JP2013112668A - Vaccine composition for prevention and/or treatment of alzheimer's disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vaccine composition for prevention and/or treatment of Alzheimer's disease.SOLUTION: The vaccine composition for prevention and/or treatment of Alzheimer's disease includes fusion protein having an amino acid sequence in which a single unit of or a repeated plurality of units of 5 to 15 continuous amino acid sequences derived from an N-terminal part of amyloid β peptide is/are inserted into an amino acid sequence of seed storage protein.

Description

  The present invention provides a vaccine composition for the prevention and / or treatment of Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disease in which causative substances such as amyloid β accumulate in the brain and cause damage to nerve cells. While the number of Alzheimer's disease patients is expected to increase with the aging of society, there are almost no preventive drugs or therapeutic drugs, and the development of new preventive drugs, therapeutic drugs, vaccines, etc. is desired.
The major amyloid β protein produced in the brain of Alzheimer's disease patients consists of 40 to 43 amino acids produced by cleavage of amyloid precursor protein by γ-secretase, and amyloid plaques (also called senile plaques) are amyloid β and its surroundings. It is an aggregate composed of microglia, fibrous astroglia and dystrophic neurites. Currently, the most promising pathologic hypothesis of Alzheimer's disease is the amyloid cascade hypothesis, which is caused by the formation of amyloid plaques due to aggregation and deposition of amyloid β. Based on this hypothesis, vaccine immunotherapy is attracting attention as a new treatment for Alzheimer's disease. This vaccine therapy is a method of removing amyloid β in the brain by immunological technique, and it was reported that amyloid β42 was administered intramuscularly to a disease model-transgenic mouse together with an adjuvant, and a decrease in amyloid deposition in the brain was confirmed. (Patent Document 1 and Non-Patent Document 1).
In addition, although the synthesized amyloid β42 is administered intramuscularly with an adjuvant and anti-amyloid β antibody that recognizes amyloid is detected in the serum of the subject, the side effect of meningoencephalitis is a problem in clinical trials (non- Patent Document 2).
Therefore, there is a need for a vaccine composition for preventing and / or treating Alzheimer's disease without side effects.

WO 99/27944

Schenk D. et al, Nature 400: 173-177, 1999 Hock C. et al., Nat. Med. 8: 1270-1275, 2002

  An object of the present invention is to provide a new vaccine composition for the prevention and / or treatment of Alzheimer's disease.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that 5-15 consecutive amino acid sequences derived from the N-terminal portion of the amyloid β peptide are repeated in a single or plural number to store seeds. The present inventors have succeeded in finding a vaccine composition for the prevention and / or treatment of Alzheimer's disease including a fusion protein (modified seed storage protein) having an amino acid sequence inserted in the amino acid sequence of the protein, and completed the present invention. It was.

That is, the present invention includes the following.
(1) A fusion protein having an amino acid sequence in which 5 to 15 consecutive amino acid sequences derived from the N-terminal portion of amyloid β peptide are inserted into the amino acid sequence of a seed storage protein by repeating single or plural A vaccine composition for preventing and / or treating Alzheimer's disease.
(2) The vaccine composition according to (1), wherein the seed storage protein is any one of soybean 11S globulin A1aB1b subunit, kidney bean arserin, and rice prolamin.
(3) The vaccine composition according to (1) or (2), which produces amyloid-specific antibody production when administered to an animal.
(4) The vaccine composition according to (1) or (2), which causes a significant decrease in soluble and / or insoluble amyloid β1-42 associated with amyloid plaques in the brain when administered to animals.
(5) The vaccine composition according to (1) or (2), wherein administration to an animal suffering from Alzheimer's disease characterized by loss of cognitive memory ability results in restoration of cognitive memory ability.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the new vaccine composition with respect to Alzheimer's disease, and the prevention and treatment of Alzheimer's disease are attained.

The figure which shows the measurement result of the anti- amyloid (beta) antibody titer in mouse | mouth serum by a vaccine composition. The figure which shows the memory disorder improvement effect by a waterway maze test. The figure which shows the amyloid accumulation amount in a mouse | mouth brain.

The present invention is described in detail below.

1. Amyloid β antigen peptide and gene encoding it Amyloid β antigen peptide in the present invention is 5 to 15, preferably 5 to 10, derived from the N-terminal portion of natural amyloid β peptide (42 amino acid residues). Peptides consisting of consecutive amino acid residues are exemplified. Specifically, a peptide in a region containing the 1st to 15th amino acids (SEQ ID NO: 1) from the N-terminus can be preferably used, and preferably a peptide consisting of the 1st to 5th amino acids (SEQ ID NO: 2) Or the peptide which consists of a 4th-10th amino acid (sequence number 3) can be used, More preferably, the peptide which consists of a 4th-10th amino acid can be used.
Moreover, although these peptides may be single, they can also be used as a series of connected peptides in which a plurality, for example, 2 to 15, preferably 2 to 5, are repeated.
Since the base sequence encoding the gene for amyloid β antigen peptide is known (GenBank accession No. AB113349), DNA encoding amyloid β peptide is isolated from the cDNA library by screening based on this base sequence information. can do. Further, DNA encoding amyloid β peptide can also be prepared by chemical synthesis.

2. Wild-type seed storage protein and gene encoding the same Examples of the seed storage protein in the present invention include proteins derived from crop seeds having a dietary experience in daily eating habits. For example, each subunit constituting soybean 11S globulin, each subunit constituting soybean 7S globulin, kidney bean arserin, rice prolamin, rice globulin, and seed crop proteins of other crops. Preferred wild type seed storage proteins include A1aB1b subunit of soybean 11S globulin, α subunit or β subunit of 7S globulin, with A1aB1b subunit of soybean 11S globulin being more preferred.
In the present invention, the wild-type seed storage protein means a seed storage protein before the Aβ antigen peptide is inserted, and the amino acid sequence of SEQ ID NO: 5, 23 or 25, or 80% or more, preferably 90% with this amino acid sequence. As mentioned above, the protein containing the amino acid sequence which has 95% or more identity (identity) more preferably is mentioned. As long as the wild-type seed storage protein maintains the function as a seed storage protein, one or more (preferably 1-20, more preferably 1-10) amino acid sequences of SEQ ID NO: 5, 23, or 25 are used. Or more preferably 1 to 5) amino acid substitutions, insertions, additions and / or deletions may be present.
Here, the identity (%) of amino acid sequences refers to the identity (%) of the maximum amino acid sequence obtained by aligning two amino acid sequences to be compared with a gap as necessary. . Alignment for the purpose of determining amino acid sequence identity can be performed using a variety of methods well known to those skilled in the art, such as publicly available such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Commercially available software such as available computer software, Gene Works 2.5.1 software (Teijin System Technology Co., Ltd.), and GENETIX-WIN (Software Development Co., Ltd.) can also be used.

  The gene encoding the wild type seed storage protein is not particularly limited as long as it has a sequence encoding the wild type seed storage protein as described above. For example, it has the base sequence of SEQ ID NO: 4, 22 or 24. Genes are exemplified. As long as it has a function as a seed storage protein, it may be a gene that hybridizes with a complementary sequence of SEQ ID NO: 4, 22, or 24 under stringent conditions. Here, stringent conditions include, for example, conditions of washing at 68 ° C., 0.1 × SSC, 0.1% SDS.

3. Vaccine composition in which Aβ antigen peptide is inserted into wild-type seed storage protein The vaccine composition of the present invention has 5 to 15 consecutive amino acid sequences derived from the N-terminal portion of amyloid β peptide. Repetitively, it includes a fusion protein (modified seed storage protein) having an amino acid sequence inserted into the amino acid sequence of the seed storage protein.
Here, the fusion protein can be preferably obtained by inserting a gene encoding an amyloid β antigen peptide into a variable region coding part of a gene encoding a wild type seed storage protein so that no frame shift occurs.

  Here, "inserting a gene encoding amyloid β antigen peptide so as not to cause a frame shift" is a sequence obtained by removing the amino acid sequence of amyloid β antigen peptide from the amino acid sequence of the modified seed storage protein, The amyloid β antigen peptide is inserted into the variable region of the wild-type seed storage protein so that it is identical to the amino acid sequence of the wild-type seed storage protein. In addition to the insertion of the gene encoding the β antigen peptide, this also includes that a part of the base sequence encoding the variable region is replaced with the gene encoding the amyloid β antigen peptide.

In the present invention, the variable region of the wild-type seed storage protein maintains a stable three-dimensional structure equivalent to the wild-type seed storage protein even when an amino acid sequence derived from a foreign gene is inserted therein. Refers to a region that can maintain the properties of a wild type seed storage protein.
For example, when the seed storage protein is the A1aB1b subunit of soybean 11S globulin comprising the amino acid sequence of SEQ ID NO: 5, amino acid numbers 20-2 of SEQ ID NO: 5
8 region (variable region I), amino acid number 111 to 128 region (variable region II), amino acid number 198 to 216 region (variable region III), amino acid number 268 to 315 region (variable region IV), amino acid number Five of the 490 to 495 regions (variable region V) are known as variable regions.

In addition, the wild-type seed storage protein has a certain sequence identity with the amino acid sequence of SEQ ID NO: 5 in which one or more amino acid substitutions, insertions, additions and / or deletions exist in the amino acid sequence of SEQ ID NO: 5. A region corresponding to amino acid numbers 20 to 28 of SEQ ID NO: 5, a region corresponding to amino acid numbers 111 to 128, a region corresponding to amino acid numbers 198 to 216, and amino acid numbers 268 to 315. The region corresponding to, and the region corresponding to amino acid numbers 490 to 495 are variable regions.
Here, the “corresponding amino acid sequence” for a specific amino acid sequence refers to the alignment of two amino acid sequences to be compared with a gap as necessary so that the maximum amino acid sequence identity (%) can be obtained. The term “alignment” refers to the other partial amino acid sequence corresponding to one specific partial amino acid sequence. Such an amino acid sequence can be easily specified by those skilled in the art.

When a plurality of variable regions are present in the wild type seed storage protein, the modified seed storage protein can be prepared by inserting an amyloid β antigen peptide into one or a plurality of variable regions.
For example, when the soybean 11S globulin A1aB1b subunit containing the amino acid sequence of SEQ ID NO: 5 is used as the wild-type seed storage protein, the variable regions II, III, IV, Either V can be selected, but it is more preferable to insert in III or IV. In addition, two or more of the variable regions II, III, IV, and V can be selected as the variable region into which the amyloid β antigen peptide is inserted. For example, it can be simultaneously inserted into two regions III and IV. It can be inserted into three regions II, III, and IV simultaneously, or can be simultaneously inserted into four regions II, III, IV, and V. Here, the gene encoding the amyloid β antigen peptide needs to be introduced so that no frame shift occurs in the base sequence encoding the wild type seed storage protein.

  In addition, when the gene encoding the kidney bean Arserin 5 containing the amino acid sequence of SEQ ID NO: 23 is used as the gene encoding the wild-type seed storage protein, since the variable region is not known, the gene with the A1aB1b subunit It is necessary to estimate the variable region by comparing the DNA sequence to confirm the disordered region, comparing the amino acid sequence and the three-dimensional structure with other similar storage proteins, and confirming the sequence gap and the structural difference. is there. A region encoding amino acid numbers 149 to 150 (variable region A) and / or a region encoding amino acid numbers 250 to 251 (variable region B) specified by such estimation is encoded in the Alzheimer's disease vaccine. It is preferable to insert a gene.

  Further, the gene encoding the wild type seed storage protein is identical to the amino acid sequence of SEQ ID NO: 23, wherein one or more amino acid substitutions, insertions, additions and / or deletions are present in the amino acid sequence of SEQ ID NO: 23 A region encoding an amino acid sequence corresponding to amino acid numbers 149 to 150 of SEQ ID NO: 23, a region encoding an amino acid sequence corresponding to amino acid numbers 250 to 251 It is preferable to insert a gene encoding an Alzheimer's disease vaccine into.

Further, when a gene encoding a rice prolamin containing the amino acid sequence of SEQ ID NO: 25 is used as a gene encoding a wild-type seed storage protein, the three-dimensional structure and the variable region are not known. Compared to other similar storage proteins,
It is necessary to estimate the variable region by confirming the gap structure. It is preferable to insert a gene encoding an Alzheimer's disease vaccine into a region (variable region a) encoding amino acid numbers 110 to 111 of SEQ ID NO: 25 specified by such estimation.

  In addition, the gene encoding the wild type seed storage protein is identical to the amino acid sequence of SEQ ID NO: 25 in which one or more amino acid substitutions, insertions, additions and / or deletions are present in the amino acid sequence of SEQ ID NO: 25 In the case where it contains a nucleotide sequence encoding an amino acid sequence having the property, it is preferable to insert a gene encoding an Alzheimer's disease vaccine into a region encoding an amino acid sequence corresponding to amino acid numbers 110 to 111 of SEQ ID NO: 25.

4). Production of vaccine composition in which amyloid β antigen peptide is inserted There is no limitation on the method for producing the vaccine composition of the present invention, either by a conventional peptide synthesis method or in advance by a conventional peptide synthesis method. It can be prepared by binding peptides synthesized in the above. Moreover, the said peptide can be synthesize | combined according to the protocol of an apparatus using the peptide synthesizer marketed from each manufacturer.

  In addition, the fusion protein of the present invention can be prepared by recombinant DNA technology. For example, DNA encoding the amino acid sequence of a fusion protein designed using PCR, restriction enzymes, etc. is prepared, and this is inserted into a vector that contains a promoter that functions in the host and is capable of autonomous growth, and is inserted into E. coli, Bacillus subtilis , Actinomycetes, yeast and other microorganisms, animals and plants, and their cells or tissues, etc., to be transformed into transformants or transgenic animals and plants, cultured and cultivated, and then the fusion protein of the present invention Can be collected and purified by an appropriate method.

  In addition, the fusion protein of the present invention can be produced by a recombinant plant. Plant species include, for example, Asteraceae, Brassicaceae, Cucurbitaceae, Apiaceae, Rosaceae, Vitaceae, Vaccinium, Papaya Family (Caricaceae), Legume Family (Fabaceae), Walnut Family (Juglandaceae), Redwood Family (Chenopodiaceae), or Solanum Family (Solanaceae), Convolvulaceae, Gramineae (Poaceae), or Dioscoreaceae, or Plant species belonging to the family Convolvulaceae, more specifically lettuce, chicory, mugwort, broccoli, cabbage, radish, horseradish, mustard, cucumber, melon, pumpkin, chayote, carrot, honeybee, celery, apple, plum, Japanese apricot, peach, strawberry, raspberry, almond, pear, loquat, grape, cranberry, bilberry, blueberry, papaya , Alfalfa, soybean, walnut, spinach, tomato, peppers, sweet potato, rice, maize, wheat, barley, rye, Japanese yam, potato and the like.

The introduction of the gene encoding the modified seed storage protein into the host can be performed by various methods already reported and established, including the Agrobacterium method, PEG method, electroporation method, particle gun It is preferable to introduce a vector for gene introduction using a method, whisker ultrasonic method or the like.
In particular, when produced by a recombinant plant, a plant body in which the fusion protein of the present invention is expressed, for example, seeds, foliage, fruits, tubers and the like, is processed as it is, and is for oral consumption containing the fusion protein of the present invention. It is also optional to use it as a composition.

5. Administration Method of Vaccine Composition to Animals By administering the vaccine composition produced as described above to an animal individual, an immune response to amyloid β is induced, thereby reducing amyloid β deposition.
The vaccine composition of the present invention can be administered by any of the conventional routes such as subcutaneous, oral, intramuscular, parenteral or enteral routes. For example, it is possible to use a method of administration by oral ingestion or injection into a body cavity such as subcutaneous, intradermal, intramuscular, intravascular, intraperitoneal or intrathoracic cavity, a catheter, or drip.

  Examples of animals to which the vaccine composition of the present invention is administered include mammals including humans and other vertebrates.

  When administering the vaccine composition of the present invention, an adjuvant (a substance or a composition of a substance that can enhance an immune response to an immunogen) that is usually used in the formulation of a vaccine can be formulated. And cholera toxin B and saponin system.

The dose of the vaccine composition can be appropriately set according to conditions such as the age, sex, body weight, symptom, route of administration, etc. of the animal to be administered, but generally it is converted into the dose of amyloid β peptide. It is in the range of 0.1 to 3000 μg / kg body weight per day, preferably in the range of 1 to 1000 μg / kg body weight. The above dose may be administered at intervals of several days, or may be administered at intervals of several weeks to several months, for example, 1 to 12 weeks. In addition, the number of administration is suitably from several to several tens, but is not particularly limited.
The method of the present invention induces an immune response against amyloid β. The immune response to amyloid β can be confirmed by production of anti-amyloid β antibody, decrease in the amount of amyloid β in brain tissue, decrease in amyloid β deposition, and the like.

  The production of anti-amyloid β antibody can be examined by detecting anti-amyloid β antibody in blood, and the antibody level can be measured, for example, by ELISA (enzyme-linked immunosorbent assay). In ELISA, for example, antigen is adsorbed on a microplate, antiserum is prepared, the prepared antiserum is diluted 2-fold (starting solution 1: 1000), and the diluted antiserum is added to the plate for antigen-antibody reaction. To do. Then, for color development, the antibody of the immunized animal is reacted with a heterologous antibody labeled with peroxidase enzyme to obtain a secondary antibody. When the absorbance is ½ of the maximum color absorbance, the antibody titer can be calculated based on the antibody dilution factor.

  The amyloid β level in brain tissue can be measured using, for example, an extract of brain tissue and a Biosource ELISA kit.

The effect of reducing amyloid β deposition (amyloid plaques) can be measured, for example, by the following method. Treat brain tissue sections with 70% formic acid, inactivate endogenous peroxidase with 5% H ₂ O ₂ , react the sections with anti-Aβ antibody, and perform DAB staining with peroxidase-labeled secondary antibody . After staining, the area of the Aβ accumulation portion can be measured by observation with a microscope. It can be determined that the amyloid β deposition level has decreased if the proportion of the area of the accumulation portion is reduced as compared with the case where the vaccine composition of the present invention is not administered.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

  In addition, the procedure of the experimental operation performed in the following examples is in accordance with the method described in “Molecular Cloning” 2nd edition (J. Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) unless otherwise specified. .

Example 1 Construction of expression plasmid of modified soybean protein inserted with amyloid β antigen peptide (Aβ1-15 antigen peptide)
An expression plasmid for expressing a gene encoding a modified A1aB1b containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 known as β-amyloid antigenic determinant (hereinafter abbreviated as Aβ15) in soybean seeds was constructed.
An oligonucleotide (sense strand, SEQ ID NO: 6) in which two nucleotide sequences encoding Aβ15 are linked in tandem with two lysine residues sandwiched between them (sense strand, SEQ ID NO: 6) Was synthesized using the custom DNA commissioned synthesis service of Fasmac Co., Ltd. (the sense strand is referred to as AB15F, and the antisense strand is referred to as AB15R). Each 100 pmol of AB15F and AB15R was phosphorylated with T4 Polynucleotide Kinase (manufactured by Takara Bio Inc.) in the presence of ATP at a final concentration of 1 mM, mixed after reaction, and heated at 94 ° C. for 10 minutes. Then, it was gradually cooled to 37 ° C. over 1 hour and annealed. In this way, a double-stranded DNA fragment encoding a peptide (Aβ15 × 2) in which two Aβ15s are connected with two lysine residues in between was obtained.
A plasmid containing the vector part using the plasmid pBSK-A1aB1b as a template, in which the cDNA of a known A1aB1b gene (GenBank accession No. AB113349) has been cloned into the SmaI site of pBluescriptII SK (-) (Stratagene) encodes A1aB1b Amplification using PCR so that the specific variable region of the gene is at the 5 'end and 3' end, and modify the resulting DNA fragment and the double-stranded DNA fragment encoding Aβ15x2 A plasmid containing the gene encoding type A1aB1b was prepared. A specific method is shown below.
A primer set consisting of the primer pair of SEQ ID NOs: 8 and 9 (PS-2 primer set) to be inserted into the variable region III of the gene encoding A1aB1b of SEQ ID NO: 4 was prepared.
The base sequence region of SEQ ID NO: 4 into which DNA encoding Aβ15 × 2 is inserted using the above primer set is referred to as PS-2 region.
PCR was performed using 10 ng of pBSK-A1aB1b as a template. In PCR, 50 μl of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes and 1 cycle, denaturation reaction was performed at 94 ° C. for 30 seconds, annealing was performed at 57 ° C. for 30 seconds, and extension reaction was performed at 68 ° C. 25 cycles were performed with 5 minutes as one cycle. The reaction solution was 200 μM dNTP mixed solution, 1.5 mM MgSO 4 solution, 1 μM each of the above primers, 1 unit of KOD-Plus- (manufactured by Toyobo Co., Ltd.), KOD-Plus-Ver. Contains 2 buffers. Thereafter, PCR was performed using the same composition unless otherwise specified, except for the primers.
50 fmol of the DNA fragment thus obtained and 150 fmol of the double-stranded DNA fragment encoding the above Aβ15 × 2 were subjected to a ligation reaction at 16 ° C. for 40 minutes using a DNA ligation kit (manufactured by Takara Bio Inc.). . The reaction product was transformed into E. coli DH5α competent cell (Takara Bio Inc.) to obtain a plurality of transformed E. coli. Plasmid DNA was extracted and purified from the obtained Escherichia coli, and the base sequence of the gene encoding the modified A1aB1b (hereinafter referred to as A1aB1bMK1) was confirmed using DNA sequencing service of Fasmac Co., Ltd.

Example 2 Construction of expression plasmid of modified soybean protein inserted with amyloid β antigen peptide (Aβ4-10 antigen peptide)
An expression plasmid for expressing a gene encoding modified A1aB1b containing a peptide consisting of the amino acid sequence of SEQ ID NO: 3 known as β amyloid antigenic determinant (hereinafter abbreviated as Aβ7) in soybean seeds was constructed.
Custom DNA entrusted by Fasmac Co., Ltd. for oligonucleotide (sense strand, SEQ ID NO: 11) consisting of three nucleotide sequences encoding Aβ7 linked in tandem (sense strand, SEQ ID NO: 10) and its complementary sequence (antisense strand, SEQ ID NO: 11) Synthesis was performed using a synthesis service (the sense strand is called 410F and the antisense strand is called 410R). Subsequently, unless otherwise specified, oligonucleotides were synthesized using the company's custom DNA commissioned synthesis service. Each 100 pmol of 410F and 410R was phosphorylated with T4 Polynucleotide Kinase (manufactured by Takara Bio Inc.) in the presence of ATP at a final concentration of 1 mM, mixed with each reaction solution, and heated at 94 ° C. for 10 minutes. Then, it was gradually cooled to 37 ° C. over 1 hour and annealed. In this way, a double-stranded DNA fragment encoding a peptide in which three Aβ7s were linked (Aβ7 × 3) was obtained.
A plasmid containing the vector part using the plasmid pBSK-A1aB1b as a template, in which the cDNA of a known A1aB1b gene (GenBank accession No. AB113349) has been cloned into the SmaI site of pBluescriptII SK (-) (Stratagene) encodes A1aB1b The DNA fragment obtained by amplification using PCR so that the specific variable region of the gene is at the 5 ′ end and 3 ′ end, and the double-stranded DNA fragment encoding (Aβ7 × 3) are ligated. Thus, a plasmid containing the gene encoding the modified A1aB1b was prepared. A specific method is shown below.
Primer set (PS-1, SEQ ID NO: 12, 13), primer set (PS-2, SEQ ID NO: 8, 9) for insertion into variable regions II, III and IV of the gene encoding A1aB1b of SEQ ID NO: 4 A total of three primer sets (primer set (PS-3, SEQ ID NOs: 14 and 15)) were prepared.
PCR was performed using 10 ng of pBSK-A1aB1b as a template. In PCR, 50 μl of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes and 1 cycle, denaturation reaction was performed at 94 ° C. for 30 seconds, annealing was performed at 57 ° C. for 30 seconds, and extension reaction was performed at 68 ° C. 25 cycles were performed with 5 minutes as one cycle. The reaction solution was 200 μM dNTP mixed solution, 1.5 mM MgSO ₄ solution, 1 μM each of the above primers, 1 unit of KOD-Plus- (manufactured by Toyobo Co., Ltd.), KOD-Plus-Ver. Contains 2 buffers.
50 fmol of each DNA fragment thus obtained and 150 fmol of the double-stranded DNA fragment encoding the above (Aβ7 × 3) were ligated for 40 minutes at 16 ° C. using a DNA ligation kit (manufactured by Takara Bio Inc.). Went. The nucleotide sequence of the gene encoding the modified A1aB1b (hereinafter referred to as A1aB1bM1) prepared in this way was confirmed.

Example 3 Production of Modified Soy Protein by E. coli A1aB1bMK1 and A1aB1bM1 prepared above were expressed in E. coli, and expression plasmids for producing the respective proteins were constructed.
After phosphorylating the A1aB1bMK1 and A1aB1bM1 gene DNA fragments, cutting with NcoI and XhoI in advance, blunting with T4 DNA polymerase (Takara Bio), and dephosphorylating with CIAP (Takara Bio) A ligation reaction was performed with a commercially available E. coli expression vector pET-21d vector (NOVAGEN). By analyzing the base sequence of the obtained clone, a clone in which the A1aB1bMK1 and A1aB1bM1 genes were linked in the forward direction was selected.
Thus, E. coli expression vectors pETA1aB1bMK1 and pETA1aB1bM1 in which a 6 × His tag was ligated downstream of the genes encoding A1aB1bMK1 and A1aB1bM1 were constructed.
Commercially available Escherichia coli AD494 strain (manufactured by NOVAGEN) transformed with pETA1aB1bMK1 and pETA1aB1bM1 prepared by the above method as antibiotics in a known LB medium supplemented with 15 mg / l kanamycin and 50 mg / l carbenicillin at 37 ° C., 120 rpm For 18 hours. 5% (volume / volume) of the pre-cultured bacterial solution in a modified LB medium in which 15 mg / l kanamycin and 50 mg / l carbenicillin were added as newly prepared antibiotics and the NaCl concentration was changed to 1M After adding and culturing at 37 ° C. and 120 rpm for 2 hours, IPTG was added at a final concentration of 1 mM, and the culture was continued at 20 ° C. and 50 rpm.
After culturing, the cells were collected by centrifugation. As a result, about 10 g of cells were collected from the 6 L culture solution.
The obtained bacterial cells were subjected to extraction of a soluble protein fraction using a commercially available BugBuster Protein Extraction Reagent (manufactured by NOVAGEN). The extracted protein was purified by a commercially available HisBind Purification Kits (manufactured by NOVAGEN). The purified protein was desalted by dialysis overnight with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and a buffer solution.
Thus, 96 mg of pETA1aB1bMK1 and 60 mg of pETA1aB1bM1 could be obtained.

Example 4 Production of Modified Soy Protein by Recombinant Soybean In order to express the gene encoding A1aB1bMK1 in a seed-specific manner, the gene encoding A1aB1bMK1 obtained above, the known soybean promoter Gy1P and the terminator Gy1T are known. pUHG vector (Y. Kita, K. Nishizawa, M Takahashi, M. Kitayama, M. Ishimoto. (2007) Genetic improvement of somatic.
embryogenesis and regeneration in soybean and transformation of the improved breeding lines. Plant Cell Reports 26: 439-447) and expression plasmids were constructed.
In order to obtain a DNA fragment encoding A1aB1bMK1 (SEQ ID NO: 17) among the above, PCR was performed using a primer set consisting of the oligonucleotide pairs of SEQ ID NOs: 18 and 19. In PCR, 50 μl of reaction solution was used per reaction, denaturation reaction was performed at 94 ° C. for 2 minutes and 1 cycle, denaturation reaction was performed at 94 ° C. for 30 seconds, annealing was performed at 57 ° C. for 30 seconds, and extension reaction was performed at 68 ° C. 25 cycles were performed with 2 minutes as one cycle. Thus, a DNA fragment of the A1aB1bMK1 gene was obtained. The DNA fragment, promoter DNA fragment, and terminator DNA fragment of the A1aB1bMK1 gene were phosphorylated and ligated with a pUHG vector that had been previously cleaved with SmaI and dephosphorylated with CIAP (Takara Bio). From the obtained clones, clones correctly linked in the order of the Gy1P promoter, the gene encoding A1aB1bMK1, and the Gy1T terminator were selected by analyzing the nucleotide sequence. Thus, a plant transformation vector pUHGA1aB1bMK1 in which a gene encoding A1aB1bMK1 was expressed in a seed-specific manner was constructed.

  Similarly, a plant transformation vector pUHGA1aB1bM1 for constructing a seed-specific expression of a gene encoding A1aB1bM1 (SEQ ID NO: 21) correctly linked in the order of a Gy1P promoter, a gene encoding A1aB1bM1, and a Gy1T terminator was constructed.

  These plant transformation vectors were introduced into soybean cells by a known method, and A1aB1bMK1 and A1aB1bM1 proteins were expressed and many transformed seeds accumulated in seeds could be obtained. Accumulation of A1aB1bMK1 and A1aB1bM1 proteins in seeds was confirmed by a detection method by Western blotting using a known amylod β antibody.

Purification method of A1aB1bM1 protein Ten transformed seeds that accumulate A1aB1bM1 protein produced in Example 4 above were pulverized by a (1.92 g) mill. Ten times the volume of pulverized hexane and pulverized seeds were mixed and degreased by shaking and stirring at room temperature for 1 hour. After removing hexane by centrifugation at 15000 rpm at room temperature for 30 minutes, the seed pulverized product was air-dried at room temperature. The weight after air drying was 1.8 g. 0.4 M NaCl, 35 mM sodium phosphate (pH 7.6), 10 mM 2-mercaptoethanol, 1% protease inhibitor cocktail (Protease Inhibitor Cocktail for plant cell)
and 20 ml of a protein extraction buffer consisting of DMSO solution and DMSO solution (manufactured by SIGMA) and a defatted seed pulverized product were mixed, and the seed protein was extracted by shaking and stirring at room temperature for 1 hour. 16ml of supernatant was collected by centrifugation at 15000rpm, room temperature for 30 minutes.
The protein concentration was measured by DC protein assay kit II (manufactured by BioRad). As a result, the concentration was 16.8 mg / ml, and 269 mg of seed protein was extracted. This seed protein solution was subjected to gel filtration chromatography. The conditions are as follows. Column: Sephacryl S-300 26 mm ID x 60 cm (GE Healthcare), flow rate: 2 ml / min, buffer: 0.4 M NaCl, 35 mM sodium phosphate (pH 7.6), 0.02% sodium azide, 10 mM 2− Mercaptoethanol, fraction collection: 3 min / fra. Each fraction was subjected to Western blotting in the same manner as in Example 4, and the A1aB1bM1 fraction was identified. In this way, 48 ml of a purified A1aB1bM1 protein solution having a concentration of 1 mg / ml was recovered. The collected A1aB1bM1 protein was analyzed by Western blotting using the E. coli produced A1aB1bM1 protein purified in Example 3 as a standard. As a result, the purity was 100%.

Example 5 Measurement of anti-amyloid β antibody titer in mouse serum using vaccine composition Commercially available 2-month-old mice (BALB / c) were used in each test group, and 50 μg / ml of A1aB1bMK1 and A1aB1bM1 obtained in Example 3 were used. The mice were injected subcutaneously 5 times every other week at a dose of 1 animal / dose. As an adjuvant, AbISCO-100 (manufactured by ISCONOVA) was used at 12 μg / animal / dose. Blood was collected from the mice 10 weeks after the start of administration, and the amount of anti-Aβ42 antibody in the serum was measured. 100 μl of Aβ1-42 synthetic peptide (manufactured by Peptide Institute, Inc.) (5 μg / mL) was adsorbed to each well of a 96-well plate for ELISA (manufactured by Greiner), and Blocker Casein in PBS (manufactured by Thermo SCIENTIFIC) After blocking with, mouse serum was added and detected with a peroxidase-labeled anti-mouse IgG antibody (Thermo SCIENTIFIC). 1-step Ultra TMB-ELISA (Thermo SCIENTIFIC) was used as a detection reagent. The antibody titer was evaluated by measuring absorbance (OD450) with a RAINBOW sunrise microplate reader (manufactured by TECAN).
The results are shown in FIG. Compared with the control group (A1aB1b (wild type seed storage protein) administration group), the absorbance was high in all the individuals in the treatment group, and a marked increase in antibody titer was observed.

Example 6 Behavioral Test by Oral Administration of Vaccine Composition
In the behavioral test carried out in this example, a known Morris water maze test was conducted. 2-month-old APP transgenic mouse TgCRND8 (Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695. (2001) Chishti MA, Yang DS, Janus C, Phinney AL, Horne P, Pearson J, Strome R, Zuker N, Loukides J, French J, Turner S, Lozza G, Grilli M, Kunicki S, Morissette C, Paquette J, Gervais F, Bergeron C, Fraser PE, Carlson GA, George-Hyslop PS, Westaway D., THE JOURNAL OF BIOLOGICAL CHEMISTRY 276 (24): 21562-70) was used to examine the cognitive function recovery effect of the vaccine composition A1aB1bM1 of the present invention. The mice were divided into two groups of 8 mice, and after preliminary training, one group was an A1aB1bM1 administration group, and the other group was an A1aB1b control group not containing amyloid β antigen. To the administration group, A1aB1bM1 was orally administered at a dose of 0.5 mg / animal / dose once a week for a total of 16 times (4 months). The A1aB1b control group was performed in the same manner.
In the test, a circular platform was placed at the bottom of a circular pool (diameter: 150 cm, depth: 45 cm) so as to be hidden in water, and the mouse was memorized for the position of the platform. The swimming test was conducted for 5 consecutive days once a day every month. In this test, the swimming time until reaching the platform after the start of swimming was measured.
The results are shown in FIG. In the A1aB1bM1 administration group, a significant improvement was observed in the time to reach the platform compared to the control group, and it became clear that the learning disorder of the diseased mice was improved.

Example 7 Influence on Brain Amyloid Transgenic mice orally administered with the above A1aB1bM1 or A1aB1b were dissected 4 months after administration, and the frontal cortex, parietal lobe, and hippocampal cortex were analyzed with 4% paraformaldehyde. And then embedded in paraffin wax to prepare brain tissue sections. To detect amyloid β protein or amyloid plaques using the tissue, tissue sections were treated with 70% formic acid and endogenous peroxidase was inactivated with 5% H ₂ O ₂ . Next, the tissue section was reacted with a rabbit anti-pan-Aβ antibody (1000-fold diluted), a peroxidase-labeled secondary antibody was added, and DAB staining was performed. The stained sections were observed using a 3CCD camera connected to a microscope, the area of the amyloid β accumulation portion in each region was measured, and the area ratio (index) was calculated.
The amyloid β deposition index is shown in FIG. It was confirmed that administration of A1aB1bM1 significantly reduced amyloid plaques compared to the control (A1aB1b administration group).

Claims (5)

A fusion protein having an amino acid sequence in which 5 to 15 consecutive amino acid sequences derived from the N-terminal portion of the amyloid β peptide are inserted into the amino acid sequence of a seed storage protein in a single or multiple repetition, A vaccine composition for preventing and / or treating Alzheimer's disease. The vaccine composition according to claim 1, wherein the seed storage protein is any of soybean 11S globulin A1aB1b subunit, kidney bean arserin, and rice prolamin. 3. A vaccine composition according to claim 1 or 2 that results in the production of amyloid specific antibodies when administered to an animal. The vaccine composition according to claim 1 or 2, which causes a significant decrease in soluble and / or insoluble amyloid β1-42 associated with amyloid plaques in the brain when administered to an animal. The vaccine composition according to claim 1 or 2, wherein administration to an animal suffering from Alzheimer's disease characterized by loss of cognitive memory ability results in restoration of cognitive memory ability.