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Anti-Abeta42 oligomer single-chain antibody and gene for coding single-chain antibody

A single-chain antibody and oligomer technology, applied in the field of genes encoding the single-chain antibody, to achieve the effect of reducing toxicity and broad application prospects

Inactive Publication Date: 2015-04-01
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention solves the technical problem that existing antibodies can bind to all forms of Aβ42 including Aβ42 monomers without specificity, and uses genetic engineering antibody technology to successfully screen out antibodies that specifically bind to Aβ42 oligomers but not to Aβ42 monomeric and fiber-bound single-chain antibodies

Method used

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  • Anti-Abeta42 oligomer single-chain antibody and gene for coding single-chain antibody
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  • Anti-Abeta42 oligomer single-chain antibody and gene for coding single-chain antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Preparation of Aβ42 oligomers and Aβ42 fibers

[0025] Aβ42 monomer (purchased from Sigma, USA) was dissolved in ice-cold hexafluoroisopropanol (HFIP) to a concentration of 1 mg / mL, sonicated in an ice-water bath for 10 min, dried in vacuum, and frozen at -20°C. When using, first dissolve the Aβ42 monomer with dimethyl sulfoxide (DMSO) to a concentration of 1 mg / mL, then dilute Aβ42 to a concentration of 10 μM in phosphate buffer (pH 7.4, 50 mM), and the final concentration is 10 μM , respectively incubated at 37°C for 12h to form the aggregated state of Aβ42 oligomers and incubated for 3 days to form the aggregated state of mature fibers. All Aβ42 aggregation states were confirmed by electron microscopy. Due to the irreversibility of Aβ42 to form aggregates, the oligomers and fibers of Aβ42 are prepared on-site and stored at -80°C for short-term use.

Embodiment 2

[0026] Example 2 Screening of positive clones

[0027] (1) RNA extraction and reverse transcription of peripheral blood lymphocytes to synthesize cDNA

[0028] Separation and extraction of peripheral blood lymphocytes: 10 mL of human peripheral blood blood samples were collected with dipotassium ethylenediaminetetraacetic acid (EDTA-2K) anticoagulant tubes, and balanced salt buffer (D'Hanks) without calcium and magnesium ions (purchased from Bi Yuntian Biotechnology Research Institute) was diluted with 1:1 volume, and added Ficoll-diatrizoate lymphocyte separation medium (purchased from American sigma company) with the ratio of diluted blood sample and lymphocyte separation medium=2:1 (volume ratio) . Centrifuge at 2000rpm / min for 20min at room temperature. After centrifugation, the mononuclear cell layer is aspirated. The cells were washed with D'Hanks and centrifuged at 1000rpm / min for 10min to obtain peripheral blood lymphocytes.

[0029] Peripheral blood lymphocyte RNA...

Embodiment 3

[0055] Example 3 DNA sequence determination of single chain antibody MO6 gene

[0056] The plasmid pET28a-MO6 was extracted and submitted to Shanghai Sangon Sequencing Department for sequence determination. The results are as follows: figure 1 (PET28a-MO6 carrier sequencing spectrum, wherein the gene encoding MO6 is nucleotide 38-808). The measured effective MO6 gene sequence was compared with the immunoglobulin gene sequence in GeneBank, and the results proved that the obtained MO6 clone was a DNA sequence encoding scFv, the start codon was ATG, and the stop codon was It is TGA (its effective nucleotide sequence is shown in SEQ ID NO.4), which includes the DNA sequence encoding the antibody heavy chain variable region (VH) and light chain variable region (VL), and the deduced amino acid sequence (SEQ ID NO.1, SEQ ID NO.2) have a typical antibody variable region structure.

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PUM

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Abstract

The invention relates to the technical field of gene engineering antibody, specifically relates to a single-chain antibody for diagnosis and treatment of Alzheimer's disease (senile dementia) and a gene for coding the single-chain antibody, and more specifically, provides a human-derived anti-Abeta42 oligomer single-chain antibody comprising a heavy chain variable region, a light chain variable region and a connecting peptide sequence, and the amino acid sequence is shown as SEQ ID NO.3. The single-chain antibody can specifically recognize and bind Abeta42 oligomer, reduces Abeta42 oligomer level, effectively inhibits the cytotoxicity of the Abeta42 oligomer, and can effectively penetrate an in-vitro blood brain barrier model. The invention also provides the gene for coding the single-chain antibody, and the nucleotide sequence is as shown in SEQ ID NO.4.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering antibodies, in particular to a single-chain antibody that specifically recognizes and binds to beta-amyloid (Aβ42) oligomers and a gene encoding the single-chain antibody. Background technique [0002] Alzheimer's disease (AD) is the most common neurodegenerative disease, and its pathological features are progressive accumulation and deposition of Aβ42, loss of synapses and neurofibrillary tangles, clinically manifested as attenuation of memory function And cognitive impairment, accompanied by various neurological symptoms and behavioral disorders. There are many views on the pathogenesis of AD, among which the theory of Aβ42 toxicity is generally accepted. [0003] A large number of studies have proved that Aβ42 oligomers formed by the aggregation of Aβ42 monomers are the main pathogenic factors causing AD. However, most of the antibodies used in the diagnosis or treatment of AD in ...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/63A61K39/395A61P25/28
Inventor 张应玖张媛
Owner JILIN UNIV
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