Conformation-specific recombinant A beta 1-42-like oligomer antigen as well as preparation method and application thereof

An oligomer and antigen technology, applied in the fields of biopharmaceuticals and genetic engineering, can solve the problems of inability to improve cognitive ability, inability to effectively neutralize oligomers, and failure to achieve the therapeutic effect of improving cognitive ability.

Active Publication Date: 2020-02-07
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These completed clinical trials have not achieved the therapeutic effect of improving cognitive ability. The possible reason is that these vaccines mainly produce non-specific antibodies against Aβ oligomers after immunization, and cannot effectively neutralize the effects on synaptic function and neurons. More toxic oligomers (Meli G, et al., Nature Communications, 2014; 5(5):3867.), so that cognitive ability cannot be improved

Method used

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  • Conformation-specific recombinant A beta 1-42-like oligomer antigen as well as preparation method and application thereof
  • Conformation-specific recombinant A beta 1-42-like oligomer antigen as well as preparation method and application thereof
  • Conformation-specific recombinant A beta 1-42-like oligomer antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Construction of recombinant prokaryotic expression vector and expression and purification of recombinant Aβ1-42-like oligomer antigen in Escherichia coli

[0067] 1. Design and synthesis of 6×(Aβ1-15-Th) and 12×(Aβ1-15-Th) genes

[0068] According to the codon degeneracy, six copies of the tandem 6×(Aβ1-15-Th) gene (see SEQID No.1) and 12 copies of the tandem 12×(Aβ1-15-Th) gene (see SEQ ID No.2), directly synthesized and cloned into the pMD18-T(TaKaRa)T vector, named pMD18-6×(Aβ1-15-Th) and pMD18-12×(Aβ1-15-Th), respectively encoding 6×(Aβ1-15-Th) and 12×(Aβ1-15-Th) (see SEQ ID No.3 and SEQ ID No.4 for the amino acid sequence), each Aβ1-15-Th is connected with a GS flexible peptide ( figure 1 Middle A). In each Aβ1-15-Th molecule, Aβ1-15 is the B-cell epitope of Aβ1-42 molecule, the sequence is DAEFRHSGYEVHHQ; Th (DAEFRHDSGYEVHHQAKFVAAWTLKAAAQYIKANSK FIGITE) is the helper T-cell epitope, including the universal DR helper T-cell epitope PADRE sequence (AKA...

Embodiment 2

[0079] Example 2. Recombinant Aβ1-42-like oligomer antigen induces normal mice to produce a high level of Th2 anti-Aβ antibody response

[0080] Using the recombinant Aβ1-42-like oligomer antigen protein 6×(Aβ1-15-Th) and 12×(Aβ1-15-Th) expressed and purified in Example 1 as the immunogen, ie subunit vaccine, to immunize mice, to test its immunogenicity. The specific method is as follows: C57 / BL6 mice (8 weeks old, female, SPF level, Experimental Animal Center of Military Medical Research Institute) were randomly divided into 3 groups, 8 mice in each group, and 5 μg of recombinant protein was immunized, while the control group (Control) Immunize with PBS without recombinant protein, and immunize four times in total. Before immunization, the antigen was diluted to a final concentration of 10% (V / V) aluminum adjuvant (Alhydrogel TM , Brenntag Biosector, Frederikssund, Denmark). Intramuscular injection of 100 μl per mouse was used for immunization. For booster immunization, th...

Embodiment 3

[0084] Example 3. Recombinant Aβ1-42-like oligomer antigen subunit vaccine immunizes 3×Tg AD model mice to induce high levels of anti-Aβ antibodies

[0085] The present invention further evaluates the immunogenicity and immunotherapeutic effect of the recombinant Aβ1-42-like oligomer antigen subunit vaccine by 3×Tg AD model mice (for the scheme, see figure 1 Middle D). The specific scheme is as follows. The AD model mice are 3×Tg AD model mice, which are knocked in the presenilin protein PS1 M146V Genetic mice containing Tau were microinjected P301L and APP Swe Constructed a pathological model mouse of AD capable of expressing these three proteins. The disease course of this model mouse will show a certain degree of progression with the growth of the age of the mouse, and it will occur at the age of 6 months. Intracellular Aβ deposition, the pathological characteristics of synaptosomal Tau can be observed at the age of 9 months, compared with other single transgenic mice, i...

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Abstract

The invention discloses a conformation-specific recombinant A beta 1-42-like oligomer antigen as well as a preparation method and an application thereof. The recombinant A beta 1-42-like oligomer antigen is 6 or 12 beta amyloid peptide B cell epitope A beta 1-15 and two helper T cell epitope Th chimeric molecules which are connected in series. The recombinant 6 / 12*(A beta1-15-Th) chimeric antigenimmune serum antibody disclosed by the invention can be specifically combined with an A beta oligomer, the toxicity is neutralized, a high-level Th2 type anti-A beta antibody can be generated by immunizing common and model mice with low dosage and few times, no harmful T cell immune reaction aiming at A beta 1-42 exists, the content of A beta and the oligomer in the brain of an AD mouse is reduced, the related synaptic protein level is up-regulated, and the learning and memory ability is improved. Therefore, the recombinant A beta 1-42-like oligomer antigen targeting the structural characteristics of the A beta oligomer prepared by the invention has great potential and application prospect in AD prevention.

Description

technical field [0001] The invention relates to the technical fields of biopharmaceuticals and genetic engineering, in particular to a conformation-specific recombinant Aβ1-42-like oligomer antigen, a preparation method and application thereof. Background technique [0002] Alzheimer's disease (Alzheimer's disease, AD), also known as senile dementia, was first discovered by German doctor Alzheimer Alois in 1906. It is a chronic long-term neurodegenerative disease of the elderly. Cognitive and memory impairment due to deletion. According to statistics, the incidence rate of dementia in the elderly aged 60 to 80 is about 4%, while the incidence rate of elderly aged over 80 is as high as 20% to 40%. It is the most common disease among all dementias. According to the latest report of the World Health Organization, there are more than 50 million dementia patients worldwide, and it is expected that this number will triple by 2050. More than 150 million, nearly 10 million new cas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12A61K39/00A61P25/28
CPCA61K39/0007A61P25/28C07K14/4711
Inventor 余云舟杨志新陆健昇王荣
Owner ACADEMY OF MILITARY MEDICAL SCI
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