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Recombinant beta-amyloid peptide B cell epitope polypeptide chimeric antigen and preparation method and application thereof

A chimeric antigen and epitope polypeptide technology, applied in the fields of biopharmaceuticals and genetic engineering, can solve the problems of low immunogenicity and difficult to achieve the expected effect, and achieve the effect of mature technology, good immunogenicity and avoiding expensive costs.

Active Publication Date: 2013-10-30
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since Aβ is a small molecule polypeptide with low immunogenicity, it is difficult to achieve the expected effect when Aβ is used as an immunogen alone

Method used

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  • Recombinant beta-amyloid peptide B cell epitope polypeptide chimeric antigen and preparation method and application thereof
  • Recombinant beta-amyloid peptide B cell epitope polypeptide chimeric antigen and preparation method and application thereof
  • Recombinant beta-amyloid peptide B cell epitope polypeptide chimeric antigen and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 16A

[0034] Example 16 Obtaining of Aβ15, 6Aβ15-T and THc Genes

[0035] According to the codon degeneracy, six copies of the 6Aβ15 gene (see SEQ ID No.1 in the sequence listing) were artificially synthesized to encode the AβB cell epitope polypeptide antigen (amino acids 1 to 15, DAEFRHDSGYEVHHQ, referred to as Aβ15) in tandem repetition. The direct synthetic clone was named pMD18-6Aβ15 in the pMD18-T(TaKaRa)T vector, encoding 6Aβ15 (see SEQ ID No.18 in the sequence listing for the amino acid sequence). Each Aβ15 was connected with a KL or GS flexible peptide, starting from 9 nucleotides (ATG), all the way to CAG (7th last nucleotide). Among them, DAEFRHDSGYEVHHQ is Aβ15, KL (AAGCTT) is the enzyme cutting site introduced to provide a site for the introduction of carrier molecules at the N-terminus of Aβ15 in the next step, and GS is a flexible peptide.

[0036] Then artificially synthesized the recombinant polypeptide chimeric antigen 6Aβ15-T gene (see SEQ ID No. 2), directly cl...

Embodiment 2

[0039] Example 2 Construction of prokaryotic expression vector and expression and purification of recombinant polypeptide antigen in Escherichia coli

[0040] 1. Construction of recombinant prokaryotic expression vector

[0041] The plasmids pMD18-6Aβ15 and pMD18-6Aβ15-T obtained in Example 1 were double-digested with EcoR I and Xho I and EcoR I and Not I, respectively, and the target fragments with a length of about 320 and 350 bp were respectively recovered with a DNA recovery kit, and compared with the The prokaryotic expression vector pTIG-Trx (see patent: ZL2007100895882) was ligated with the same enzyme, and the ligated product was transformed into Escherichia coli (E.coli) DH5α competent cells, positive clones were screened, plasmids were extracted, sequenced, and the sequence and insertion position were obtained The correct recombinant prokaryotic expression vectors were named pTIG-Trx-6Aβ15 and pTIG-Trx-6Aβ15-T, respectively.

[0042]The 6Aβ15-T and the toxin fragmen...

Embodiment 3

[0055] Example 3 Recombinant Polypeptide Chimeric Antigen 6Aβ15-T Induces Common Mice to Produce High Levels of Anti-Aβ Antibody

[0056] Using the purified recombinant protein 6Aβ15 and 6Aβ15-T expressed in Example 2 as the immunogen, i.e. the subunit vaccine, to immunize mice to detect its immunogenicity, and the artificially synthesized Aβ15 dissolved in PBS (the amino acid sequence is DAEFRHDSGYEVHHQ , with a purity of more than 90%, synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) and Aβ42 (the amino acid sequence is DAEFRHDSGYEVHHQKLVFFAED VGSNKGAIIGLMVGGVVIA, with a purity of more than 90%, synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) polypeptide antigen for comparison. The specific method is as follows: Balb / c puppies (8 weeks old, female, SPF grade, purchased from the Experimental Animal Center of the Academy of Military Medical Sciences) were randomly divided into 5 groups, with 8 animals in each group, and the...

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Abstract

The invention discloses a recombinant beta-amyloid peptide B cell epitope polypeptide chimeric antigen and a preparation method and application thereof. The recombinant polypeptide chimeric antigen is a polypeptide antigen Abeta1-15 comprising 5-6 serial beta-amyloid peptide B cell epitopes or a dipolymer constituted by the polypeptide antigen. The recombinant polypeptide chimeric antigen further comprises auxiliary Pan-DR Helper T Cell Epitopes (PADRE) or further comprises toxin segment carrier molecules. In the invention, genes of targeted beta-amyloid peptide B cell epitope polypeptide chimeric antigen are synthesized manually, expression is performed by using a prokaryotic expression system, and a purified recombinant polypeptide chimeric antigen protein can be taken as a subunit vaccine for immunoprophylaxis and treatment of Alzheimer's disease.

Description

technical field [0001] The invention relates to the technical fields of biopharmaceuticals and genetic engineering, in particular to recombinant β-amyloid peptide B cell epitope polypeptide chimeric antigen, its preparation method and application. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive dysfunction and memory loss as the main clinical features, and is the main form of senile dementia. The latest statistics show that about 27 million people in the world are affected by AD, while the number of AD patients in my country is as high as 10 million, and it is increasing at a rate of 300,000 every year, which has caused a heavy economic burden to society and families. At present, there are mainly acetylcholinesterase inhibitors and NMDA (N-methyl-D-asparagine) antagonists in the clinical treatment of AD, but these drugs can only alleviate the symptoms of patients, but cannot effectively treat AD. [0003] Amyl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/17A61K39/00A61P25/28
Inventor 余云舟孙志伟王双仇玮祎
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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