Senile dementia recombinant protein vaccine and preparation method thereof

A recombinant protein and protein vaccine technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problem of unfavorable B cell antigen epitope exposure, difficult chemical modification of synthetic peptides, and impact on antigen space. structure and other problems, to avoid high difficulty and high cost, enhance immunogenicity, and avoid the effect of space rigid structure

Inactive Publication Date: 2008-12-10
LIVZON PHARM GRP INC
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Problems solved by technology

[0003] There are still many unsatisfactory aspects of the second-generation AD vaccines reported in recent literature: 1) Most of the antigens prepared by researchers are chemically synthesized, and the chemical modification of synthetic peptides is difficult, costly, and not high in purity
2) When researchers prepare multivalent vaccines containing Aβ N-terminal B-cell epitopes, most of them connect the B-cell epitope fragment directly with a new T-cell epitope, or directly connect the B-cell epitope in series, It is easy to affect the spatial structure of the antigen, which is not conducive to the exposure of the B cell epitope of Aβ

Method used

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  • Senile dementia recombinant protein vaccine and preparation method thereof
  • Senile dementia recombinant protein vaccine and preparation method thereof

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preparation example Construction

[0022] 1 recombinant plasmid pQE-4×Aβ 15 preparation of

[0023] 1.1 Design of tetravalent B cell antigen epitope fragment 4×Aβ inserted into pQE-30 15 The sequence is: Aβ 1-15 +GG+Aβ 1-15 +GSSG+Aβ 1-15 +GG+Aβ 1-15

[0024] 1.2 Recombinant plasmid pQE-4×Aβ 15 Construct

[0025] 1.2.1 Aβ 1-15 +GG+Aβ 1-15 +GSSG(Sac I)(2×Aβ 15 -1, the first bivalent Aβ 1-15 gene) fragment primer design

[0026] P1 Primer: 5'-CG GAT GCA GAA TTC CGA CAT GAT TCAGGA TAT GAA GTT CAT CAT CAA GAT GCA GAA TTC CGACAT GAT TCA GGA TAT GAA GTT CAT CAT CAA GG G -3'. The bold italics are the glycine-linked peptides, and the front and back of the glycine-linked peptides are respectively Aβ 15 Base sequences, the bolded BamHI and Sac I restriction sites, the underlined bases for primer pairing.

[0027] P2 Primer: 5'- C CC TTG ATG ATG AAC -3'; Bold is the Sac I restriction site, and the underline is the base paired by the primer;

[0028] 1.2.2GSSG(Sac I)+Aβ 1-15 +GG+Aβ 1-15 (2×Aβ ...

example example 2

[0044] Example 2 Recombinant 4×Aβ 15 Observation of immunology, pathology and behavior of Tg2576 transgenic mice after protein vaccination.

[0045] 1 Materials and methods

[0046] 1.1 Experimental animals Twelve 12-month-old Tg2576 transgenic mice (Tg2576 transgenic mice are products of Taconic, USA, and were successfully bred in our laboratory). After feeding to 12 months old, they were randomly divided into 2 groups: 4×Aβ 15 group and PBS control group, with 6 rats in each group.

[0047] 1.2 Immunization with recombinant 4×Aβ 15 The protein was mixed with an equal amount of adjuvant MF59 (5ml squalene, 0.5ml Tween80, 0.5ml span85, 94ml PBS to make a 100% mixture, mixed and emulsified for 15min) and fully emulsified to prepare recombinant 4×Aβ 15 The protein vaccine was inoculated with 100 μg / time subcutaneous multi-point injection in six 12-month-old Tg2576 mice, each injection was 100 μL. After the first inoculation, it was boosted two weeks later, and then inoculate...

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Abstract

The invention relates to a recombinant multivalent Beta-amyloid 1-15(ABeta1-15) protein vaccine of senile dementia and a preparation method thereof. The proposal is adopted as following: a plurality of oligonucleotide primers are synthesized, two or more than two bivalent ABeta1-15 gene sections containing flexion connection peptide gene are obtained by gene amplification and are cloned into pQE-30 prokaryotic expression plasmid, and inducible expression by bacillus and purification are carried out, thus preparing the folding bivalent ABeta1-15 recombinant protein vaccine which can express flexibility. In the invention, the multivalent folding ABeta1-15 is selected as the immunogen, thus avoiding the spacing rigescent structure of multi-copy ABetaB cell epitope, leading the ABeta1-15 epitope to be exposed easily, and improving immunogenicity. Meanwhile, the molecular weight of immunogenicity is increased, and the possibility of degradation is lowered. The prokaryotic expression system is selected for preparing recombinant protein, thus avoiding the shortcomings of high difficulty and high cost of chemosynthesis.

Description

technical field [0001] The invention relates to a vaccine and a preparation method thereof, in particular to a recombinant multivalent β-amyloid 1-15 (Aβ) for senile dementia 1-15 ) protein vaccine and its preparation method. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized clinically by progressive memory and loss of acquired knowledge until the patient loses the ability to live. With the increase of the number of cases, AD brings a heavy burden to the family and society, and is an important social and medical health problem. Regarding the treatment of AD, there is no ideal method at present. The existing treatment strategies mainly focus on relieving symptoms, but do not prevent the pathological process of AD, and their clinical efficacy is not satisfactory. Aβ is the main component of senile plaques in the pathological changes of senile dementia, which consists of 39-42 amino acids. Recent studies have shown that the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00C12N15/12C12N15/70C07K14/47A61P25/28
Inventor 姚志彬
Owner LIVZON PHARM GRP INC
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