suitable for the treatment that resembles this class chronic disease of AD.Once more, from present obtained experimental result, though evident in efficacy, do not reach optimal degree yet, still not very high as the titre of specific antibody, still have part senile plaque formation etc.Therefore, the immunization therapy of AD requires further improvement.The development trend of AD immunization therapy is in the world at present: (1) strengthens A β
42The immunogenicity of peptide vaccine further improves the titre of specific antibody; (2) improve immunization route and method; (3) reduce complexity and the cost of operating, be convenient to a large amount of preparations, be extensive use of.
Summary of the invention
According to an aspect of the present invention, a kind of new genetic vaccine is provided, this vaccine contains the recombinant DNA sequence or derivatives thereof of coding choleratoxin B subunit and A β fragments of peptides, also contain with this recombinant DNA molecules effectively be connected also can be in eukaryotic cell the promoter of expressed fusion protein (choleratoxin B subunit and A β fragments of peptides).Simultaneously, successfully secrete from express cell in order to make fusion rotein, holding at 5 ' of recombinant DNA molecules also provides a targeting sequencing, the terminal signal peptide of coding N-.The fusion rotein nucleotide sequence of coding choleratoxin B subunit and A β fragments of peptides as shown in Figure 4.
Another aspect of the present invention provides a kind of recombinant DNA molecules, and it contains the gene of the fusion rotein of a kind of choleratoxin B subunit of coding and A β fragments of peptides, and this fusion rotein can bring out body and produce anti-beta amyloid antibody.
Another aspect of the present invention provides a kind of recombinant viral vector that comprises recombinant DNA molecules of the present invention, this recombinant viral vector coding can effectively be induced amyloid beta is produced the choleratoxin B subunit of immunne response and the fusion rotein of A β fragments of peptides, is used for described recombinant DNA molecules is imported body cell.The viral vector that is fit to comprises adeno-associated virus, adenovirus, herpesvirus, retrovirus, vaccinia virus, fowlpox virus, sindbis virus or bird pox virus.
Another aspect of the present invention provides a kind of new pharmaceutical composition that further develops that is used to prevent or suppress Alzheimer.This pharmaceutical composition comprises a kind of fused protein, is called CB-A β.This protein C B-A β is made up of choleratoxin B subunit-joint-A β fragments of peptides.This protein C B-A β can be recombinant DNA molecules expression product in prokaryotic cell in some pharmaceutical compositions, also can be eukaryotic cell such as zymic expression product.
It should be understood that fusion rotein of the present invention is not limited only to be formed by choleratoxin B subunit and A β peptide.In some pharmaceutical compositions, fusion rotein also can be immunoglobulin-joint-A β peptide; In some pharmaceutical compositions, fusion rotein also can be bacteriotoxin-joint-A β peptide of transforming; In some pharmaceutical compositions, fusion rotein also can be HBsAg-joint-A β peptide of HBV; In some pharmaceutical compositions, fusion rotein also can be lipid or liposome-A β peptide.
The present invention also provides the pharmaceutical composition of the recombinant DNA molecules of the present invention that contains encoding fusion protein (choleratoxin B subunit-joint-A β peptide) and suitable gene delivery carrier.Described compositions is applicable to multiple route of administration, is used to prevent or suppress further developing of Alzheimer.That the route of administration of this pharmaceutical composition comprises is oral, nasal feeding, Intradermal, subcutaneous, intramuscular, part or intravenous administration.
Brief Description Of Drawings
Fig. 1 represents the mechanism of amyloid beta generation and the approach of beta-amyloyd precursor protein (APP) enzymolysis.Amyloid beta is the fragments of peptides that produces by after beta-amyloyd precursor protein (APP) process beta-secretase (BACE) and the gamma-secretase cracking.Simultaneously, Fig. 1 has also shown the process that APP degrades after alpha-secretase enzyme and gamma-secretase cracking.
Fig. 2 is illustrated in the aminoacid sequence that APP produces the amyloid beta zone.Arrow represent α-, β-or the cleavage site of gamma-secretase.Amyloid beta is the fragments of peptides that contains 39～43 amino acid residues.
Fig. 3 represents the constructing technology route of genetic vaccine, mainly comprise CB and A β 42DNA the clone, be connected, processes such as the packing of the structure of order-checking, plasmid vector, virus and results.Particular content is with reference to embodiment 1.
Fig. 4 represents the DNA sequence of encoding fusion protein in the gene recombinaton dna molecular, contains 510 nucleotide altogether and forms.
Fig. 5 represents the aminoacid sequence of gene recombinaton dna molecular expression product-fusion rotein, is made up of 170 amino acid residues.
Fig. 6 represents tiring of peripheral blood anti-beta amyloid antibody behind gene recombinaton immune vaccine immunity PDAPP transgenic mice and the non-transgenic mice.(A) treatment group PDAPP transgenic mice; (B) prevention group PDAPP transgenic mice; (C) treatment group non-transgenic mice; (D) prevention group non-transgenic mice.
Fig. 7 represents the neutralization of the anti-A β of serum IgG antibody, at rAAV/CB-A β
42During 1: 10 dilution factor of immune serum, can the part in and A β
42Neurotoxic effect.
Fig. 8 represents that treatment group PDAPP transgenic mice searched for the average latency of hidden platform (Mean ± SEM) in 1～6 day in water maze laboratory.A. non-processed group and AAV/GFP group.B.rAAV/A β
42Group.C.rAAV/CB-A β
42Group.Wherein im represents intramuscular injection, and in represents nasal feeding, and oral represents to irritate stomach (following respectively scheme identical).
Fig. 9 represents that prevention group PDAPP transgenic mice searched for the average latency of hidden platform (Mean ± SEM) in 1～6 day in water maze laboratory.A. non-processed group and AAV-GFP group.B.AAV-CB-A β
42Three kinds of immunization routes.
Figure 10 represent respectively to organize in treatment group (A) and the prevention group (B) the PDAPP transgenic mice in exploring experiment in the TQ quadrant the shared percentage ratio of search time.
*P＜0.05,
*Compare with non-treatment group or AAV-GFP group in P＜0.01.
Figure 11 represent treatment group (A) and prevention group (B) respectively organize mice in exploring experiment in 30 seconds through the number of times of position of platform.
Figure 12 represents the average latency of treatment group PDAPP transgenic mice in the visible platform water maze test (Mean ± SEM).A. non-processed group and AAV/GFP group.B.rAAV/CB-A β
42Group.C.rAAV/A β
42Group.
Figure 13 represents the average latency of prevention group PDAPP transgenic mice in the visible platform water maze test (Mean ± SEM).A. non-processed group and AAV/GFP group.B.rAAV/CB-A β
42Group.
Figure 14. A amyloid beta immunohistochemistry detects (cerebral cortex) in the treatment group PDAPP transgenic mice cerebral tissue.A.Control.B.rAAV/A β 42 intramuscular injection group C.rAAV/CB-A β 42 intramuscular injection groups.
Figure 15. A amyloid beta immunohistochemistry detects (hippocampus) in the treatment group PDAPP transgenic mice cerebral tissue.A.Control.B.rAAV/A β 42 intramuscular injection group C.rAAV/CB-A β 42 intramuscular injection groups.
Figure 16. A amyloid beta immunohistochemistry detects (cerebral cortex) in the prevention group PDAPP transgenic mice cerebral tissue.A.Control.B.rAAV/GFP intramuscular injection group C.rAAV/CB-A β 42 intramuscular injection groups.
Figure 17. A amyloid beta immunohistochemistry detects (hippocampus) in the prevention group PDAPP transgenic mice cerebral tissue.A.Control.B.rAAV/GFP intramuscular injection group C.rAAV/CB-A β 42 intramuscular injection groups.
Figure 18. treatment group PDAPP transgenic mice cerebral tissue Congo red histochemistry detects (cerebral cortex).A.Control.B.rAAV/A β 42 intramuscular injection group C.rAAV/CB-A β 42 intramuscular injection groups.
Figure 19. treatment group PDAPP transgenic mice cerebral tissue Congo red histochemistry detects (hippocampus).A.Control.B.rAAV/A β 42 intramuscular injection group C.rAAV/CB-A β 42 intramuscular injection groups.
Figure 20. prevention group PDAPP transgenic mice cerebral tissue Congo red histochemistry detects (cerebral cortex).A.Control.B.rAAV/A β 42 intramuscular injection group .C.rAAV/CB-A β 42 intramuscular injection groups.
Figure 21. prevention group PDAPP transgenic mice cerebral tissue Congo red histochemistry detects (hippocampus).A.Control.B.rAAV/A β 42 intramuscular injection group .C.rAAV/CB-A β 42 intramuscular injection groups.
Figure 22. treatment group PDAPP transgenic mice cerebral tissue GFAP (Glial fibrillaryacidic protein GFAP) immunohistochemistry detects.A.Control.B.rAAV/A β 42 intramuscular injection group C.rAAV/CB-A β 42 intramuscular injection groups.
Figure 23. prevention group PDAPP transgenic mice cerebral tissue GFAP immunohistochemistry detects.A.Control.B.rAAV/A β 42 intramuscular injection group C.rAAV/CB-A β 42 intramuscular injection groups.
Specific embodiments
Genetic vaccine of the present invention can be used for prevention and treatment Alzheimer (A amyloid beta deposition characteristics disease is arranged).Recombinant DNA molecules of the present invention can be expressed a peptide species such as choleratoxin B subunit-joint-A β peptide, is meant that it contains the nucleotide sequence of transcribing and translate adjusting information, and this sequence is to be connected with the nucleotide sequence of this peptide species of coding effectively.Effectively connection is meant a kind of like this connection, and the connected mode of promptly regulating DNA sequence and expressing gene DNA sequence can cause expression of gene.In a word, the needed control band of gene expression comprises a kind of promoter region and a kind of DNA sequence, and this sequence will become the initial signal of protein synthesis when being transcribed into RNA.This zone will generally include 5 '-non-coding sequence that those participations are transcribed and translated.In one embodiment of the invention, used promoter for example is the CMV promoter, and this promoter is applicable to various kinds of cell, can impel the expression of genes of interest in various kinds of cell.
For the recombinant DNA molecules of the present invention that has coding choleratoxin B subunit and A β fragments of peptides being imported in patient's body, need the different carriers that is used for gene delivery, be used in combination with this recombinant DNA molecules.The carrier that is used for gene delivery comprises viral vector (as gland relevant viral vector), lipid/liposome, the part of cell surface receptor etc.This recombinant DNA molecules need with the gene delivery carrier combinations together, for example the recombinant DNA molecules of gene delivery carrier in some viral vector and encoding fusion protein is integrated among the DNA of viral vector or is packaged in the virion; Some lipids or liposome gene transport vehicle can be wrapped in recombinant DNA molecules wherein; The part of some cell surface receptors combines with coupling or alternate manner with recombinant DNA molecules as the gene delivery carrier.Therefore, term " combination " comprises modes such as integration or packing, compound, coupling.
Adeno-associated virus (adeno-associated virus, AAV) be in the pollutant of tissue culture, to separate at first, found afterwards to be separated to a kind of non-pathogenic coinfection factor (Blacklow et al in childhood infection adenovirus stage of attack, 1986), be called adeno-associated virus or adeno-associated virus.AAV is the single-stranded DNA viruses that a kind of parvovirus belongs to, and has the 4.7kb genome.AAV is a kind of replication-defective virus, needs helper virus (being generally adenovirus or herpesvirus) coinfection just can effectively duplicate, and finishes its life cycle.Under the situation that lacks the helper virus infection, AAV becomes the form of hiding, and carries out stable integration with altofrequency, normally at No. 19 chromosomal specific site (Kotinet al, 1992).The AAV genome is checked order, finds that integrating necessary, unique sequence for AAV is the reverse terminal repeat (ITR) of 145 nucleotide.Clone's capacity of AAV is approximately 4.7Kb (Muzyczka, 1992).Since this virus have self superior characteristics (non-pathogenic, widely host cell, efficiently integration ability, heat-resisting, acidproof, can efficiently express for a long time) make it be especially suitable for use as the gene delivery carrier.
Utilize the recombinant DNA molecules of AAV packing, in case be transported in patient's body, AAV is permissive cell and is incorporated on the cell chromosome, expressed fusion protein (choleratoxin B subunit-joint-A β peptide), justacrine is to the extracellular, by antigen-presenting cell discern, engulf, processing and antigen presentation effect, give T and bone-marrow-derived lymphocyte with antigen presentation, bring out immune response, produce anti-beta amyloid antibody.This antibody can enter the neuronal cell external environment in the cerebral tissue, combines with solubility or accumulative amyloid beta, forms amyloid beta-antibody complex.Antibody promotes it to engulf amyloid beta-antibody complex by the opsonic action of microgliacyte surface Fc receptor in cerebral tissue, to remove the deposition of amyloid beta, perhaps amyloid beta-antibody complex is eliminated out the central nervous system by the Excretion of superior sagittal sinus arachnoid villi, thereby has avoided the deposition of amyloid beta in cerebral tissue and the neurotoxic effect that is brought out by it.
Genetic vaccine of the present invention and pharmaceutical composition are not only applicable to obviously develop into the patient with Alzheimer disease pathologic feature, and be applicable to that the general population prevents the generation of Alzheimer, make the general population obtain this popularity is had the disease immunity of destructive power, particularly suffer from the patient of mongolism (Down ' s syndrome) or have the crowd that familial Alzheimer associated gene mutation is easy to develop into Alzheimer.Best drug delivery route is subcutaneous, Intradermal, intramuscular, oral, nasal feeding or vein.
Can be used to produce or pharmaceutical compositions with the gene delivery carrier combinations recombinant DNA molecules that contains antigen-4 fusion protein gene together, this pharmaceutical composition comprises the recombinant DNA molecules of needed effective dose.For example, when the gene delivery carrier is the AAV carrier, is used for the treatment of or prevents the suitable dosage of virion in the pharmaceutical composition of Alzheimer to be approximately 5 * 10
4To 1 * 10
12Individual virion.When with a kind of part of cell surface receptor during as the gene delivery carrier, the amount of the bonded dna molecular of employed part is approximately 0.5～100 μ g.If during as the gene delivery carrier, the amount of employed dna molecular is approximately 1～500 μ g with lipid or liposome.
In the present invention, employed term definition is as follows:
Term " PDAPP transgenic mice " has been meant transfection APP
V717IThe C57 mice of gene mutation body, this mice just began to have behavioristics to change in the time of 3～5 months, occurred similar Alzheimer patient's pathological change in the time of 11～13 months in the cerebral tissue, the deposition of tangible amyloid were arranged, the deposition of senile plaque.
Term " non-transgenic mice " is meant untransfected APP
V717IThe C57 normal mouse of gene mutation body.
Term " APP
V717I, APP
V717GAnd APP
V717F" refer to respectively sport isoleucine, glycine and phenylalanine by proteic 717 sites of APP of people's app gene coding by original valine.
Term " A β peptide " is meant the A β fragments of peptides that contains amino acid residue 1-39,1-40,1-41,1-42 and 1-43, mainly comprises A β
39, A β
40, A β
41, A β
42With A β
43" A β peptide " also comprises A β
39, A β
40, A β
41, A β
42With A β
43Derivant.A β
41, A β
40With A β
39With A β
42Difference be that they lack Ala, Ala-Ile and the Ala-Ile-Val of C-end respectively.A β
43With A β
42Difference be that it has added a Thr at the C-end.
Term " amyloid beta (amyloid β protein) " comprises A β at the first-class term " A β peptide " that is same as of the meaning
39, A β
40, A β
41, A β
42With A β
43Peptide.
Term " immunne response " is meant replying of create antagonism amyloid beta antibody and/or A β specific T-cells or its secretory product in patient's body of receiving treatment.
Term " adjuvant " refer to when being used in combination with antigen can enhancing body to antigenic immunne response, but the material of the former immunne response that when use separately, do not create antagonism.Adjuvant can be replied by several machine-processed enhance immunity, comprises assembling lymphocyte, activation B cell and/or T cell, activating macrophage.
Term " patient " comprises people and other mammalian subject of accepting preventative or therapeutic treatment.
Term " fusion rotein " in this article refers to and contains a kind of new fused protein, is called CB-A β.This new protein (CB-A β) is made up of choleratoxin B subunit-joint-A β fragments of peptides.In a preferred embodiment, described joint aminoacid sequence is glycine-proline-glycine-proline.
Term " recombinant DNA molecules " is meant nucleotide sequence and the derivant thereof that contains coding choleratoxin B subunit and A β fragments of peptides.
Term " derivant " is meant and contains the nucleotide sequence that coding keeps people's amyloid beta biological function or active polypeptide basically.Perhaps be meant and keep people's amyloid beta biological function or active polypeptide basically.
Term " promoter sequence " be meant dna molecular can with the position of RNA polymerase specific bond, just make the position of transcribing beginning.
Term " gene delivery carrier " is meant any being applicable to intravital technology of the importing of dna molecular and/or material.
AAV genetic vaccine of the present invention is after giving the patient, it is a kind of fusion protein molecule of ectopic expression in patient's body, bring out the antibody that body produces anti-beta amyloid, this antibody can enter the neuronal cell external environment in the cerebral tissue, combine with solubility or accumulative amyloid beta, form amyloid beta-antibody complex.Antibody promotes it to engulf amyloid beta-antibody complex by the opsonic action of microgliacyte surface Fc receptor in cerebral tissue, to remove the deposition of amyloid beta, perhaps amyloid beta-antibody complex is eliminated out the central nervous system by the Excretion of superior sagittal sinus arachnoid villi, thereby has avoided the deposition of amyloid beta in cerebral tissue and the neurotoxic effect that is brought out by it.With relevant amyloid, i.e. A β in the pathological process of Alzheimer
39, A β
40, A β
41, A β
42With A β
43Be mainly to remove target.Thus, genetic vaccine of the present invention can be used for prevention or treatment Alzheimer, perhaps is used to suppress Alzheimer and further develops.
Utilize genetic vaccine of the present invention or pharmaceutical composition, can prevent or remove the deposition of amyloid beta in the neuronal cell external environment, thereby prevent neuronic infringement and lose, and with this key point as prevention or treatment Alzheimer.The present invention has also avoided long-term repeated use need pass through the problem that pharmaceutical preparation brought of blood brain barrier.In addition, experiment shows, the structure Design of fusion rotein of the present invention makes two polypeptide that are connected by joint keep activity separately in vivo with the selection of joint, thereby for genetic vaccine of the present invention and fusion rotein prepayment or treat Alzheimer assurance is provided.
Can more easily understand content of the present invention by consulting following embodiment, these embodiment are just for further specifying, and do not mean that qualification scope of the present invention.
The structure of genetic vaccine
The constructing technology route of genetic vaccine as shown in Figure 3.At first, by polymerase chain reaction (PCR) with A β 42DNA fragment from human brain tissue cDNA library, middle clone comes out, and amplimer is 5 '-GGTCCTGGTCCTGATGCAGAATTCCGACATG AC-3 ' and 5 '-GGAAGATCTTTACTACGCTATGACAACACCGCCC-3 '.By the RT-PCR technology gene clone of choleratoxin B subunit (CB) is come out, amplimer is 5 '-CCGGGGTACCCCACCATGATTAAATTAAAATTTGGTG-3 ' and 5 '-CTGCATCAGGACCAGGACCATTTGCCATACTAATTGCG-3 '.Utilize overlapping pcr with CB and A β then
42DNA couples together, and the centre adds one section linker DNA (sequence is GGTCCTGGTCCT).The aminoacid short chain of one section flexibility of this linker DNA coding makes the choleratoxin B subunit (CB) and the A β that express
42Fragments of peptides can be kept biologic activity separately.With CB-A β
42Genetic fragment is packed into and is checked order behind the pGEM-T Easy plasmid.Sequencing result as shown in Figure 4.To have CB-A β
42The pGEM-TEasy plasmid of dna fragmentation with Kpn I and Bgl II double digestion after, reclaim and to contain CB-A β
42Dna fragmentation; Simultaneously,, behind Kpn I and Bgl II double digestion, reclaim big fragment, obtain containing the pSNAV carrier segments of sticky end universal AAV plasmid vector pSNAV (making up) by this yuan Zhenyang gene company limited.With the pSNAV carrier segments behind the enzyme action with contain CB-A β
42Dna fragmentation connects into pSNAV-CB-A β
42Plasmid.Then, conversion, a small amount of that connects product extracted plasmid DNA purification.
Made up pSNAV-A β simultaneously
42Plasmid, and with pSNAV-A β
42Plasmid and pSNAV-GFP plasmid are as pSNAV-CB-A β
42The contrast of plasmid.To be that Wu Xiaobing etc. is previous make up the pSNAV-GFP plasmid (Chinese patent application number: 99119038.6, denomination of invention: the structure of serial universal adenovirus accompanying virus carriers and purposes).PSNAV-A β
42Only contain A β in the plasmid
42Dna fragmentation, and added the targeting sequencing of an AAP in its front, but do not contain the CB gene DNA fragment.Inserted green fluorescent protein (GFP) gene in the pSNAV-GFP plasmid.
RAAV/CB-A β
42The packing of virus and the mensuration of titre thereof
PSNAV-CB-A β with the foregoing description 1 acquisition
42Plasmid is packaged into and contains CB-A β
42The reorganization AAV virus of expression cassette (rAAV/CB-A β 42 viruses), it is genetic vaccine of the present invention, adopt be the patent of being applied for such as Wu Xiaobing method (Chinese patent application number: 98120033.8, denomination of invention: be used for generation of the global function helper virus that recombinant adeno-associated virus produces and uses thereof; Chinese patent application number: 99119039.4, denomination of invention: the recombinant adeno-associated virus production method and the purposes that can be used for large-scale production; Chinese patent application number: 99123723.4, denomination of invention: a kind of rapidly and efficiently the separation and the method and the purposes of purification of Recombinant adenovirus correlated virus).Concrete steps are as follows:
Step 1.rAAV/CB-A β
42The foundation of viral vector cell strain
Use pSNAV-CB-A β
42Plasmid transfection bhk cell (being provided by this yuan Zhenyang gene company limited) obtains rAAV/CB-A β through selecting to cultivate
42Viral vector cell strain BHK/pSNAV-CB-A β 42.
Bhk cell 37 ℃ of cultivations of RPMI1640 culture fluid that contain 10% hyclone.With pSNAV-CB-A β
42Plasmid digests behind the 24hr with liposome lipofectamine (GIBCO BRL) transfection bhk cell, and 1: 2～5 go down to posterity.Adding G418800 μ g/ml selects to cultivate.Can form obvious resisting cell clone after 10 days.Cell clone is chosen the continuation cultivation and carried out frozen guarantor's kind.The viral vector cell of the cloning cultivation that is obtained is provided with global function helper virus HSV1-rc (providing by this yuan Zhenyang gene company limited), treat that cell goes out poison back harvesting and culture fluid thereof, SDS-PAGE electrophoresis detection result shows three characteristic electrophoresis bands of AAV virus, the Southern results of hybridization shows that this rAAV virus packets contains CB-A β 42 nucleotide sequences, and this rAAV virus is rAAV/CB-A β
42Virus.Measure the titre of rAAV/CB-A β 42 viruses.Pick out the high cell strain of rAAV virus titer as AAV-GFP viral vector cell strain.
The viral vector cell that the cloning of above-mentioned acquisition is cultivated continues at 37 ℃ with the RPMI1640 culture fluid that contains 10% hyclone and is cultured to and rolls the flask culture scale in a large number, after cell reached some, HSV1-rc infected the cell of amplification culture with the global function helper virus.Treat that cell goes out poison back harvesting and culture fluid thereof, adopt following step the recombinate separation and the purification of rAAV/CB-A β 42 viruses: 1) chloroform smudge cells, deactivation HSV helper virus and make a large amount of cell protein degeneration precipitations; 2) handle cell pyrolysis liquid with degraded nucleic acid with DNaseI and RNase; 3) add NaCl and impel rAAV to separate, centrifugal removal cell debris with cell debris; 4) precipitate rAAV with PEG/NaCl; 5) remove foreign protein and remaining PEG with the chloroform extracting; 6) dialysis desalination; 7) be further purified rAAV with density or affinity chromatography.
The titer determination of rAAV/CB-A β 42 viruses: the method with Roche company digoxin dna marker and detection kit (Cat.No.1093657) is carried out, plasmid pSNAV-CB-A β 42 plasmids with serial dilution are standard control, carry out southern hybridization, the hybridization signal and the standard control of sample compared, quantitatively.The result shows that this rAAV virus packets contains CB-A β
42Nucleotide sequence.The titre of rAAV virus is 2 * 10
12-13Virion/ml.
RAAV/CB-A β
42The electrophoresis purity of virus is measured: adopt the SDS-PAGE method, the application of sample amount is 5 μ g, gel carries out coomassie brilliant blue staining behind the electrophoresis, sees AAV-2 coat protein VP1, VP2, three master tapes of VP3, with calculating the percentage ratio that VP1, VP2, VP3 Tot Prot account for total protein concentration after the scanner scanning.The result shows three characteristic electrophoresis bands of AAV virus.
RAAV/A β
42The packing of virus and the assay method and the rAAV/CB-A β of titre thereof
42Virus is identical.
Peripheral blood A β specific IgG antibodies titre detects
With packaged virus respectively by oral, nasal feeding and intramuscular injection, immune 1 monthly age and 11 monthly age PDAPP transgenic mice (PDAPP
V717ITransgenic mice is referring to document: Qin Chuan, Chang Yang etc.The foundation of Alzheimer transgenic animal model.Dissect journal; 2000; 31 (2): 144-147) with C57 non-transgenic mice.After immunity 1,2,5,12 month, gather the mice peripheral blood, separation of serum.Adopt the ELISA method to detect the titre of anti-amyloid beta antibodies in the serum.As shown in Figure 6, rAAV/CB-A β
42No matter immune mouse still is the prevention group in the treatment group, no matter be at transgenic mice or the non-transgenic mice, all can produce the anti-A β IgG antibody of high titre, in the time of immune 2 months, the highest (titre of antibody is 5 for the titre of anti-A β IgG antibody in the serum, 000-15 between 000, is equivalent to the antibody titer that 10 times of rAAV/A β, 42 immune corresponding mices produce).After 12 months, still can detect the anti-A β IgG antibody of high titre in treatment group immunity 5 months and the immunity of prevention group; RAAV-A β
42Only produce low-down anti-A β IgG antibody (titre of antibody is between 200-500) behind the immune transgenic mice; RAAV/CB-A β
42Between immune transgenic mice and non-transgenic mice, do not have evident difference, and the titre that produces anti-A β IgG antibody behind the rAAV-A β 42 immune non-transgenic mices is apparently higher than transgenic mice group (P＜0.05); No matter still all do not detect anti-A β IgG antibody (antibody titer is lower than 1: 64) in rAAV-GFP immune group and the non-processed group periphery serum the non-transgenic mice at transgenic mice.Also find in addition, the titre that antibody produces between three kinds of approach of rAAV/CB-A β 42 immune mouses is different, the intramuscular injection group is when first and second month, and the titre of antibody is apparently higher than filling stomach group and nasal feeding group, and no significant difference between filling stomach group and the nasal feeding group.RAAV/CB-A β
42Immune mouse, the titre of anti-A β IgG antibody is lower than prevention group (P＜0.05) in the treatment group peripheral blood.
The anti-A β of serum IgG antibody is to the neutralization of the beta mediated neurocyte toxic action of A
SH-SY5Y human neuroblastoma cell (buying the cell centre in China Concord Medical Science University) is contained 10 by every hole 100 μ l
4Individual cell inoculation is in 96 orifice plates, and culture medium is serum-free medium DMEM/F12 (purchasing the Gibicol company in the U.S.).(U.S. Sigma company) is diluted to 0.12 μ M with A β 42 peptides, hatches for 1 week for 37 ℃, makes it produce cellosilk.Then, by 10: 1 and 50: 1 and immune serum mixing, hatch 24h for 7 ℃.Simultaneously, do contrast with NIS.Each sample is done two multiple holes.This reactant mixture is added in the culture hole that contains SH-SY5Y human neuroblastoma cell, 37 ℃, hatched 2 days.The survival rate of cell adopts the MTT experiment to detect.Every hole adds the MTT of the 5mg/ml of 15 μ l, hatches 4 hours for 37 ℃.Careful sucking-off culture supernatant, every hole adds DMSO 200ul, fully blows and beats with liquid-transfering gun.On full-automatic microplate reader (the precious biotech firm of the U.S. produces), use detection wavelength 570nm to measure optical density (OD.) value.
Testing result shows, rAAV/CB-A β
42The serum of immune mouse can the part in and A β
42The neurotoxic effect to SH-SY5Y human neuroblastoma cell of mediation.As shown in Figure 7, at rAAV/CB-A β
42During 1: 10 dilution factor of immune serum, can the part in and A β
42Neurotoxic effect (70% cell survival rate), when 1: 50 dilution factor, then do not have obviously in and A β
42Neurovirulent effect.NIS does not then have obvious effect.
(water maze laboratory) detects in the behavioristics of immune mouse
(1) hidden platform experiment (Hidden water maze)
In order to study of the improvement effect of rAAV/CB-A β 42 genetic vaccines, at first adopt the hidden platform experimental evaluation of Morris water maze respectively to organize the learning and memory ability (Morris RGM.1981) of mice to cognition of PDAPP transgenic mice and memory.In experimentation, incubation period, detection range and speed that mice is searched for hidden platform have been write down.Respectively organize the search speed there was no significant difference between the mice by analysis, have very significantly dependency between incubation period and the detection range, so, with incubation period of mice search platform as the index of estimating mice space learning and memory ability.As shown in Figure 8, the treatment group although respectively organize there are differences between incubation period of mice search platform, all has tangible shortening incubation period in 6 days training process.The average latency that adopts post hoc to analyze each group is found rAAV/CB-A β
42There is not evident difference (p＞0.05) between three kinds of immunization routes of immunity PDAPP transgenic mice, same rAAV/A β
42Between three kinds of immunization routes, there is not evident difference (p＞0.05) yet.Therefore, we are with rAAV/CB-A β
42Intramuscular injection immune group and rAAV/A β
42Intramuscular injection immune group, rAAV-GFP intramuscular injection group and non-processed group are carried out statistical analysis, and the result shows rAAV/CB-A β
42The average latency of intramuscular injection immunity PDAPP transgenic mice group was significantly shorter than rAAV/GFP group (p＜0.01) and non-processed group (p＜0.01) in the time of the 5th～6 day.And rAAV/A β
42All do not have evident difference with AAV-GFP group (p＞0.05) and non-processed group (p＞0.05) incubation period of immune transgenic mice group in 6 days.Equally in the time of the 5th～6 day, rAAV/CB-A β
42The average latency (24.2s) and rAAV/A β of immunity PDAPP transgenic mice group
42Group (30.5s) also has evident difference (p＜0.05).Each is organized between the non-transgenic mice did not all have evident difference in 1～6 day in experiment.
In order further to study rAAV/CB-A β
42Whether genetic vaccine can have preventive effect to the cognition and the memory impairment of PDAPP transgenic mice, with rAAV/CB-A β
42Genetic vaccine be used for 1 the monthly age mice, after 12nd month, observe mice behavioristics index.Because rAAV/A β
42Genetic vaccine finds in PDAPP transgenic mice body to produce anti-amyloid beta antibodies in preliminary experiment titre is very low, and this vaccine do not have tangible improvement in the treatment group to the behavioristics of transgenic mice, so we do not add rAAV/A β in the prevention group
42Group.
In the prevention group, as shown in Figure 9, we find rAAV/CB-A β in the time of the 6th day
42The average latency of immunity PDAPP transgenic mice obviously shortens than rAAV/GFP group (p＜0.05) and non-processed group (p＜0.05).Each is organized between the non-transgenic mice did not all have evident difference (p＞0.05) in 1～6 day in experiment.
(2) explore experiment (Probe test)
Because mice is in the hidden platform process of search, have mice once in a while, unconscious or running at random appear on the stage behind the platform, in order to eliminate this randomness, we take platform away from the pond, allow mice search platform 60 seconds in the pond, observe mice and containing quadrant (the Target quadrant of platform originally, TQ) search time in and through the position of platform number of times, by percentage ratio that compares search time in the TQ quadrant and the number of times that passes through platform, estimate mice space learning and memory ability.Simultaneously, consider that mice may be owing to the behavior that influences the mice back in the position of platform search less than platform, so, we add up preceding 30 seconds mices in the TQ quadrant search time and through the position of platform number of times as the index of estimating mice space learning and memory ability.
The demonstration of exploration experimental result, in the treatment group, rAAV/CB-A β
42The learning and memory of immune mouse makes great efforts to be significantly improved, and rAAV/A β
42There is not evident difference between the space learning of immune mouse and memory ability and rAAV/GFP group and the non-processed group.Shown in Figure 10 .A, rAAV/CB-A β
42The percentage ratio of search time is apparently higher than AAV-GFP group, non-processed group and rAAV/A β in 30 seconds inherent TQ quadrants of immune mouse
42Group.RAAV/CB-A β
42The percentage ratio of intramuscular injection immune mouse search time in the TQ quadrant is 37.5%, and the rAAV/GFP group is 27.2%, non-processed group 25.9%.AAV-GFP group and non-processed group and rAAV/CB-A β
42Evident difference (p＜0.05) is all arranged between the intramuscular injection immune group.RAAV-A β 42
42The percentage ratio of intramuscular injection immune mouse search time in the TQ quadrant for and average number of times through platform be respectively 28.9%, with rAAV/CB-A β
42Evident difference (p＜0.05) is also arranged between the intramuscular injection immune group.And and all do not have evident difference (p＞0.05) between rAAV/GFP group and the non-processed group.Equally, in the prevention group, shown in Figure 10 .B, rAAV/CB-A β
42The percentage ratio of mice immunized search time in the TQ quadrant is also all apparently higher than rAAV/GFP group and non-processed group (p＜0.05).
Shown in Figure 11 .A, rAAV/CB-A β 42 intramuscular injection immune group average number of times through the position of platform in 30 second search time is (1.70 ± 0.35), apparently higher than rAAV/GFP group (0.49 ± 0.30) (P＜0.01) and non-processed group (0.55 ± 0.21) (P＜0.01).RAAV/A β
42Intramuscular injection immune group average number of times through the position of platform in 30 second search time is 0.66 ± 0.25, and does not have evident difference (all P＞0.05) between rAAV/GFP group and the non-processed group, also is less than rAAV/CB-A β significantly
42Intramuscular injection immune group (P＜0.01).Each is organized between the non-transgenic mice percentage ratio of search time in 30 seconds inherent TQ quadrants and is not had evident difference through the number of times of position of platform.
In the prevention group, shown in Figure 11 .B, rAAV/CB-A β 42
42Mice immunized number of times through the position of platform in exploring experiment is also all organized and non-processed group (p＜0.05) apparently higher than rAAV/GFP.
(3) visible platform water maze laboratory (Visible water maze)
After finishing " probe test ", we plug in a marker post above platform, and chequered with black and white, very eye-catching, mice is held discovery very much.Simultaneously above marker post, put a white bead, prevent that the image tracing system from causing that to the identification of marker post experiment stops.Visible platform water maze laboratory result demonstration, in the treatment group, rAAV/CB-A β in the time of the 4th day
42The average latency of respectively organizing mice of immunity significantly is shorter than other each group, but rAAV/CB-A is β
42There is not evident difference between three kinds of immunization routes of genetic vaccine.As shown in figure 12, rAAV/CB-A β
42The intramuscular injection immune group is 15.1 ± 1.1s at the 4th day average latency, and rAAV/GFP group, non-processed group and rAAV/A β
42The intramuscular injection group is respectively 19.4 ± 3.1s, 21 ± 3.6s and 19.9 ± 3.3s, finds rAAV/CB-A β through repeating variance analysis
42Intramuscular injection immune group and AAV-GFP group, non-processed group and rAAV/A β
42Has significant difference (the P value is all less than 0.05) between the intramuscular injection group.
In the prevention group, as shown in figure 13, rAAV/CB-A β in the time of the 4th day
42All there is not evident difference between the average latency of respectively organizing mice of immunity and rAAV/GFP group and the non-processed group, and rAAV/CB-A β
42There is not evident difference between three kinds of immunization routes of genetic vaccine.We discover that further the average latency of searching for visible platform between PPDAPP transgenic mice and the non-transgenic mice in experiment in the 4th day does not have evident difference (P＞0.05).
Detect discovery rAAV/CB-A β by these several water maze laboratories
42Genetic vaccine immunity PDAPP transgenic mice is respectively organized mice with other and is compared, and its learning and memory ability improves significantly.
ELISA detects the content of A amyloid beta in the cerebral tissue
In order further to confirm rAAV/CB-A β
42The effect of genetic vaccine a in reducing PDAPP transgenic mice cerebral tissue, we have adopted people A β in the highstrung ELISA method detection by quantitative mouse brain tissue
40With A β
42Content.The method can detect total people A β in the cerebral tissue
40With A β
42Content, comprise solubility and sedimentary.But this method can not detect mice A β
40Or A β
42, therefore, when detecting non-transgenic mouse brain tissue extract, no detection signal.The testing result demonstration, in the treatment group, rAAV/CB-A β
42A β in the immunity PDAPP transgenic mice cerebral tissue
40Or A β
42Content all be starkly lower than other each group.RAAV/CB-A β
42Do not have evident difference between three kinds of immunization routes, we are with rAAV/CB-A β
42Intramuscular injection group and other each group are carried out statistical analysis.The result shows, rAAV/CB-A β
42A β in the intramuscular injection group cerebral tissue
42Average content (253.6 ± 32.7ng/g cerebral tissue weight in wet base) be starkly lower than non-processed group (432.8 ± 51.3ng/g), rAAV/GFP group (415.9 ± 61.3ng/g) and rAAV/A β
42Intramuscular injection group (413.5 ± 56.1ng/g) (the p value is all less than 0.05).Equally, rAAV/CB-A β
42A β in the intramuscular injection group cerebral tissue
40Average content (756.4 ± 112.5ng/g) also be starkly lower than be lower than non-processed group (954.6 ± 156.3ng/g), rAAV/GFP group (960.4 ± 154.9ng/g) and rAAV/A β
42Intramuscular injection group (925.3 ± 147.8ng/g) (the p value is all less than 0.05).
Equally, in the prevention group, we find A β in the PDAPP transgenic mice cerebral tissue
40With A β
42Content be starkly lower than the treatment group.Though rAAV/CB-A β
42Do not have evident difference between three kinds of immunization routes, but rAAV/CB-A is β
42A β in the immunity PDAPP transgenic mice cerebral tissue
40With A β
42Content all be starkly lower than other each group.Wherein, rAAV/CB-A β
42A β in the intramuscular injection immune group PDAPP transgenic mice cerebral tissue
40Content be 90.1 ± 13.2ng/g, A β
42Content be 40.2 ± 10.3ng/g, all be starkly lower than non-processed group (A β
40Be 130.6 ± 17.9ng/g, A β
42Be 51.9 ± 10.2ng/g) and rAAV/GFP group (127.8 ± 18.6ng/g, A β 42 are 55.3 ± 13.5ng/g) (the p value is all less than 0.05).
Embodiment 7
Amyloid beta immunohistochemical staining in the PDAPP mouse brain tissue
In order further to study the deposition conditions of amyloid beta in the PDAPP transgenic mice cerebral tissue, we utilize A β monoclonal antibody specific 3D6 (purchasing the Chemicon company in the U.S.) to do immunohistochemical staining.As Figure 14 and shown in Figure 15, in the treatment group, all can see tangible amyloid beta deposition region being arranged, a large amount of sophisticated senile plaques in the cerebral cortex and the hippocampus of non-processed group PDAPP mouse brain tissue.Obviously reduce the amyloid beta deposition region in the rAAV/CB-A β 42 immune group mouse brain tissues, though also can see senile plaque, the diameter of senile plaque is obviously little than non-processed group, can't see big senile plaque.And do not discover a marked discrepancy between rAAV/GFP group and interior amyloid beta deposition conditions of 42 groups of mouse brain tissues of rAAV/A β and the non-processed group.We utilize NIHimage J gradation of image scanning quantitative analysis software (http:rsb.info.nih.gov/nih-image) analysis respectively to organize the percentage ratio of amyloid beta deposition region in the PDAPP mouse brain tissue.The result shows, rAAV/CB-A β
42The immune group mouse brain organizes the meansigma methods of cerebral cortex and hippocampus amyloid beta to be starkly lower than non-processed group, rAAV/GFP group and rAAV/A β
42Group.As Figure 16 and shown in Figure 17, in the prevention group, in non-processed group and rAAV/GFP group PDAPP mouse brain tissue, also can see tangible amyloid beta deposition region being arranged, a small amount of sophisticated senile plaque, do not discover a marked discrepancy between two groups, and rAAV/CB-A β
42Obviously reduce the amyloid beta deposition region in the immune group mouse brain tissue, can't see sophisticated senile plaque.Utilize gradation of image scanning quantitative analysis respectively to organize the percentage ratio of amyloid beta deposition region in the PDAPP mouse brain tissue.The result shows, rAAV/CB-A β
42The meansigma methods of immune group mouse brain cortex and hippocampus amyloid beta is starkly lower than non-processed group and rAAV/GFP group.
Embodiment 8
The PDAPP mouse brain is organized the Congo red histological stain
As Figure 18 and shown in Figure 19, in the treatment group, all can see having in the cerebral cortex of non-processed group and rAAV/A β 42 immune group PDAPP mouse brain tissues and hippocampus and significantly have a liking for the Congo red neurocyte in a large number.The cerebral cortex of rAAV/CB-A β 42 immune group mouse brain tissues and hippocampus are had a liking for Congo red neurocyte number and are obviously reduced.Show that neurocyte is obviously reduced by the damage of a.Equally, in the prevention group, as Figure 20 and shown in Figure 21, the cerebral cortex of rAAV/CB-A β 42 immune group mouse brain tissues and hippocampus are had a liking for Congo red neurocyte number and are obviously reduced than non-processed group and rAAV/GFP group.
Embodiment 9
The immunohistochemical staining of astrocyte hypertrophy situation in the PDAPP mouse brain tissue
As shown in figure 22, in the treatment group, non-processed group and rAAV/A β 42 immune group group PDAPP mouse brains organize cerebral cortex all can see the positive astrocyte hypertrophy of a large amount of significantly GFAP, and rAAV/CB-A β
42The positive astrocyte hypertrophy of very weak GFAP is only arranged in the immune group PDAPP mouse brain tissue.Show activatory reactive astrocyte decreased number in the cerebral tissue, the inflammatory reaction degree reduces in the cerebral tissue.Equally, in the prevention group, as shown in figure 23, the positive astrocyte number of the cerebral cortex GFAP of rAAV/CB-A β 42 immune group mouse brain tissues obviously reduces than non-processed group and rAAV/GFP group.
CB-A β
42With A β
42The expression and purification of albumen in prokaryotic cell
At first, by polymerase chain reaction (PCR) amplification CB-A β
42With A β
42Dna fragmentation (concrete steps are with embodiment 1), and they are respectively charged in pET30a and the pET42a prokaryotic expression carrier.Then, be transformed in e. coli bl21 (DE3) expression strain, induce, can obtain fusion rotein by IPTG.With fusion rotein by His Trap purification column purification in addition, then, with the thrombin Xa factor respectively with CB-A β
42Albumen and A β
42Fragments of peptides cuts down, and purification in addition, after the lyophilizing, and-20 ℃ of preservations.
Embodiment 11.CB-A β
42Albumen and A β
42The detection of peptide based immunogens
Freeze dried CB-A β with embodiment 10 acquisitions
42Albumen and A β
42Peptide is dissolved in respectively in the phosphate buffer (PBS), under the situation of no adjuvant, and subcutaneous immune Balb/c mice, result's demonstration, after immunity one month, CB-A β
42The protein immunization group can produce the anti-beta amyloid antibody (antibody titer is 1: 10,000～20,000) of high titre, and A β
42The peptide immune group only produces low-down anti-beta amyloid antibody (antibody titer is 1: 200～500).Show CB-A β thus
42Albumen also can be as the medicine of prevention and treatment Alzheimer.
Claims (11)
1, a kind of genetic vaccine that is used for the treatment of and prevents Alzheimer, this vaccine contains the recombinant DNA molecules of the fusion rotein of coding choleratoxin B subunit and A β fragments of peptides, that effectively be connected with this recombinant DNA molecules and can be in eukaryotic cell the promoter of expressed fusion protein, and the targeting sequencing that is positioned at the terminal signal peptide of 5 ' end coding N-of described recombinant DNA molecules, wherein said A β fragments of peptides is A β
42
2, genetic vaccine as claimed in claim 1, wherein encode choleratoxin B subunit and A β fragments of peptides fusion rotein nucleotide sequence as shown in Figure 4, described promoter is the CMV promoter, and described targeting sequencing is the targeting sequencing of choleratoxin B subunit.
3, a kind of recombinant DNA molecules, the fusion rotein of its coding choleratoxin B subunit and A β fragments of peptides, described A β fragments of peptides is A β
42
4, recombinant DNA molecules as claimed in claim 3, it has nucleotide sequence as shown in Figure 4.
5, a kind of fusion rotein, it is held to the C end from N and is made up of choleratoxin B subunit, joint A β fragments of peptides and A β fragments of peptides, and described A β fragments of peptides is A β
42, the aminoacid sequence of described joint is glycine-proline-glycine-proline.
6, fusion rotein as claimed in claim 5, it has sequence shown in Figure 5.
7, a kind of recombinant viral vector, it comprises the recombinant DNA molecules of claim 3 or 4, and described viral vector is selected from adeno-associated virus, adenovirus, herpesvirus, retrovirus, vaccinia virus, fowlpox virus, sindbis virus or bird pox virus.
8, recombinant viral vector as claimed in claim 7, it is the adeno-associated virus that comprises the recombinant DNA molecules of claim 3 or 4.
9, a kind of pharmaceutical composition, it comprises the recombinant DNA molecules of claim 3 or 4 and suitable gene delivery carrier.
10, a kind of pharmaceutical composition, it comprises the fusion rotein and the materia medica acceptable carrier of claim 5 or 6.
11, the application of the fusion rotein of claim 3 or 4 recombinant DNA molecules or claim 5 or 6 in the medicine of preparation treatment and prevention Alzheimer.
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| PCT/CN2003/001019 WO2004050876A1 (en) | 2002-11-29 | 2003-12-01 | A recombinant adenovirus relating gene vaccine used to therapy and prevent alzheimers disease, and use thereof |
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|---|---|---|---|---|
| DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
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| WO2005014041A2 (en) * | 2003-07-24 | 2005-02-17 | Novartis Ag | Use of an amyloid beta dna vaccine for the treatment and/or prevention of amyloid diseases |
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| WO2010050585A1 (en) * | 2008-10-31 | 2010-05-06 | ディナベック株式会社 | Vector for treating alzheimer's disease |
| CN104744591B (en) | 2010-04-15 | 2022-09-27 | Abbvie德国有限责任两合公司 | Amyloid beta binding proteins |
| CN105348387B (en) | 2010-08-14 | 2020-08-25 | Abbvie 公司 | Amyloid beta binding proteins |
| CN103071160A (en) * | 2011-10-24 | 2013-05-01 | 四川百利药业有限责任公司 | Gene vaccine for Alzheimer's disease |
| KR20170040782A (en) * | 2014-05-16 | 2017-04-13 | 비트루비안 바이오메디칼, 인코포레이티드 | Diagnostic test and treatment/prevention of alzheimer's disease |
| CN107034198B (en) | 2015-07-15 | 2021-03-09 | 长春百克生物科技股份公司 | Chimeric norovirus P particle and preparation and application thereof |
| CN105597108A (en) * | 2015-12-30 | 2016-05-25 | 广州凯拓生物科技开发有限公司 | Construction and application of Complexin 1/2 overexpression recombinant vector |
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| CN1281366A (en) * | 1997-12-02 | 2001-01-24 | 神经实验室有限公司 | Prevention and treatment of amyloidogenic disease |
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| CA2449663A1 (en) * | 2001-06-07 | 2002-12-12 | Wyeth Holdings Corporation | Mutant forms of cholera holotoxin as an adjuvant |
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| CN1281366A (en) * | 1997-12-02 | 2001-01-24 | 神经实验室有限公司 | Prevention and treatment of amyloidogenic disease |
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| 霍乱毒素B亚基融合蛋白表达系统的构建. 曹诚等.生物化学杂志，第10卷第5期. 1994 * |
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| Publication number | Publication date |
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| AU2003289631A8 (en) | 2004-06-23 |
| WO2004050876A1 (en) | 2004-06-17 |
| CN1504240A (en) | 2004-06-16 |
| AU2003289631A1 (en) | 2004-06-23 |
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