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Composite vaccine for Alzheimer's disease prevention and treatment, and preparation method thereof

A technology for senile dementia and vaccines, applied in chemical instruments and methods, drug combinations, gene therapy and other directions, can solve problems such as side effects and low antibody titers, and achieve the effects of simple production methods, easy industrial production and low cost.

Active Publication Date: 2013-01-30
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For this reason, researchers have proposed different solutions in continuous research and discussion, for example, adding non-steroidal anti-inflammatory drugs, antiviral drugs or hormones to prevent inflammation during vaccine treatment. In addition, There is also the application of Aβ subunit vaccines to remove the toxic fragments of Aβ, which can suppress adverse cellular reactions while producing antibodies; but the above methods have their disadvantages, such as hormones and other inhibitors also have side effects, subunit vaccines, The antibody titers produced are not very high, etc.

Method used

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  • Composite vaccine for Alzheimer's disease prevention and treatment, and preparation method thereof
  • Composite vaccine for Alzheimer's disease prevention and treatment, and preparation method thereof
  • Composite vaccine for Alzheimer's disease prevention and treatment, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Preparation of Alzheimer's Disease Aβ42 Nucleic Acid Vaccine

[0067] 1) Construction of eukaryotic expression plasmid for Alzheimer's disease Aβ42

[0068] Using the plasmid Abeta 42-C3d3 (provided by Dr. McArgo Janie) as a template, primer P1: 5'-AAAGGATCCATGGATGCAGAATTCC-3' and primer P2: 5'-GCCTCTAGATTACGCTATGACAACA-3', (in primer 1 and primer 2, respectively The Aβ42 gene was amplified by PCR under the guidance of BamHI recognition site and XbaI recognition site). Reaction system: 1 μL plasmid template, 10 pmol each of primer 1 and primer 2, 500 mM KCl, 100 mM Tris-HCl (pH8.4), 1.5 mM MgCl 2 , 100μg / mL BSA, 1mM dNTPs, 2.5U Taq DNA polymerase, the total volume is 25μL; the reaction conditions are: denaturation at 94°C for 30 seconds, renaturation at 60°C for 30 seconds, extension at 72°C for 30 seconds, a total of 30 cycles; The amplified DNA fragments in 1.5% liposugar gel electrophoresis were recovered and connected to the pMD18-T cloning vector; the c...

Embodiment 2A

[0071] Example 2 Prokaryotic expression of Aβ42 protein

[0072] 1) Construction of prokaryotic expression plasmid for Alzheimer's disease Aβ42

[0073] Using Abeta 42-C3d3 as a template, in primer 1: 5'-AAAGGATCCATGGATGCAGAATTCC-3' and primer 2: 5'-GCCGTCGACTAACGCTATGACAACA-3' (in primer 1 and primer 2, BamHI recognition site and SalI recognition site were introduced respectively) The Aβ42 gene was amplified by PCR under the guidance of the guide; the recovered target fragment was connected to the pMD18-T vector, and after the correct identification by enzyme digestion, the target fragment was subcloned into the pET28a vector. same. The result is as image 3 As shown, A is the result of double digestion of pMD18T-Aβ42 plasmid with BamHI and SalI, B is the result of double digestion of pET28a-Aβ42 plasmid with BamHI and SalI, and the sequence analysis result is correct.

[0074] Using pMD18T-Aβ42 as a template, in primer 1: 5'-AAAGGATCCATGGATGCAGAATTCC-3' and primer 2: 5'-G...

Embodiment 3A

[0077] Example 3 ELISA detection and T cell response detection of antibody production after Aβ42 protein vaccine and DNA vaccine co-immunized with different mouse species

[0078] Select 6-8 weeks old mice of Balb / c and C57BL / 6 species for immunization, and detect whether the combined immunization of Aβ42 protein vaccine and DNA vaccine can cause immunosuppression by detecting antibody IgG and T cell proliferation responses, and At the same time, it does not affect the production of antibodies.

[0079] 1) ELISA detection of antibodies produced after Aβ42 protein vaccine and DNA vaccine were co-immunized with Balb / c and C57BL / 6 mice

[0080] Divide 16 6-8 week-old BALB / c or C57BL / 6 female mice into 4 groups, 4 in each group; the first group was intramuscularly injected with 50 microliters of PBS solution containing 50 micrograms of pVAX1-Aβ42 plasmid DNA; Group subcutaneous immunization contained 50 micrograms of one copy of Aβ42 protein, 1 / 2 volume of Freund's complete adjuv...

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Abstract

The invention belongs to the field of biological products, and relates to a composite vaccine for Alzheimer's disease prevention and treatment, wherein the composite vaccine is prepared by mixing a nucleic acid vaccine encoding Abeta42 and a protein gene engineering vaccine. Experiment results show that: Abeta42 protein antigen expressed through prokaryotic expression and pVAX-Abeta42 plasmid are adopted to co-immunize, such that the Alzheimer's disease protein vaccine in the prior art is improved, high level anti-Abeta antibody IgG can be produced, inhibition on T cell response can be induced, and the inhibition can be maintained for a fairly long time. In addition, the antibody generated through co-immunizing and for Abeta can be combined with Abeta protein fibers and nature Abeta protein precipitate in APP / PS1 Alzheimer's disease transgene incidence mice brain so as to provide Abeta precipitate removing capacity; and the vaccine is a potential, effective, and side effect-free vaccine, and can be used for Alzheimer's disease prevention and treatment.

Description

technical field [0001] The invention belongs to the field of biological products, and relates to a compound vaccine for preventing and treating senile dementia and a preparation method thereof. Background technique [0002] Dementia is a brain disorder that is caused by abnormal degeneration of the brain. According to statistics, most of the patients are elderly. The clinical manifestations are: due to the decline of brain function, the patients are affected in terms of memory, calculation, learning, understanding, and even language, judgment, and sense of direction. The disease not only suffers from the patient himself, but also has a very negative impact on his family and even the whole society. With the increase of the elderly, the number of patients with Alzheimer's disease is also increasing. Alzheimer's disease (AD) is a common form of degenerative dementia. At present, there are more than 25 million AD patients in the world. Due to the characteristics of its onset...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K48/00A61P25/28A61P37/02
CPCA61K38/00A61K31/7088C07K14/4711C07K16/00A61K45/06C07H21/00A61K39/0005C07K16/18A61K31/70A61P25/28A61P37/02
Inventor 王宾于杨朱贤主王爽
Owner FUDAN UNIV
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