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Antibody to beta-amyloid peptide and application thereof

A technology of amyloid peptide and antibody, which is applied in the field of disease medicine and monoclonal antibody of β-amyloid peptide, which can solve the problems of low antibody affinity and inability to solve the source of human B cells

Inactive Publication Date: 2013-08-14
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When obtaining humanized antibodies, the latest method is to use phage antibody library technology to replace the Fab fragment of mouse monoclonal antibody with a fully humanized antibody through chain replacement. This form of antibody has 100% human antibody sequence, but it cannot To solve the source of immunized human B cells, the resulting antibodies often have low affinity, and this method needs further improvement

Method used

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  • Antibody to beta-amyloid peptide and application thereof
  • Antibody to beta-amyloid peptide and application thereof
  • Antibody to beta-amyloid peptide and application thereof

Examples

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Embodiment 1

[0034] Example 1: Preparation and Identification of Anti-Aβ42 Monoclonal Antibody:

[0035] Aβ1-42 polypeptide 100μg / 0.1ml PBS (0.01M, PH7.2) was mixed with an equal volume of Freund's complete adjuvant (CFA) as an immunogen, mixed with each other through a syringe until emulsified, and subcutaneously in the abdomen (6 points) ) to immunize female BALB / c mice for 5-6 weeks, and inject 200 μl per mouse; after the basic immunization, repeat the injection 5 times every two weeks to boost the immunization and monitor changes in serum antibody levels; select the sensitized mice with the highest titer , Three days before cell fusion, Aβ1-42 polypeptide 50μg / 0.1ml saline was injected into the tail vein, and cell fusion was carried out 10 days later. Spleen single cells were routinely isolated, and spleen cells from immunized mice (1×10 8 ) and SP2 / 0 myeloma cells (1×10 7 ) and mix, centrifuge at 1000rpm for 5 minutes, suck up the supernatant and flick the bottom of the tube to loos...

Embodiment 2

[0036] Example 2: Amplification and sequencing of the variable region of the anti-Aβ42 monoclonal antibody

[0037] Adopt BD SMART TM The 5'-RACE method in the RACE cDNA kit was used to obtain the variable region coding sequence of the anti-Aβ1-42 mouse monoclonal antibody mZ1, and the primers were designed as follows:

[0038] GSP-L1: 5'-CTATGAATTCTCAACACTCATTCCTGTTGAAGC-3'

[0039] GSP-L2: 5'-CTATGAATTCTCAGRRACAKTCWGCASGRGACA-3'

[0040] GSP-H: 5'-CTATGAATTCTCATTTACCAGGAGAGTGGGAGAG-3'

[0041] One-step extraction of 1×10 with TRIZOL 7 Total RNA of hybridoma mZ1. According to the 5'-RACE kit instructions (BD Company), the first strand was amplified with 5'-CDS and BD SMART II oligo as primers, and then poly(C) was added to the 3' end of the first strand cDNA After tailing, the mouse antibody gene sequence was amplified with the universal primer UPM and the gene-specific primer GSP. The reaction conditions are: 94°C for 30 seconds, 72°C for 3 minutes, 5 cycles; 94°C for...

Embodiment 3

[0046] Example 3: Construction of Humanized Chimeric Monoclonal Antibody Expression Vector

[0047] The heavy and light chain variable regions of the mouse monoclonal antibody mZ1 were recombined with the human IgG antibody constant regions by recombinant PCR to form a full-length antibody. At the same time, the rare amino acids at the end of the heavy and light chains were mutated when designing primers, and the pre-designed primers were used. The restriction site of the enzyme was connected into the expression vector of PCDNA3.1(+) and PCDNA3.1(-), wherein the restriction site at the 5' end was Hind III, and the restriction site at the 3' end was XbaI. The designed primers were as follows:

[0048] Light chain variable region forward primer VLf (23bp): 5'-cccAAGCTTcaccATGTCCTCTGCTCAGTTCCTTGG-3'

[0049] Light chain variable region reverse primer VLb (31bp): 5'-TGCAGCCACAGTTTTGATCTCTACCTTGGTG-3'

[0050] Light chain constant region forward primer CLf (31bp): 5'-GTAGAGATCAAAc...

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Abstract

The invention relates to an antibody to beta-amyloid peptide and application thereof. The inventor invents the antibody to the beta-amyloid peptide, wherein the sequence of a heavy chain variable-region is shown as SEQ ID No:3 and the sequence of a light chain variable-region is shown as SEQ ID No:4, a hybridoma secreting the antibody, an antibody humanized from the antibody and an antibody further improved to reduce human anti-mouse antibody reaction, polynucleotide encoding the antibodies, and application of the antibodies in preparing medicines for treating Alzheimer disease. The antibodies have high specificity and affinity and do not cause the human anti-mouse antibody reaction..

Description

technical field [0001] The present invention relates to a novel antibody and its use. Specifically, the present invention relates to a monoclonal antibody to β-amyloid polypeptide, the humanization of the antibody, and the use of the antibody in medicines for treating diseases such as Alzheimer's disease. Background technique [0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system characterized by progressive memory impairment and mental decline in the elderly. Senile plaques, neurofibrillary tangles, and neuronal loss are the three main pathological features of AD. The main component of senile plaques is the fibers formed by the aggregation of β-amyloid peptide (Aβ), and Aβ is considered to be the main pathogenic substance that causes neuronal damage and cognitive function decline. [1] . Aβ mainly includes two molecules, Aβ40 and Aβ42, which are composed of 39-43 amino acids. It is generally believed that Aβ is produced by co-cleavage w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N5/20C12N5/10A61K39/395A61P25/28
Inventor 梁平常德张建华
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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