Anti-alpha-synuclein monoclonal antibody and application thereof

A technology of synuclein and monoclonal antibody, applied in application, antibody, anti-animal/human immunoglobulin, etc., can solve the problem of undetermined efficacy of antibody

Active Publication Date: 2019-08-27
CHANGCHUN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, most antibodies reflect the function of the antibody only by verifying the combination with α-synuclein monomer or polymer (such as using ELISA method, immunohistochemistry method, western blotting method, etc.), but the efficacy of the antibody in medicine is not determined. Undetermined, only a small number of antibodies have been validated for efficacy in animals, but only at the beginning of research

Method used

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  • Anti-alpha-synuclein monoclonal antibody and application thereof
  • Anti-alpha-synuclein monoclonal antibody and application thereof
  • Anti-alpha-synuclein monoclonal antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0136] The preparation of embodiment 1 people, monkey, mouse α-synuclein protein

[0137] 1. Human, monkey and mouse α-synuclein gene synthesis and expression vector construction

[0138] The amino acid sequences of human, monkey and mouse α-synuclein full-length proteins were respectively fused with the connecting peptide-7his amino acid sequence, and the amino acid sequences of human, monkey and mouse α-synuclein were shown as SEQ ID NO.61 and SEQ ID NO. 62 and shown in SEQ ID NO.63. Design the coding nucleotide sequence corresponding to the full-length fusion protein of human, monkey, and mouse α-synuclein (α-synuclein-connecting peptide-7his), add a HindIII restriction site and Kozak sequence GCCGCCACC at the 5' end, and add a Kozak sequence GCCGCCACC at the 3' end Add stop codon TAG and EcoRI restriction site, and amplify the DNA encoding α-synuclein fusion protein by PCR, and clone it into pUC57simple vector to obtain human, monkey, and mouse pUC57simple-α-synuclein-con...

Embodiment 2

[0148] The preparation of embodiment 2 hybridoma cells

[0149] 1. Immunization of mice

[0150] The human α-synuclein-connecting peptide-7his fusion protein prepared in Example 1 was used as the antigen, the antigen and the adjuvant were mixed at a volume ratio of 1:1, and the immunogen human α-synuclein was emulsified. For the first immunization, the antigen was emulsified with complete Freund's adjuvant, and after 2 weeks, the second immunization was started, and the antigen was emulsified with incomplete Freund's adjuvant, and injected intraperitoneally. The amount of antigen injected per mouse was 150 μg, and the volume of each injection was for 200 μL.

[0151] Three days after the third immunization, the mice were subjected to orbital blood collection, and a small amount of blood samples were taken for serum titer detection. After the serum titer titer was detected to be 1:210000 or above by indirect ELISA method, the mice were boosted with immunization .

[0152] 2....

Embodiment 3

[0175] Example 3 Monoclonal antibody preparation and identification

[0176] 1. Preparation of antibodies by in vitro culture method

[0177] Resuscitate the hybridoma cell line prepared in Example 2 by resuscitating the DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin and culturing in vials. After the confluence of the cells is about 90%, carry out Subculture and expand the culture to a total of about 200 mL of cell culture supernatant.

[0178] 2. Antibody Purification

[0179] Collect the cell supernatant cultured for about 7 days, measure the volume (about 200mL), add NaCl to make the NaCl in the supernatant 2.5M, vacuum filter through a 0.22μm mixed cellulose microporous filter membrane, store at 4°C, and Protein A and chromatography for antibody purification. Loading: the cell culture supernatant containing 2.5M NaCl was filtered through a 0.22μm filter membrane, concentrated to 30ml, and then loaded directly; washing: pH 7.4, 2.5M PBS,...

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Abstract

The invention relates to the technical field of antibody drugs, and specifically relates to an anti-alpha-synuclein monoclonal antibody and application thereof. The anti-alpha-synuclein monoclonal antibody provided by the invention can specifically bind to monomers and aggregates of alpha-synuclein, has high affinity to monomers and aggregates of a human alpha-synuclein, has no affinity to beta-synuclein and gamma-synuclein, can effectively inhibit the polymerization of the monomers of alpha-synuclein, promote microglial cells to clear formed alpha-synuclein aggregates, and protect neuronal cells from the toxicity of the alpha-synuclein aggregates, and can be used to prevent, treat and diagnose alpha-synuclein-related diseases and conditions such as Parkinson's disease, Lewy body dementia,Alzheimer's disease with Parkinson's disease, pure autonomic failure, and multiple system atrophy.

Description

technical field [0001] The invention relates to the technical field of antibody medicines, in particular to monoclonal antibodies against α-synuclein and applications thereof. Background technique [0002] 1. α-synuclein (α-synuclein) [0003] α-synuclein (α-synuclein) is a member of the synuclein family, which consists of a group of proteins with a molecular weight of about 14KD, including β-synuclein and γ-synuclein in addition to α-synuclein. Human α-synuclein is encoded by the SNCA gene located on the fourth chromosome. It contains 140 amino acids and is divided into three structural domains, which are the N-terminal KTKEGV repeat region (1-60aa), the middle NAC region (61-95aa) and the C Proline-rich region (96-140aa) at the end. α-synuclein is widely expressed in brain tissue, accounting for about 1% of the total cytoplasmic protein in brain cells, especially highly expressed in neocortex, hippocampus, substantia nigra, thalamus and cerebellum, and also expressed in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/20C12N15/13A61K39/395A61P25/00A61P25/16A61P25/28
CPCC07K16/18A61P25/00A61P25/16A61P25/28C07K2317/565A61K2039/505
Inventor 滕国生高冷殷玉和吴丛梅孙尧牛晓辉张凤英
Owner CHANGCHUN UNIV OF TECH
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