CN101491683A - Development of new-generation gene-therapy immuno-therapy for alzheimer's disease - Google Patents
Development of new-generation gene-therapy immuno-therapy for alzheimer's disease Download PDFInfo
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Abstract
It is possible to realize gene therapy with immunotherapy for Alzheimer's disease with a sustaining effect compared to antibody investment and a higher antibody capacity compared to a single chain antibody. An integrated anti-[beta]-amyloid antibody or a fragmented carrier is expressed, where the carrier contains a promoter sequence, a coding sequence of an immunoglobulin heavy chain or its fragment of anti-[beta]-amyloid antibody, for coding a self-processing peptide sequence and coding a sequence of an immunoglobulin heavy chain of anti-[beta]-amyloid antibody. Prevention of Alzheimer's disease with the carrier as an active ingredient and therapeutic medication are included.
Description
Technical field
The present invention relates to a kind of gene therapy immunotherapy that can be used for Alzheimer's disease importing the carrier of the proteic antibody gene of directed against amyloid-beta or its modified form.
Background technology
The main pathological characters of human Alzheimer (AD) has senile plaque and nerve fiber to change, in case they further develop, then can be observed the minimizing and the brain atrophy of neurocyte.Well-known senile plaque is because the accumulation of the amyloid-beta (A β) that the receptor amyloid precursor protein (APP) from cell membrane cuts off out causes.In addition, in recent years, be not only insoluble A β, the neurocyte toxicity of solubility A beta peptide aggregation body also is identified, and has caused great concern (non-patent literature 1).At a kind of AD curative that Japan is given the ratification, be a kind of precursor of cholinesterase inhibitor, the nerve transmission that this medicine is covered the loss only suppresses symptom, but does not reach radical cure.
Therefore, reduce in order to reach that to cause the A β of AD basic reason be purpose, " A β vaccine therapy " (non-patent literature 2) that imagination relies on antibody to remove to a matter of self.This vaccine therapy is with the administration of adjuvant muscle synthetic A β 42 peptides.In calendar year 2001, the clinical trial of this vaccine (II phase) begins.The result has confirmed the senile plaque disappearance, confirms that tool has certain effect, and has also confirmed to have 6% patient that the side effect of meningoencephalitis has taken place but then, and 1 routine death is arranged.Because the appearance of this side effect, clinical trial is stopped.At present, the side effect of vaccine is considered to two reasons.One thinks that the A β antibody of administration has activated microgliacyte, and perhaps this is associated with meningoencephalitis.Another thinks owing to have strong immune activation effect in the contained adjuvant in the vaccine, and causes cellular immunity such as T cell, and the result react A β and APP reactivity Th1 type CD4 in some patients
+The T Premeabilisation of cells in brain, this fact perhaps relevant (non-patent literature 3) with meningitis.Therefore, the safe and vaccine that can reduce A β effectively of exploitation is desired.
Tamura etc. use through synthetic A β peptide mice immunized, and preparation produces the hybridoma (non-patent literature 4) at the antibody of each epi-position of A β.The antibody of hybridoma production is cut off with pepsin, remove the Fc zone relevant, be purified into F (ab ') 2 with activating microgliacyte.This anti-A β F (ab ') 2 antibody in the abdominal cavity that is administered into the AD model mice (Tg2576) that A β antibody excess expresses a week, are carried out the research of AD prevention and therapeutic effect.After using this antibody,, confirm that senile plaque disappears according to the immunostaining of mouse brain section.In addition, with sandwich ELISA method, A β quantitative results in the insoluble brain of solubility is determined that A β is owing to antibody reduces in the brain.According to above content, not by cytophagy, successfully removed A β by the Fc receptor.Yet the therapy by passive immunity carries out must constantly add antibody for lasting effect is arranged, and this is a difficult point of Tamura subject study.
In addition, it is reported that the adeno-associated virus that has the anti-A β light chain antibody of encoding is dropped in the AD model mice brain, and the deposition of amyloid reduces, even and also observe the expression (non-patent literature 5) of anti-A β light chain antibody after 1 year in administration.But, in general, the comparison of light chain antibody and complete antibody, the antibody ability is low.
[non-patent literature 1]
Rakez?Kayed?et?al，.Science(2003)300：486-489
[non-patent literature 2]
Dale?Schenk?et?al.，Nature(1999)400：173-177
[non-patent literature 3]
James?A.R.Nicoll?et?al.，Nature?med(2003)9：448-452
[non-patent literature 4]
Y.Tamura?et?al.，Neurobiology?of?Disease?20(2005)541-549
[non-patent literature 5]
K.Fukuchi?et?al.，Neurobiology?of?Disease?23(2006)502-511
Summary of the invention
Target of the present invention is to make the gene therapy and the immunization therapy of the Alzheimer of more lasting than antibody administration effect, higher than monoclonal antibody body antibody intensity become possibility.
Therefore, address the above problem, the present invention the first, is conceived to expect the adeno-associated virus (AAV) of long-term gene expression.Because AAV can gene expression stably in cell, even therefore single-dose also can expect to have secular effect.In addition, AAV is to cystic fibrosis, and hemophilia and other disease are carried out clinical research, and the Vectors in Gene Therapy very high as safety is widely used.In addition, owing to do not need adjuvant, can expect that viral vector perhaps can suppress Th1 type CD4
+The T cell activation.Secondly, note the serum type of AAV.According to the inventor all the year round about the real achievement of AAV study on the carrier, known the affinity difference of the gland relevant viral vector pair cell of different serotypes.The present inventor determines to use at the highest AAV 1 type carrier of muscle cell expression efficiency.
The inventor extracts mRNA and by reverse transcription, obtains anti-amyloid beta antibodies cDNA (Fig. 1) from producing the anti-amyloid beta antibodies hybridoma.In order to suppress the activity of microgliacyte, remove the CH3 zone from the complete antibody gene, preparation CH3 deficiency anti-amyloid beta antibodies gene.In addition, for the dividing a word with a hyphen at the end of a line property of antibody in brain of confirming to express endways, add histidine mark (Fig. 2) at the antibody gene C-terminal.On transport vehicle, gene is directed in cultured cell with these antibody clonings, and the expression of anti-amyloid beta antibodies and function confirm with Western blot method and ELISA method.The anti-amyloid beta antibodies gene of making is imported the AAV genome, and preparation has the AAV carrier (Fig. 1) of anti-amyloid beta antibodies.With the muscle administration that the AAV carrier is given mice, anti-amyloid beta antibodies energy and the combination of A β peptide have confirmed this point with the ELISA method in the blood that gives expression to.The present invention finishes by these understanding.
Main points of the present invention are as follows.
(1) the directed against amyloid-beta protein antibodies of The expressed or segmental carrier, this carrier contains promoter sequence, the heavy chain immunoglobulin of directed against amyloid-beta protein antibodies or segmental coded sequence, and the sequence of the light chain immunoglobulin of the sequence of the self-processed peptide of encoding and coding directed against amyloid-beta protein antibodies.
(2) also contain the polyA sequence (1) record carrier.
(3) also contain the ITR sequence (1) or (2) record carrier.
(4) be connected with the carrier of each record of (1)-(3) of processing protease enzyme action part at the 5 ' end of sequence of the self-processed peptide of coding.
(5) processing protease is the carrier of (4) record of furin.
(6) self-processed peptide is the carrier of each record of (1)-(5) of 2A for sign indicating number.
(7) sequence of the light chain immunoglobulin of the heavy chain immunoglobulin of coding directed against amyloid-beta protein antibodies or its fragments sequence and coding directed against amyloid-beta protein antibodies all contains the carrier of each record of (1)-(6) of secretory signal sequence.
(8) fragment of the heavy chain immunoglobulin of directed against amyloid-beta protein antibodies comprises Variable Area, contains the constant zone of part or does not contain (1) in constant zone carrier to (7) each record fully.
(9) contain the carrier of (8) record of expression CH3 deficiency antibody of removing the segmental coded sequence in CH3 zone from the heavy chain immunoglobulin of directed against amyloid-beta protein antibodies.
(10) carrier is the carrier of gland relevant viral vector (1)-(9) each record.
(11) adeno-associated virus is the carrier of 1 type (10) record.
(12) comprise (1)-carrier of (11) each record prevents and/or treats medicine as the Alzheimer's disease of effective ingredient.
The effect of invention
The present invention has developed treating and/or preventing property of the AD vaccine that adopts reorganization AAV carrier.This vaccine can be determined the effectiveness of animal pattern.In addition, the continuous expression of high in vivo antibody of tiring is possible.
Description of drawings
The experimentation of [Fig. 1] expression embodiment.
The antibody structure of producing among [Fig. 2] expression embodiment by reorganization AAV expression.
[Fig. 3] detects anti-amyloid beta antibodies by the 293T that imports antibody gene.
The H chain monomer of the IIA2 that discovery is purified into is positioned at about 50KDa, and L chain monomer is positioned near about 30KDa.Cytolysate and cell conditioned medium are all found H chain, L chain, if remove among the pW1HF2AL of CH3, the H chain belt is positioned at about 45KDa, and is littler than the molecular weight of matched group.Thereby, according to the expression that antibody also is described by the gene of having removed the CH3 zone.In the supernatant of cytolysate, do not find, but about 80KDa, confirmed band.This is predicted to be, H chain Furin2A L chain is expressed by the state that connects, and is subjected to the processing of self in the cell exocrine process, is secreted into cell after forming dimer.
The structure of the reorganization AAV that produces among [Fig. 4] expression embodiment
The administration of reorganization AAV to the Balb/C Mus and the plan of the blood sampling of mice are given in [Fig. 5] expression.
[Fig. 6] carried out the special IgG1 detection of antibodies of A β from the serum of the Balb/c that dropped into AAV HF2AL.Depend on the viral vector input amount, anti-amyloid beta antibodies concentration rises in the blood.3.0 * 10
11Anti-amyloid beta antibodies amount in the vg AAV HF2AL infected group, in administration 4 all bleeding from anus rises gradually, and peak reaches 939 μ g/mL.Afterwards, antibody concentration reduces lentamente, even but can also maintain 432 μ g/mL after the administration 24 weeks.In addition, 3.0 * 10
10In the vg HF2AL infected group, peak only reaches 222 μ g/mL, becomes 157 μ g/mL 24 weeks after 8 weeks.In 3.0 * 109vg HF2AL infected group, 24 week back peaks only reach 28 μ g/mL.
[Fig. 7] used the detection of the senile plaque of anti-amyloid beta antibodies
(result) by having used the immunostaining of the anti-A β of rabbit 1-40 polyclonal antibody, the anti-A β of rabbit 1-42 polyclonal antibody or IIA2, detected senile plaque near the Hippocampus cerebral cortex in the immunostaining of the brain section that uses 13 months big Tg2576.And, used AAVHF2AL to infect the supernatant of HEK293, by immunostaining, detected senile plaque at identical position.Thereby, can think that the antibody and the deutero-antibody of hybridoma that are given expression to by AAV have equal antigenic specificity.
[sequence table]
＜serial number 1 〉
The DNA sequence of the total length of the heavy chain immunoglobulin of serial number 1 expression mice.
＜serial number 2 〉
The aminoacid sequence of the total length of the immune heavy chain immunoglobulin of serial number 2 expression mices.
＜serial number 3 〉
Remove the DNA sequence in CH3 zone in the total length of the heavy chain immunoglobulin of serial number 3 expression mices.
＜serial number 4 〉
Remove the aminoacid sequence in CH3 zone in the total length of the heavy chain immunoglobulin of serial number 4 expression mices.
＜serial number 5 〉
The DNA sequence of the total length of the light chain immunoglobulin of serial number 5 expression mices.
＜serial number 6 〉
The aminoacid sequence of the total length of the light chain immunoglobulin of serial number 6 expression mices.
＜serial number 7 〉
The DNA sequence of the secretion signal of the heavy chain immunoglobulin of serial number 7 expression mices.
＜serial number 8 〉
The aminoacid sequence of the secretion signal of the heavy chain immunoglobulin of serial number 8 expression mices.
＜serial number 9 〉
The DNA sequence of the secretion signal of the light chain immunoglobulin of serial number 9 expression mices.
＜serial number 10 〉
The aminoacid sequence of the secretion signal of the light chain immunoglobulin of serial number 10 expression mices.
＜serial number 11 〉
The DNA sequence of the 2A in serial number 11 presentation code foot and mouth disease viruses source.
＜serial number 12 〉
The aminoacid sequence of the 2A in serial number 12 expression foot and mouth disease virus sources.
＜serial number 13 〉
The DNA sequence at serial number 13 expression Furin enzyme action positions.
The aminoacid sequence at expression Furin enzyme action position.RAKR
＜serial number 14 〉
The aminoacid sequence of serial number 14 expression people A β.
＜serial number 15～36 〉
The DNA sequence of the primer that serial number 15～36 expressions are used in an embodiment.
The specific embodiment
Following embodiment of the present invention is elaborated.
The invention provides the directed against amyloid-beta protein antibodies or the segmental carrier of The expressed, this carrier contains promoter sequence, heavy chain immunoglobulin or its fragments sequence of coding directed against amyloid-beta protein antibodies, the sequence of the light chain immunoglobulin of the sequence of the self-processed peptide of encoding and coding amyloid-beta antibody.
Insert the promoter sequence of carrier of the present invention, as long as the energy proteic antibody of The expressed directed against amyloid-beta or its fragment, any promoter sequence all is fine, and for example, can be the promoter that mammals is used.Can exemplify many application as CMV promoter and CAG promoter etc.In addition, also can be cell specificity promotor.
Insert heavy chain immunoglobulin or its segmental coded sequence of the directed against amyloid-beta protein antibodies of carrier of the present invention, it can be the sequence that contains the zone of the antibody combining site in the immunoglobulin heavy chain variable region of coding directed against amyloid-beta protein antibodies, can illustration the total length of coding heavy chain immunoglobulin, the Fab zone, by the zone in total length removal CH3 zone, the sequence of light chain antibody etc.For fear of the meningoencephalitis that causes because of the microgliacyte activation, preferably remove the fragments sequence of the heavy chain immunoglobulin in the coding Fc zone (all or part of) relevant with activating microgliacyte.An example of the total length of the heavy chain immunoglobulin of the directed against amyloid-beta protein antibodies of encoding murine is shown in serial number 1.Its aminoacid sequence is shown in serial number 2.In addition, (annotate: heavy chain immunoglobulin CH zone is divided into these three parts of CH1～CH3 to remove the CH3 zone from the heavy chain immunoglobulin total length of the directed against amyloid-beta protein antibodies of mice.The CH3 zone is positioned at the C-end of heavy chain.) an example of segmental coded sequence be shown in serial number 3.Its aminoacid sequence is shown in serial number 4.Total length by the heavy chain immunoglobulin of insertion directed against amyloid-beta protein antibodies in carrier of the present invention is removed the segmental coded sequence in CH3 zone, and the directed against amyloid-beta protein antibodies of CH3 zone deletion form is expressed.
The coded sequence of light chain immunoglobulin that inserts the directed against amyloid-beta protein antibodies of carrier of the present invention can be the sequence of total length of the immune globulin light chain of coding directed against amyloid-beta protein antibodies.An example of the sequence of the total length of the light chain immunoglobulin of the amyloid-beta antibody of encoding murine is shown in serial number 5.Its aminoacid sequence is shown in serial number 6.
The sequence of the light chain immunoglobulin of the heavy chain immunoglobulin of optimized encoding directed against amyloid-beta protein antibodies or its fragments sequence and coding directed against amyloid-beta protein antibodies all contains secretory signal sequence.An example of the directed against amyloid-beta protein antibodies heavy chain immunoglobulin secretory signal sequence of mice is shown in serial number 7.Its aminoacid sequence is shown in serial number 8.In addition, an example of the secretory signal sequence of the directed against amyloid-beta protein antibodies light chain immunoglobulin of mice is shown in serial number 9.Its aminoacid sequence is shown in serial number 10.
The sequence of the heavy chain immunoglobulin of coding directed against amyloid-beta protein antibodies or its fragments sequence and coding light chain can follow these steps to preparation.At first, preparation produces the hybridoma of directed against amyloid-beta protein antibodies, extracts total RNA from this hybridoma.With commercial reagents box (SMART
TMRACE cDNA Amplification Kit terminal gene amplification kit (Clontech.Mountain View.USA) preparation contains the cDNA library of full length antibody gene, utilize and be added on mRNA3 ' the few dT of the complementary primer of polyA and and the complementary primer of mRNA5 ' carry out the gene fragment amplification (RACE method) of antibody by PCR.Like this, can obtain the to encode sequence of total length of heavy chain immunoglobulin and light chain.In addition, in order to obtain the heavy chain immunoglobulin fragments sequence, utilize 5 of desired zone ' terminal complementary primer and with 3 ' terminal complementary primer, can carry out the antibody gene fragment amplification by PCR.The sequence of the coding heavy chain immunoglobulin that obtains in such a way or its fragments sequence and coding light chain immunoglobulin also contains conventional secretion sequence.The forecasting software of excretory signal sequence available signal sequence (http://bp.nuap.nagoya-u.ac.jp/sosui/) is retrieved.
The heavy chain immunoglobulin of directed against amyloid-beta protein antibodies or its fragment can be any classes among IgG, IgM, IgD, IgE or the IgA, but preferred IgG.In addition, the antibody by vector expression of the present invention or its fragment be from the mankind, mice, and rabbit, sheep and other animals all are fine, but also can be the chimeric antibodys that comes from animal beyond people and the people, humanized antibody.The preparation method of chimeric antibody and humanized antibody is many known.
Inserting the sequence of the coding oneself processed peptide of carrier of the present invention, as long as can The expressed directed against amyloid-beta protein antibodies or its fragment, can be the sequence of any self-processed peptide of coding, for example, and the sequence illustration of available code 2A.2A expresses more employing internal ribosome entry site (IRES) simultaneously for two kinds of genes usually.Yet, by utilizing the gene expression of IRES, be positioned at the gene in IRES downstream, lower than the expression of gene level that is positioned at the upstream.
Because there are two complete forms of heavy chain light chain in vivo in IgG antibody, when antibody gene was expressed, chain H and L chain must be that equivalent is expressed in cell.In addition, by configuration H chain and L chain before and after the 2A sequence, can make the equivalent antibody gene express (Jianmin Fang eta1., nature biotechnology (2005) 23:584-589) simultaneously.So-called 2A is the self-processed peptide from foot and mouth disease virus (FMDV).Be known that in virus to exist between the P1 and P2 protein, participate in producing the generation of maturase P1 and P2.After P1 and P2 express as 1 ORF simultaneously, in the distolateral generation oneself processing of the C-of 2A type.(details is not clear).Various 2A sequences and 2A sample sequence are known, and the 2A sequence of foot and mouth disease virus (foot and mouth diseasevirus) is shown in Figure 11.Its aminoacid sequence is shown in serial number 12.
Adjacent with the coded sequence of self-processing polypeptides, it is relatively good to connect processing protease enzyme action part.Processing protease enzyme action part preferably is connected 5 ' end of the coded sequence of self-processing polypeptides.As long as directed against amyloid-beta protein antibodies or its fragment that processing protease enzyme action position can The expressed, any processing protease enzyme action position of then encoding can, for example, can exemplify the furin restriction enzyme site.Furin is that a member in the amino acid whose protease of the base family is cut off in identification, and the part is present in the Golgi body.The DNA sequence at Furin enzyme action position is shown in serial number 13.
Carrier of the present invention, it is reasonable inserting a polyA sequence again.Rely on and insert the polyA sequence, can make the mRNA stabilisation of transcribing.
Carrier of the present invention, by under the control of controlling elements such as promoter, the mode that complete directed against amyloid-beta protein antibodies or its fragment are expressed, the gene of the proteic antibody of insertion directed against amyloid-beta in carrier.
Carrier is the nucleic acid molecules that is transported to the host as the different DNA of target, plasmid, and cosmid, phage, phasmid, virus, medium such as YAC and BAC are as carrier.Carrier is used for the making in library, and clone's enforcement is expressed the DNA product that inserts in host cell.According to DNA size of inserting and the purpose of inserting, can select suitable media.For the directed against amyloid-beta protein antibodies or the fragment of The expressed, preferred plasmid (using) and virus (gene therapy is used) at host cell expression.
Using plasmid vector as expressing, can be to have mammal to use promoter, multiple clone site, and polyadenylic acid, the carrier of ITR can exemplify pW1 etc.
When in gene therapy, using carrier, the preferred virus carrier.As viral vector adeno-associated virus (AAV), retroviral vector, adenovirus vector, slow virus carrier, sendai virus vector etc. are arranged.
AAV is to the vesicle cystic fibrosis, and diseases such as hemophilia are being carried out clinical research, is extensively used as safe gene therapy vector.In addition, this viral vector, because do not need adjuvant, therefore perhaps expectation can suppress Th1 type CD4
+The activation of T cell energy.AAV is not considered to the virus of pathogenic, lacks independently multiplication capacity, duplicates the miscellaneous function that depends on adenovirus and herpesvirus etc.Known, for the miscellaneous function of adenovirus, E1A, E1B, E2A, E4, the VA gene is essential.The known AAV that can infect the mankind has the serotype of 1 to 9 type.2,3,5 and 9 types all come from the mankind, and 2 types have deep research, are fully utilized as carrier.The inventor has known the affinity difference (Xin, K-Q.et al., J.Virol.80:11899-910,2006) of the AAV carrier pair cell of different serotypes.In the following embodiments, use the highest AAV1 type carrier of expression in muscle cell.The AAV genome is the DNA with strand of about 5kB, and only about half of virion has the genome of (+) chain DNA, and all the other have (-) DNA chain.The only about half of of 5 ' end is the zone that is called " rep ", and coding is with virus replication and transcribe relevant Rep protein.Rep coding is from the big Rep (Rep78 and Rep68) of p5 promoter transcriptional start and translation and transcribe four albumen of the little Rep of translation (Rep52 and Rep40) allusion quotation from the p19 promoter.Half is to be called as " cap " zone to 3 ' end approximately, coding structure protein VP1, these three capsid proteins of VP2 and VP3.The mRNA of capsid protein is transcribed by the p40 promoter.AAV genomic two terminal ITR (reverse terminal repetition) hairpin structures that are called as 145 nucleotide that exist it is believed that this ITR zone is very important when host cell chromosome is integrated.
In order to make reorganization AAV carrier, can carry out transfection to transform HEK293 cell (cell) by calcium phosphate method through the human embryos nephridial tissue source that the E1AE1B of adenovirus transforms.For example, preparation 1) is retained in the ITR at the two ends of wild type AAV, insert the purpose different DNA betwixt (among the present invention, promoter sequence, heavy chain immunoglobulin or its fragments sequence of coding directed against amyloid-beta protein antibodies, processing protease enzyme action position, the sequence of self-processed peptide of encoding and the sequence of coding directed against amyloid-beta protein antibodies and light chain immunoglobulin) plasmid (AAV plasmid vector), 2) have coding virus replication and virion and form necessary virus protein (Rep, the plasmid of sequence Cap) (AAV helper plasmid), 3) has the necessary gene region (E1A that bears the adenovirus assosting effect of propagation of AAV, E1B, E2A, VA, E4 or f6) in, the plasmid (adenovirus helper plasmid) in the zone beyond E1A that the HEK293 cell has and the E1B, by transfection these three plasmids are imported in the HEK293 cell, produce reorganization AAV carrier.Commercial reagents box (for example, the AAV helper-free system of STRATAGENE company) is used in the making of reorganization AAV like this, just can finish.
Carrier of the present invention, useful as prevention and the therapeutic vaccine of AD.Therefore, the present invention also provide the directed against amyloid-beta protein antibodies that contains the handlebar The expressed or its segmental carrier as active component Alzheimer (AD) prevention and/curative.
The proteic antibody of the directed against amyloid-beta of The expressed or its segmental carrier can for example be dissolved in buffer such as PBS, normal saline, and sterilized water etc. are as required by after the filter-sterilized such as filter, by injecting experimenter's administration.In addition, in solution, can add additive (for example, disactivation agent, antiseptic, adjuvant, emulsifying agent etc.).Yet, be that directed against amyloid-beta protein antibodies or its segmental carrier of The expressed is under the situation of viral vector, do not need to add adjuvant.The proteic antibody of the directed against amyloid-beta of The expressed or its segmental carrier can veins, muscle, and the abdominal cavity, subcutaneous, mode administrations such as Intradermal also can be passed through nose, mouthful administration.
The directed against amyloid-beta protein antibodies of The expressed or the dosage of its segmental carrier, administration number of times and frequency are according to tester's symptom, age, body weight, administering mode, the difference of administration form etc. and difference, the situation of the AAV carrier of for example recombinating is that each adult is at least one time 10 per capita usually
13-15The dosage of vg can be by the lasting frequency administration of desirable effect.
[embodiment]
Explain the present invention below by embodiment, the present invention is not limited to these embodiment.
[embodiment 1]
Material and method
Experimentation is represented with Fig. 1.
(1) preparation of the hybridoma of generation anti-amyloid beta antibodies
People A β peptide (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) (serial number 14), and the 1430A peptide synthesizer of use automatization (AppliedBiosystems, CA, USA) synthetic.Peptide with complete Freund ' adjuvant emulsion, is carried out immunity by every subcutaneous subcutaneous administration 100ug of Balb/c mice.After 2 weeks of administration and incomplete Freund adjuvant equivalent supplementary immunization A β peptide together.In addition, individually dosed 100ug peptide after three week of administration.Win mouse boosting cell, prepare hybridoma by following method.
(2) separation of antibody gene
In order to obtain gene, carry out the separation of mRNA to the H chain L chain of A β antibody.From the hybridoma IIA2 that anti-amyloid beta antibodies produces, use TRIzol reagent (GIBCO BRL.N.Y.USA) to extract total RNA.Use BD SMART
TM5 ' 3 ' ends of RACE cDNA amplification kit (Clontech.Mountain View.USA), difference RACEH chain L chain, preparation cDNA.Order is undertaken by service manual.The H chain (5 ' CCC AAG CTT TTTACC AGGAGA GTG GGA GA3 ') (serial number 15) of the GSP1 of the part of the antisense of use conduct coding 5 '-RACE (RIKAKEN.Nagoya.Japan), L chain (5 ' ATAAGAATGCGG CCG CA G TCG ACG CTA ACA CTC ATT CCT GTT GA 3 ') (serial number 16) (RIKAKEN), as the coding 3 '-RACE have justice a part GSP1 H chain (5CGG GGT ACC ATG GGC AGG CTT ACT TCT TC 3 ') (serial number 17) (RIKAKEN), L chain (5 ' CCG GAA TTC ATG GAG ACA GAC ACACTC CT 3 ') (serial number 18) (RIKAKEN) obtains 5 ' 3 ' fragments of H chain L chain.Underscore partly is a restriction endonuclease recognition sequence.
1) H chain
For the ORF of the H chain that increases, prepare with the complementary primer of H chain [justice (5 ' CGG GGT ACC ATG GGC AGG CTT ACT TCT TC 3 ') (serial number 19) antisense (5 ' CCC AAG CTT TTT ACC AGG AGA GTG GGA GA 3 ') is arranged] (serial number 20) that contains the Restriction Enzyme site (RIKAKEN).Antisense primer is removed the terminal termination codon of H chain 3 ', designs.Use these primers, as template, (PCR) carries out the amplification of H chain by polymerase chain reaction with the H chain fragment of making by the RACE method.
2) L chain
ORF for the L chain that increases, preparation and 5 ' 3 ' the terminal complementary primers that contain the L chain in Restriction Enzyme site { justice (5 ' CCG GAA TTC ATG GAG ACA GAC ACACTC CT 3 ') (serial number 21) antisense (5 ' ATA AGA ATG CGG CCG CA G TCGACG CTA ACA CTC ATT CCT GTT GA 3 ') is arranged } (serial number 22) (RIKAKEN), the L chain fragment of producing by the RACE method is as template, with 1) amplification similarly.
(3) making of Furin 2A gene
Make the few DNA (5 ' CGC GCC AAG CGC GCC CCC CGT GAA GCA GAC CCT GAACTT CGA CCT GCT GAA GCT GGC CGG CGA CGT GGA GTC CAACCC CGG CCC C 3 ') (serial number 23) of the DNA sequence of the self-processed peptide 2A that connects Furin and foot and mouth disease virus.Make with the complementary primer of F2A [justice (5 ' CTA GCT AGC CGC GCC AAG CGC GCC CCC GT3 ') (serial number 24) antisense (5 ' CCG CTC GAG GGG GCC GGG GTT GGACTC CA3 ') (serial number 25) is arranged] that contains the Restriction Enzyme site (RIKAKEN).As template, use few DNA these primers to carry out the amplification of F2A by PCR.Making { has justice (5 ' CCC AAG CTT CGC GCC AAG CGC GCC CCC GT3 ') (serial number 26) antisense (5 ' CCG GAA TTC GGG GCC GGG GTT GGA CTC CA3 ') (serial number 27) } (RIKAKEN) with 5 ', 3 ' terminal complementary primer and the H chain L chain Furin 2A that increases equally.
(4) making of plasmid
Carry out Restriction Enzyme with HindIII and EcoRI and handle the Furin 2A dna fragmentation that (2) are produced, similarly be directed in HindIII and the EcoRI site of pBluescript II KS (-) phagemid.This plasmid as pBlue F2A.Use KpnI, the HindIII Restriction Enzyme is handled the H chain fragment of producing in (2), is directed in the KpnI of the F2A upstream region of gene that is positioned at pBlue F2A, the HindIII site.This plasmid as pBlue HF2A.And, use EcoRI, the NotI Restriction Enzyme is handled the L chain fragment that produce (2), is directed in the EcoRI in the F2A gene downstream of pBlue HF2A, the NotI site.And, this plasmid as p Blue HF2AL.
(5) affirmation of base sequence
The plasmid that (4) are produced, use be specific to pBluescript HF2AL gene insertion portion 5 ' side T3 zone primer (5 ' AAT TAA CCC TCA CTA AAG GG3 ') (serial number 28), be specific near the sequence the H chain 350bp primer (5 ' TCG ACT TGGTAG GAG GTA TG 3 ') (serial number 29), be specific near the primer (5 ' GGT GGA ATG GTG TAC ACC TG 3 ') (serial number 30) of the sequence the 1100bp, confirm the base sequence of H chain, F2A.And, at the L end of the chain, use and the regional special primer (5 ' TAA TAC GAC TCA CTATAG GG 3 ') (serial number 31) of 3 ' side T7 of the HF2AL gene insertion portion of p Bluescript, with special primer (5 ' GTA AAACGA CGG the CCA 3 ') sequence 32 in M13-20 zone), the primer special (5 ' CAG CAC AGT TGG GAG ATT CC 3 ') (sequence 33) with near the assortment the L chain 340bp, with near the sequence-specific primer (5 ' CCG TTT GAT TTC CAG TTT GG 3 ') (sequence 34) the 400bp, determine the L chain, the base sequence of F2A.Use ABI PRISM
TM310 (ABI) measure plasmid DNA, determine the base sequence of H chain L chain.
(6) sub-clone of antibody gene
In order to confirm the expression of antibody gene in cultured cell, the pBlueHF2AL that produces with (4) is incorporated into expression vector pW1 to antibody gene.{ H. has justice (5 ' ATA AGA ATG CGG CCG CA G TCG ACA ATG GGCAGG CTT ACT TC3 ') (serial number 35) with H chain 5 ' terminal complementary primer in preparation.Use this primer and carry out, carry out the segmental amplification of the H chain Furin a series of ORFDNA of 2AL chain with L chain 3 ' terminal complementary primer.With this dna fragmentation with the NotI Restriction Enzyme handle, the NotI site of same importing pWI, make pW1HF2AL.
(7) change of antibody gene
For the antibody of confirming to give expression to endways identifies antibody to dividing a word with a hyphen at the end of a line property, the usefulness histidine protein labelling of brain.The complementary primer of L chain of histidine gene has been added in preparation at 3 ' end.Use this primer and with the terminal complementary primer of H chain 5 ', carry out the amplification of a series of ORF dna fragmentation of H chain Furin2AL chain histidine protein.This dna fragmentation is handled with the NotI Restriction Enzyme, similarly imported the NotI site of pWI, form pW1HF2AL His.
Allow the minimizing of A β in the brain as purpose not causing receptor-mediated phagocytosis by Fc.In order to suppress the activity of microgliacyte, made the gene of having removed the CH3 deletion form antibody in CH3 zone from the antibody gene of total length.Preparation is with 3 of H chain CH2 zone ' the terminal complementary primer of making [antisense (5 ' CCC AAG CTT GCC TTT GGT TTT GGA GAT GG 3 ') (serial number 36)] (RIKAKEN), use this primer and with H chain 5 ' terminal complementary primer, carry out the amplification of the ORF dna fragmentation of H chain.This dna fragmentation is carried out Restriction Enzyme with SalIHindIII handle, be replaced by the total length H chain of pWI HFL.This plasmid as pW1HF2AL deleted CH3.
And the supposition mode of clicking in the CH2CH3 zone of antibody is carried out.The DNA base sequence of H chain is converted to aminoacid sequence.The homology search BLAST (http://au.expasy.or g/tools/blast/) of use on the Swiss-Prot of amino acid sequence database, there are the sequence of homology in retrieval and the H chain amino acid sequence of making.Therefore, select the high sequence (accession number: P01868) of sequence homology of the mice IgG1 identical with the antibody of making.Because in the sequence among the data base by the high-caliber explanation of proteinic function, regional structure etc., the therefore CH2CH3 zone of the antibody of producing according to the information prediction of P01868 etc.
(8)Western?blot
The pW1HF2AL, the pW1HF2AL His that have made with (6) (7) and as the pW1 of negative control with LipofectamineTM2000 (Invitr ogen) transfection 293T cell.With behind clean 1 time of the cell usefulness PBS (-), culture medium is changed to serum-free behind the 24 Xiao Time.Then, in the time of 48 hours, reclaim cell, carry out centrifugalize.As the cell conditioned medium sample, by in cell mass, adding the molten born of the same parents' buffer of NP-40, minute make cell solvable on ice with this supernatant in leaving standstill.The centrifugalize again of sample after leaving standstill, this supernatant is as cytolysate.In the cell conditioned medium cytolysate, add 2 * SDS respectively, heated 10 minutes, as the western sample.Use this sample the 12%Bis-Tris polyacrylamide gel to carry out electrophoresis, move at Immobilon
TMFilm.In blocking-up buffer [5% skimmed milk, PBS (-)], 4 ℃ shook 12 hours, and blocked film.Then, with film PBST[PBS (-), 0.1%Tween 20] clean.In order to detect H chain L chain, make it respectively and Anti mouse IgG1 conjugated horseradishperoxidase (Southern Biotechnology Asociates as constitutional antibody, Alabama, USA), HRPconjugated anti mouse kappa light chain antibody (BETHYLlaboratories.Montgmery.USA) reaction, clean 4 times with PBST.And make it on film and Immobilon
TM(USA) reaction is at Hyperfilm for MILLIPORE, MA for Western chemiluminescent HRP Substrate
TM(Amersham.Buckinghamshi re.UK) be sense down.The results are shown in Fig. 3.
The making of pW1LacZ
ORF for the LacZ that increases, what preparation comprised the Restriction Enzyme site terminally makes complementary primers { sense (5 ' ACG CGT CGA CAC TAT CCC GAACTT ACT 3 ') antisense (5 ' CGC GGA TCC ATG CGG CTG ATG TTG AAC TG3 ') } (RIKAKEN) with 5 ' 3 ' of LacZ, pSV-β-Galatsidase control vector (promega, W isconsin, USA), increase by PCR as template.
The LacZ dna fragmentation is carried out Restriction Enzyme with SalI and BamHI handle, be directed in SalI and the B amHI site of pW1, make pW1LacZ.
(9) making of reorganization AAV carrier
Use Calcium phosphate transfection kit (invitrogent), the pW1HF2AL that will produce with (6) (7) by calcium phosphate method, pW1HF2AL His, remove the pW1HF2AL of CH3, pW1lacZ, the AAV helper plasmid of gene that has the necessary virus protein of formation of virus replication and particle, have the gene E2AVAE4 of the necessary adenovirus of propagation of AAV or 6 adenovirus helper plasmid simultaneously gene be directed in HEK293.Then, cell is left standstill 72 hours.With this cell.At dry ice be set in 37 ℃ the couveuse, intracellular recombinant virus is reclaimed in freezing repeatedly dissolving.Again this viral solution with CsCl ultracentrifugation purified virus (AAV HF2AL, the AAV HF2AL that has deleted CH3, AAV HF2AL His, AAV LacZ).The structure of the reorganization AAV carrier of producing is shown in Fig. 4.Fig. 4 represents AAV HF2AL successively from last, has deleted the AAV HF2AL of CH3, the structure of AAV HF2AL His.
(10) administration of BALB/c Mus
To 2 months BALB/c male wister rat, the recombinant adeno-associated virus (AAV) of administration (9).AAV HF2AL respectively with every 3.0 * 10
11Vg, 3.0 * 10
10Vg, 3.0 * 10
9Vg carries out intramuscular injection, and each organizes 5 (Fig. 5).AAV LacZ presses every 1.0 * 10 as negative control
11Vg is to 5 administrations.The intramuscular administration reaches 0.4% at dorsal part subcutaneous administration (three are total to エ one Le medicine .Tokyo.Japan) in ketamine by final concentration and adds the solution that xylazine solution (バ ィ ェ Le メ デ ィ カ Le .Tokyo.Japan) forms, and administration is carried out under anesthesia.
(11) enzyme-linked immunosorbent adsorption test (ELISA)
After the administration, 3 all 4 weeks of 2 weeks in 1 week are after handling with heparin in later per 4 weeks, with capillary tube (Drummond.Broomall.USA) blood sampling.The blood of gathering 4 ℃ leave standstill 16 hours after, centrifugalize is reclaimed supernatant, the serum production sample.Carry out " ELISA " with this serum.On 96 hole microtitration plates (Nunc.N.Y.USA), with carbonate buffer solution (0.1M NaHCO
3) dissolved A β 1-13 peptide coating, left standstill 16 hours in 4 ℃.Then, add blocking-up buffer { 1% bovine serum albumin (BSA) (SIGMA), 0.05%Tween 20, PBS (-) }, 37 ℃ of blocking-up 2 hours down.After the blocking-up, clean 3 times, serum with the dilution of blocking-up buffer, is added on the plate with PBST.Plate was left standstill under 37 ℃ 2 hours.After leaving standstill, clean 3 times, add the horseradish peroxidase that combines anti-mice IgG1 (Southern Biotechnology Asociates) again, 37 ℃, left standstill 2 hours with the dilution of blocking-up buffer with PBST.After leaving standstill, clean 3 times, add color development liquid [1Tab/10mL o-phenylen dehydrochiolide (OPD) (Wako), 0.1M citric acid, 1 μ L/mL H with PBST
2O
2] color development.R.T., leave standstill 30 minutes after, add 1N H
2SO
4Stop color development.After stopping, measuring O.D.405nm with Benchmark Plus (BIO-RAD.California.USA).The result as shown in Figure 6.
(12) preparation of brain sample
Under anesthesia, cut out the brain of 13 months big Tg2576.The brain of extraction is immersed the formalin of 10% neutral buffered, and (Sigma, MO USA), leave standstill 48 hours fixing organizations.
(13) immunohistochemistry of the brain section of use Tg2576 mice
With xylene ethanol the paraffin section of Tg2576 is sloughed paraffin.(Wako, Osaka Japan), heat activation antigen in baking oven to add 100% formic acid.Reuse 0.3%H
2O
2Suppress the endogenous Peroxidase activity, (Vector Laboratories, CA USA) prevent the non-specific bond of antibody with 10%Normal Goat Searum.As 1 antibody, use the anti-A β of rabbit 1-40 polyclonal antibody, the anti-A β of rabbit 1-42 polyclonal antibody or IIA2, AAV HF2AL infects the supernatant of HEK293.Add 2 times antibody envision
+Dual Link SystemPeroxidase (Dako,, Denmark), usefulness Dako Envision+ reagent/HRP (DAB) (Dako,, Denmark) color development, logical hematoxylin carries out nuclear staining.The result as shown in Figure 7.
The possibility of industrial utilization
Carrier of the present invention can be prevented and/or the treatment vaccine as giving of Alzheimer's disease Utilize.
SEQUENCE?LISTING
＜110〉Haizheng Medicine Stock Co., Ltd., Zhejiang Prov
＜120〉to the exploitation of the gene therapy immunotherapy of new generation of Alzheimer's disease
<130>FP1F090006ZX/CNZJHZ/MY
<150>JP2008-012393
<151>2008-01-23
<160>36
<170>PatentIn?version?3.1
<210>1
<211>1329
<212>DNA
＜213〉mice (Mus musculus)
<400>1
aaagagtctg?gccctgggat?attgcagccc?tcccagaccc?tcagtctgac?ttgttctttc 60
tctgggtttt?cactgagcac?ttctggtatg?ggtgttggct?ggattcgtca?gccatcaggg 120
aagggtctag?agtggctggc?acacgtttgg?tgggatgatc?tcaagcgcta?taacccagcc 180
ctgaagagcc?gactgactat?ctccaaggat?acctccagca?gtcagatatt?tctcaggatc 240
gccagtgtgg?acactgcaga?tactgccaca?tactactgtg?ctcgacttgg?taggaggtat 300
ggtaaccctc?cctttgacta?ctggggccaa?ggcaccactc?tcacagtctc?ctcagccaaa 360
acgacacccc?catctgtcta?tccactggcc?cctggatctg?ctgcccaaac?taactccatg 420
gtgaccctgg?gatgcctggt?caagggctat?ttccctgagc?cagtgacagt?gacctggaac 480
tctggatccc?tgtccagcgg?tgtgcacacc?ttcccagctg?tcctgcagtc?tgacctctac 540
actctgagca?gctcagtgac?tgtcccctcc?agcacctggc?ccagcgagac?cgtcacctgc 600
aacgttgccc?acccggccag?cagcaccaag?gtggacaaga?aaattgtgcc?cagggattgt 660
ggttgtaagc?cttgcatatg?tacagtccca?gaagtatcat?ctgtcttcat?cttcccccca 720
aagcccaagg?atgtgctcac?cattactctg?actcctaagg?tcacgtgtgt?tgtggtagac 780
atcagcaagg?atgatcccga?ggtccagttc?agctggtttg?tagatgatgt?ggaggtgcac 840
acagctcaga?cgcaaccccg?ggaggagcag?ttcaacagca?ctttccgctc?agtcagtgaa 900
cttcccatca?tgcaccagga?ctggctcaat?ggcaaggagt?tcaaatgcag?ggtcaacagt 960
gcagctttcc?ctgcccccat?cgagaaaacc?atctccaaaa?ccaaaggcag?accgaaggct 1020
ccacaggtgt?acaccattcc?acctcccaag?gagcagatgg?ccaaggataa?agtcagtctg 1080
acctgcatga?taacagactt?cttccctgaa?gacattactg?tggagtggca?gtggaatggg 1140
cagccagcgg?agaactacaa?gaacactcag?cccatcatgg?acacagatgg?ctcttacttc 1200
gtctacagca?agctcaatgt?gcagaagagc?aactgggagg?caggaaatac?tttcacctgc 1260
tctgtgttac?atgagggcct?gcacaaccac?catactgaga?agagcctctc?ccactctcct 1320
ggtaaatga 1329
<210>2
<211>442
<212>PRT
＜213〉mice (Mus musculus)
<400>2
Lys?Glu?Ser?Gly?Pro?Gly?Ile?Leu?Gln?Pro?Ser?Gln?Thr?Leu?Ser?Leu
1 5 10 15
Thr?Cys?Ser?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?Gly?Met?Gly?Val
20 25 30
Gly?Trp?Ile?Arg?Gln?Pro?Ser?Gly?Lys?Gly?Leu?Glu?Trp?Leu?Ala?His
35 40 45
Val?Trp?Trp?Asp?Asp?Leu?Lys?Arg?Tyr?Asn?Pro?Ala?Leu?Lys?Ser?Arg
50 55 60
Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Ser?Ser?Gln?Ile?Phe?Leu?Arg?Ile
65 70 75 80
Ala?Ser?Val?Asp?Thr?Ala?Asp?Thr?Ala?Thr?Tyr?Tyr?Cys?Ala?Arg?Leu
85 90 95
Gly?Arg?Arg?Tyr?Gly?Asn?Pro?Pro?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr
100 105 110
Thr?Leu?Thr?Val?Ser?Ser?Ala?Lys?Thr?Thr?Pro?Pro?Ser?Val?Tyr?Pro
115 120 125
Leu?Ala?Pro?Gly?Ser?Ala?Ala?Gln?Thr?Asn?Ser?Met?Val?Thr?Leu?Gly
130 135 140
Cys?Leu?Val?Lys?Gly?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Thr?Trp?Asn
145 150 155 160
Ser?Gly?Ser?Leu?Ser?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln
165 170 175
Ser?Asp?Leu?Tyr?Thr?Leu?Ser?Ser?Ser?Val?Thr?Val?Pro?Ser?Ser?Thr
180 185 190
Trp?Pro?Ser?Glu?Thr?Val?Thr?Cys?Asn?Val?Ala?His?Pro?Ala?Ser?Ser
195 200 205
Thr?Lys?Val?Asp?Lys?Lys?Ile?Val?Pro?Arg?Asp?Cys?Gly?Cys?Lys?Pro
210 215 220
Cys?Ile?Cys?Thr?Val?Pro?Glu?Val?Ser?Ser?Val?Phe?Ile?Phe?Pro?Pro
225 230 235 240
Lys?Pro?Lys?Asp?Val?Leu?Thr?Ile?Thr?Leu?Thr?Pro?Lys?Val?Thr?Cys
245 250 255
Val?Val?Val?Asp?Ile?Ser?Lys?Asp?Asp?Pro?Glu?Val?Gln?Phe?Ser?Trp
260 265 270
Phe?Val?Asp?Asp?Val?Glu?Val?His?Thr?Ala?Gln?Thr?Gln?Pro?Arg?Glu
275 280 285
Glu?Gln?Phe?Asn?Ser?Thr?Phe?Arg?Ser?Val?Ser?Glu?Leu?Pro?Ile?Met
290 295 300
His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Phe?Lys?Cys?Arg?Val?Asn?Ser
305 310 315 320
Ala?Ala?Phe?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Thr?Lys?Gly
325 330 335
Arg?Pro?Lys?Ala?Pro?Gln?Val?Tyr?Thr?Ile?Pro?Pro?Pro?Lys?Glu?Gln
340 345 350
Met?Ala?Lys?Asp?Lys?Val?Ser?Leu?Thr?Cys?Met?Ile?Thr?Asp?Phe?Phe
355 360 365
Pro?Glu?Asp?Ile?Thr?Val?Glu?Trp?Gln?Trp?Asn?Gly?Gln?Pro?Ala?Glu
370 375 380
Asn?Tyr?Lys?Asn?Thr?Gln?Pro?Ile?Met?Asp?Thr?Asp?Gly?Ser?Tyr?Phe
385 390 395 400
Val?Tyr?Ser?Lys?Leu?Asn?Val?Gln?Lys?Ser?Asn?Trp?Glu?Ala?Gly?Asn
405 410 415
Thr?Phe?Thr?Cys?Ser?Val?Leu?His?Glu?Gly?Leu?His?Asn?His?His?Thr
420 425 430
Glu?Lys?Ser?Leu?Ser?His?Ser?Pro?Gly?Lys
435 440
<210>3
<211>1008
<212>DNA
＜213〉mice (Mus musculus)
<400>3
aaagagtctg?gccctgggat?attgcagccc?tcccagaccc?tcagtctgac?ttgttctttc 60
tctgggtttt?cactgagcac?ttctggtatg?ggtgttggct?ggattcgtca?gccatcaggg 120
aagggtctag?agtggctggc?acacgtttgg?tgggatgatc?tcaagcgcta?taacccagcc 180
ctgaagagcc?gactgactat?ctccaaggat?acctccagca?gtcagatatt?tctcaggatc 240
gccagtgtgg?acactgcaga?tactgccaca?tactactgtg?ctcgacttgg?taggaggtat 300
ggtaaccctc?cctttgacta?ctggggccaa?ggcaccactc?tcacagtctc?ctcagccaaa 360
acgacacccc?catctgtcta?tccactggcc?cctggatctg?ctgcccaaac?taactccatg 420
gtgaccctgg?gatgcctggt?caagggctat?ttccctgagc?cagtgacagt?gacctggaac 480
tctggatccc?tgtccagcgg?tgtgcacacc?ttcccagctg?tcctgcagtc?tgacctctac 540
actctgagca?gctcagtgac?tgtcccctcc?agcacctggc?ccagcgagac?cgtcacctgc 600
aacgttgccc?acccggccag?cagcaccaag?gtggacaaga?aaattgtgcc?cagggattgt 660
ggttgtaagc?cttgcatatg?tacagtccca?gaagtatcat?ctgtcttcat?cttcccccca 720
aagcccaagg?atgtgctcac?cattactctg?actcctaagg?tcacgtgtgt?tgtggtagac 780
atcagcaagg?atgatcccga?ggtccagttc?agctggtttg?tagatgatgt?ggaggtgcac 840
acagctcaga?cgcaaccccg?ggaggagcag?ttcaacagca?ctttccgctc?agtcagtgaa 900
cttcccatca?tgcaccagga?ctggctcaat?ggcaaggagt?tcaaatgcag?ggtcaacagt 960
gcagctttcc?ctgcccccat?cgagaaaacc?atctccaaaa?ccaaaggc 1008
<210>4
<211>336
<212>PRT
＜213〉mice (Mus musculus)
<400>4
Lys?Glu?Ser?Gly?Pro?Gly?Ile?Leu?Gln?Pro?Ser?Gln?Thr?Leu?Ser?Leu
1 5 10 15
Thr?Cys?Ser?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?Gly?Met?Gly?Val
20 25 30
Gly?Trp?Ile?Arg?Gln?Pro?Ser?Gly?Lys?Gly?Leu?Glu?Trp?Leu?Ala?His
35 40 45
Val?Trp?Trp?Asp?Asp?Leu?Lys?Arg?Tyr?Asn?Pro?Ala?Leu?Lys?Ser?Arg
50 55 60
Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Ser?Ser?Gln?Ile?Phe?Leu?Arg?Ile
65 70 75 80
Ala?Ser?Val?Asp?Thr?Ala?Asp?Thr?Ala?Thr?Tyr?Tyr?Cys?Ala?Arg?Leu
85 90 95
Gly?Arg?Arg?Tyr?Gly?Asn?Pro?Pro?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr
100 105 110
Thr?Leu?Thr?Val?Ser?Ser?Ala?Lys?Thr?Thr?Pro?Pro?Ser?Val?Tyr?Pro
115 120 125
Leu?Ala?Pro?Gly?Ser?Ala?Ala?Gln?Thr?Asn?Ser?Met?Val?Thr?Leu?Gly
130 135 140
Cys?Leu?Val?Lys?Gly?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Thr?Trp?Asn
145 150 155 160
Ser?Gly?Ser?Leu?Ser?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln
165 170 175
Ser?Asp?Leu?Tyr?Thr?Leu?Ser?Ser?Ser?Val?Thr?Val?Pro?Ser?Ser?Thr
180 185 190
Trp?Pro?Ser?Glu?Thr?Val?Thr?Cys?Asn?Val?Ala?His?Pro?Ala?Ser?Ser
195 200 205
Thr?Lys?Val?Asp?Lys?Lys?Ile?Val?Pro?Arg?Asp?Cys?Gly?Cys?Lys?Pro
210 215 220
Cys?Ile?Cys?Thr?Val?Pro?Glu?Val?Ser?Ser?Val?Phe?Ile?Phe?Pro?Pro
225 230 235 240
Lys?Pro?Lys?Asp?Val?Leu?Thr?Ile?Thr?Leu?Thr?Pro?Lys?Val?Thr?Cys
245 250 255
Val?Val?Val?Asp?Ile?Ser?Lys?Asp?Asp?Pro?Glu?Val?Gln?Phe?Ser?Trp
260 265 270
Phe?Val?Asp?Asp?Val?Glu?Val?His?Thr?Ala?Gln?Thr?Gln?Pro?Arg?Glu
275 280 285
Glu?Gln?Phe?Asn?Ser?Thr?Phe?Arg?Ser?Val?Ser?Glu?Leu?Pro?Ile?Met
290 295 300
His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Phe?Lys?Cys?Arg?Val?Asn?Ser
305 310 315 320
Ala?Ala?Phe?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Thr?Lys?Gly
325 330 335
<210>5
<211>654
<212>DNA
＜213〉mice (Mus musculus)
<400>5
attgtgctga?cacagtctcc?tccttcctta?gctgtttctc?tggggcagag?ggccaccgtc 60
tcatgcaggg?ccagccaaag?tgtcagtaca?tctaagtata?gttatctgca?ctggtaccaa 120
cagaaaccag?gacagccacc?caaagtcctc?atcgagtatt?catccaacct?agaatctggg 180
gtccctgcca?ggttcagtgg?cagtgggtct?gggacagact?tcaccctcaa?catccatcct 240
gtggaggagg?aggatactgc?aacatattac?tgtcagcaca?gttgggagat?tccgtggacg 300
ttcggtggag?gcaccaaact?ggaaatcaaa?cgggctgatg?ctgcaccaac?tgtatccatc 360
ttcccaccat?ccagtgagca?gttaacatct?ggaggtgcct?cagtcgtgtg?cttcttgaac 420
aacttctacc?ccaaagacat?caatgtcaag?tggaagattg?atggcagtga?acgacaaaat 480
ggcgtcctga?acagttggac?tgatcaggac?agcaaagaca?gcacctacag?catgagcagc 540
accctcacgt?tgaccaagga?cgagtatgaa?cgacataaca?gctatacctg?tgaggccact 600
cacaagacat?caacttcacc?cattgtcaag?agcttcaaca?ggaatgagtg?ttag 654
<210>6
<211>217
<212>PRT
＜213〉mice (Mus musculus)
<400>6
Ile?Val?Leu?Thr?Gln?Ser?Pro?Pro?Ser?Leu?Ala?Val?Ser?Leu?Gly?Gln
1 5 10 15
Arg?Ala?Thr?Val?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Thr?Ser?Lys
20 25 30
Tyr?Ser?Tyr?Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro?Lys
35 40 45
Val?Leu?Ile?Glu?Tyr?Ser?Ser?Asn?Leu?Glu?Ser?Gly?Val?Pro?Ala?Arg
50 55 60
Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Asn?Ile?His?Pro
65 70 75 80
Val?Glu?Glu?Glu?Asp?Thr?Ala?Thr?Tyr?Tyr?Cys?Gln?His?Ser?Trp?Glu
85 90 95
Ile?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Ala
100 105 110
Asp?Ala?Ala?Pro?Thr?Val?Ser?Ile?Phe?Pro?Pro?Ser?Ser?Glu?Gln?Leu
115 120 125
Thr?Ser?Gly?Gly?Ala?Ser?Val?Val?Cys?Phe?Leu?Asn?Asn?Phe?Tyr?Pro
130 135 140
Lys?Asp?Ile?Asn?Val?Lys?Trp?Lys?Ile?Asp?Gly?Ser?Glu?Arg?Gln?Asn
145 150 155 160
Gly?Val?Leu?Asn?Ser?Trp?Thr?Asp?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr
165 170 175
Ser?Met?Ser?Ser?Thr?Leu?Thr?Leu?Thr?Lys?Asp?Glu?Tyr?Glu?Arg?His
180 185 190
Asn?Ser?Tyr?Thr?Cys?Glu?Ala?Thr?His?Lys?Thr?Ser?Thr?Ser?Pro?Ile
195 200 205
Val?Lys?Ser?Phe?Asn?Arg?Asn?Glu?Cys
210 215
<210>7
<211>69
<212>DNA
＜213〉mice (Mus musculus)
<400>7
atgggcaggc?ttacttcttc?attcctgcta?ctgattgtcc?ctgcatatgt?cctgtcccag 60
gttactctg 69
<210>8
<211>23
<212>PRT
＜213〉mice (Mus musculus)
<400>8
Met?Gly?Arg?Leu?Thr?Ser?Ser?Phe?Leu?Leu?Leu?Ile?Val?Pro?Ala?Tyr
1 5 10 15
Val?Leu?Ser?Gln?Val?Thr?Leu
20
<210>9
<211>63
<212>DNA
＜213〉mice (Mus musculus)
<400>9
atggagacag?acacactcct?gctatgggtg?ctgctgctct?gggttccagg?ttccactggt 60
gac 63
<210>10
<211>21
<212>PRT
＜213〉mice (Mus musculus)
<400>10
Met?Glu?Thr?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu?Trp?Val?Pro
1 5 10 15
Gly?Ser?Thr?Gly?Asp
20
<210>11
<211>72
<212>DNA
＜213〉foot and mouth disease virus (Foot-and-mouth disease virus)
<400>11
gcgcccgtga?agcagaccct?gaacttcgac?ctgctgaagc?tggccggcga?cgtggagtcc 60
aaccccggcc?cc 72
<210>12
<211>24
<212>PRT
＜213〉foot and mouth disease virus (Foot-and-mouth disease virus)
<400>12
Ala?Pro?Val?Lys?Gln?Thr?Leu?Asn?Phe?Asp?Leu?Leu?Lys?Leu?Ala?Gly
1 5 10 15
Asp?Val?Glu?Ser?Asn?Pro?Gly?Pro
20
<210>13
<211>12
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>furin?cleavage?site
<400>13
cgcgccaagc?gc 12
<210>14
<211>42
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>14
Asp?Ala?Glu?Phe?Arg?His?Asp?Ser?Gly?Tyr?Glu?Val?His?His?Gln?Lys
1 5 10 15
Leu?Val?Phe?Phe?Ala?Glu?Asp?Val?Gly?Ser?Asn?Lys?Gly?Ala?Ile?Ile
20 25 30
Gly?Leu?Met?Val?Gly?Gly?Val?Val?Ile?Ala
35 40
<210>15
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>15
cccaagcttt?ttaccaggag?agtgggaga 29
<210>16
<211>44
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>16
ataagaatgc?ggccgcagtc?gacgctaaca?ctcattcctg?ttga?44
<210>17
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>17
cggggtacca?tgggcaggct?tacttcttc 29
<210>18
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>18
ccggaattca?tggagacaga?cacactcct 29
<210>19
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>19
cggggtacca?tgggcaggct?tacttcttc 29
<210>20
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>20
cccaagcttt?ttaccaggag?agtgggaga 29
<210>21
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>21
ccggaattca?tggagacaga?cacactcct 29
<210>22
<211>44
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>22
ataagaatgc?ggccgcagtc?gacgctaaca?ctcattcctg?ttga 44
<210>23
<211>85
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>23
cgcgccaagc?gcgccccccg?tgaagcagac?cctgaacttc?gacctgctga?agctggccgg 60
cgacgtggag?tccaaccccg?gcccc 85
<210>24
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>24
ctagctagcc?gcgccaagcg?cgcccccgt 29
<210>25
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>25
ccgctcgagg?gggccggggt?tggactcca 29
<210>26
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>26
cccaagcttc?gcgccaagcg?cgcccccgt 29
<210>27
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>27
ccggaattcg?gggccggggt?tggactcca 29
<210>28
<211>20
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>28
aattaaccct?cactaaaggg 20
<210>29
<211>20
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>29
tcgacttggt?aggaggtatg 20
<210>30
<211>20
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>30
ggtggaatgg?tgtacacctg 20
<210>31
<211>20
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>31
taatacgact?cactataggg 20
<210>32
<211>15
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>32
gtaaaacgac?ggcca 15
<210>33
<211>20
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>33
cagcacagtt?gggagattcc 20
<210>34
<211>20
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>34
ccgtttgatt?tccagtttgg 20
<210>35
<211>41
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>35
ataagaatgc?ggccgcagtc?gacaatgggc?aggcttactt c 41
<210>36
<211>29
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<223>primer
<400>36
cccaagcttg?cctttggttt?tggagatgg 29
Claims (12)
1, express the anti-β of perfect form-kind of starch body antibody or its segmental carrier, this carrier contains promoter sequence, the sequence of the light chain immunoglobulin of the sequence of the heavy chain immunoglobulin of the anti-β that encodes-kind of starch body antibody or its fragments sequence, the self-processed peptide of coding and the anti-β that encodes-kind of starch body antibody.
2, the described carrier of claim 1, it contains the polyA sequence.
3, claim 1 or 2 described carriers, it contains the ITR sequence.
4, each described carrier of claim 1～3, processing protease enzyme action position is connected with 5 ' end side of the sequence of the self-processed peptide of coding.
5, the described carrier of claim 4, described processing protease is Furin.
6, each described carrier of claim 1～5, described self-processed peptide is 2A.
7, each described carrier of claim 1～6, the sequence of the light chain immunoglobulin of the heavy chain immunoglobulin of the anti-β that encodes-kind of starch body antibody or its fragments sequence and coding anti-β-kind of starch body antibody all contains secretory signal sequence.
8, each described carrier of claim 1～7, the fragment of the heavy chain immunoglobulin of anti-β-kind of starch body antibody contains Variable Area, contains the part in constant zone or does not contain constant zone fully.
9, the described carrier of claim 8, this vector expression CH3 deficiency antibody, it contains coding is removed the CH3 zone from the heavy chain immunoglobulin of anti-β-kind of starch body antibody fragments sequence.
10, each described carrier of claim 1～9, described carrier is a gland relevant viral vector.
11, the described carrier of claim 10, described gland relevant viral vector is 1 type.
12, Alzheimer prevent and/or treat medicine, it contains each described carrier of claim 1～11 as effective ingredient.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008012393A JP2009171880A (en) | 2008-01-23 | 2008-01-23 | Development of next-generation gene therapy and immunotherapy in alzheimer's disease |
| JP2008012393 | 2008-01-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101491683A true CN101491683A (en) | 2009-07-29 |
Family
ID=40922584
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2009100011325A Pending CN101491683A (en) | 2008-01-23 | 2009-01-23 | Development of new-generation gene-therapy immuno-therapy for alzheimer's disease |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2009171880A (en) |
| CN (1) | CN101491683A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013177922A1 (en) * | 2012-05-29 | 2013-12-05 | 四川百利药业有限责任公司 | Polypeptide for treating alzheimer's disease and genetic vaccine |
| CN109414482A (en) * | 2016-05-12 | 2019-03-01 | 俄亥俄州创新基金会 | For treating the peptide and method of neurodegenerative disease |
| AU2015355126B2 (en) * | 2014-12-01 | 2019-05-02 | Inovio Pharmaceuticals, Inc. | DNA antibody constructs and method of using same |
| US11208470B2 (en) | 2012-12-13 | 2021-12-28 | The Trustees Of The University Of Pennsylvania | DNA antibody constructs and method of using same |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011126808A2 (en) * | 2010-03-29 | 2011-10-13 | The Trustees Of The University Of Pennsylvania | Pharmacologically induced transgene ablation system |
-
2008
- 2008-01-23 JP JP2008012393A patent/JP2009171880A/en active Pending
-
2009
- 2009-01-23 CN CNA2009100011325A patent/CN101491683A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013177922A1 (en) * | 2012-05-29 | 2013-12-05 | 四川百利药业有限责任公司 | Polypeptide for treating alzheimer's disease and genetic vaccine |
| US11208470B2 (en) | 2012-12-13 | 2021-12-28 | The Trustees Of The University Of Pennsylvania | DNA antibody constructs and method of using same |
| AU2015355126B2 (en) * | 2014-12-01 | 2019-05-02 | Inovio Pharmaceuticals, Inc. | DNA antibody constructs and method of using same |
| AU2015355126B9 (en) * | 2014-12-01 | 2020-03-26 | Inovio Pharmaceuticals, Inc. | DNA antibody constructs and method of using same |
| US11278619B2 (en) | 2014-12-01 | 2022-03-22 | The Trustees Of The University Of Pennsylvania | DNA antibody constructs and method of using same |
| CN109414482A (en) * | 2016-05-12 | 2019-03-01 | 俄亥俄州创新基金会 | For treating the peptide and method of neurodegenerative disease |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2009171880A (en) | 2009-08-06 |
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