CN110172098B

CN110172098B - Monoclonal antibody for resisting alpha-synuclein and application thereof

Info

Publication number: CN110172098B
Application number: CN201910446324.0A
Authority: CN
Inventors: 滕国生; 高冷; 殷玉和; 吴丛梅; 孙尧; 牛晓辉; 张凤英
Original assignee: Changchun University of Technology
Current assignee: Changchun University of Technology
Priority date: 2019-05-27
Filing date: 2019-05-27
Publication date: 2023-03-10
Anticipated expiration: 2039-05-27
Also published as: CN110172098A

Abstract

The invention relates to the technical field of antibody drugs, in particular to a monoclonal antibody for resisting alpha-synuclein and application thereof. The monoclonal antibody for resisting alpha-synuclein can specifically bind to monomers and polymers of alpha-synuclein, has higher affinity for the monomers and polymers of human alpha-synuclein, and does not have binding force for beta-synuclein and gamma-synuclein; can effectively inhibit the polymerization of alpha-synuclein monomers, promote the clearance of formed alpha-synuclein polymers by microglia, protect neuron cells from the toxicity of the alpha-synuclein polymers, and can be used for preventing, treating and diagnosing alpha-synuclein related diseases and symptoms, such as Parkinson's disease, lewy body dementia, combined Alzheimer's disease and Parkinson's disease, pure autonomic nerve failure, multiple system atrophy and the like.

Description

Monoclonal antibody for resisting alpha-synuclein and application thereof

Technical Field

The invention relates to the technical field of antibody drugs, in particular to a monoclonal antibody for resisting alpha-synuclein and application thereof.

Background

1. Alpha-synuclein (alpha-synuclein)

Alpha-synuclein is a member of the synuclein family, which consists of a group of proteins with a molecular weight of about 14kD, including beta-synuclein and gamma-synuclein in addition to alpha-synuclein. The human alpha-synuclein is coded by an SNCA gene positioned on a fourth chromosome, contains 140 amino acids and is divided into three structural domains, namely a KTKEGV repetitive region (1-60 aa) at the N end, an NAC region (61-95 aa) in the middle and a proline-rich region (96-140 aa) at the C end. Alpha-synuclein is widely expressed in brain tissue, accounting for about 1% of total cytoplasmic protein in brain cells, and is highly expressed especially in neocortex, hippocampus, substantia nigra, thalamus and cerebellum, as well as in small amounts in heart, muscle and other tissues.

Alpha-synuclein is located mainly at the presynaptic terminal of mature neuronal cells, it does not participate in the formation of synapses, but rather maintains or regulates existing synapses. Under normal physiological conditions, the functions of α -synuclein include binding fatty acids, regulating the physiological functions of certain enzymes and transporters, and playing an important role in synaptic plasticity, neurotransmitter release, and synaptic vesicle recirculation. There is evidence that α -synuclein acts as a chaperone protein during SNARE complex formation. It binds to both phospholipids and synapsin-2 of the plasma membrane via the N-and C-terminal domains, and is involved in neuronal golgi activity and vesicle trafficking. In addition, in the presynaptic area, α -synuclein affects dopamine storage by interacting with the brain vacuole and the phospholipid membrane, and regulates dopamine release by interacting with the vesicular membrane. Alpha-synuclein is also essential for the normal development of cognitive function, and experiments show that mice with the alpha-synuclein expression gene knocked out are impaired in spatial learning and working memory.

2. Alpha-synuclein related diseases

Diseases caused by abnormal synuclein are called synucleinopathies, also known as lewy body diseases. Currently, lewy body disease remains a common cause of dyskinesia and cognitive deterioration in the elderly population. Synucleinopathies are characterized by degeneration of the dopamine system, altered motility, cognitive impairment, and formation of Lewy Bodies (LBs) and/or lewy neurites. Synucleinopathies include neuronal and neurodegenerative diseases such as parkinson's disease (PD, including idiopathic parkinson's disease), lewy body dementia (DLB, also known as diffuse lewy body disease, DLBD), combined alzheimer's and parkinson's diseases, pure autonomic failure and Multiple System Atrophy (MSA). Some non-motor signs and symptoms are considered to be precursors for the development of synucleinopathies. These early symptoms include REM sleep behavior disorder (RBD), loss of smell, and constipation. In the advanced stages of disease progression (e.g. PD and DLB), however, a significant increase in soluble α -synuclein oligomers in the cerebrospinal fluid of PD and DLB patients can usually be detected, as well as insoluble fibrous precipitates formed by α -synuclein, i.e. Lewy Bodies (LBs).

Alpha-synuclein is a main component of lewy bodies and nerve inclusion bodies, is an important mechanism for causing the onset of Parkinson diseases and Lewy body diseases, and can cause rare familial Parkinson diseases due to alpha-synuclein gene mutation. Morphological and neural changes caused by alpha-synuclein overexpression in animal models of different species such as human, mouse and drosophila also confirm the important role of alpha-synuclein in the occurrence of lewy body diseases.

Abnormalities in alpha-synuclein are also associated with the pathogenesis of some Alzheimer's Disease (AD). The link between α -synuclein and AD was first to find a "non-starch-like component" (NAC) in plaques in the brains of AD patients, which component was later confirmed to be a fragment of α -synuclein. Alpha-synuclein promotes the aggregation of A beta in vitro, but does not increase the deposition of amyloid plaques in vivo. Although α -synuclein had no effect on plaque formation in vivo, it was found by comparing transgenic mice expressing either the human APP/Α β or α -synuclein gene alone with double transgenic mice that double transgenic mice exhibited loss of neuronal synapses, reduction of choline acetyltransferase positive neurons, and more severe cognitive impairment than single transgenic mice. This study also revealed that a β greatly promotes the aggregation of α -synuclein in vitro and in vivo.

3. Pathological mechanism of alpha-synuclein

(1) Aggregation of alpha-synuclein

Alpha-synuclein generally exists as a soluble monomeric protein, but is in a disordered state in solution and lacks a stable tertiary structure. Soluble α -synuclein may be degraded by ubiquitin-protease system (UPS) pathway and Chaperonin Mediated Autophagy (CMA). However, under pathological conditions, alpha-synuclein monomers tend to aggregate and polymerize into oligomers, thereby forming insoluble fibers with higher molecular weight. These highly phosphorylated and ubiquitinated insoluble fibers affect mitochondrial metabolism, reduce the autophagy efficiency of macrophages, and produce neuronal toxicity leading to neuronal cell death. The accumulation and folding of alpha-synuclein is influenced by a number of factors, including phosphorylation, mitochondrial and lysosomal dysfunction, oxidative and nitriding stress. In addition, the cleavage of the C-terminal of alpha-synuclein by synuclein also leads to the formation of oligomers of monomeric alpha-synuclein and is closely related to the severity of the disease.

(2) Alpha-synuclein removal

The clearance of alpha-synuclein in the lewy body can be performed by the proteasome. Studies have shown that loss of function of ubiquitin-protease system (UPS) or proteasome leads to aggregation of α -synuclein, inclusion body formation and neuronal degeneration in PD patients. Similarly, by inhibiting the function of proteasome or overexpressing alpha-synuclein, increased cell death will also result.

UPS-degraded proteins usually have a relatively short half-life (< 10 hours), whereas α -synuclein generally has a half-life of around 16 hours. However, some studies have not detected an increase in α -synuclein in the presence of proteasome inhibitors, probably because α -synuclein is degraded by lysosomes that degrade proteins with longer half-lives. This degradation is called chaperonin-mediated autophagy (CMA) and involves the hsc70 chaperone protein and the lysosomal membrane receptor lamp2a. Wild-type alpha-synuclein can be degraded by both UPS and CMA. However, studies on the substantia nigra of the brain of PD patients have shown that insoluble α -synuclein and α -synuclein polymers bind to proteasomes and inhibit their function. Mutations in alpha-synuclein and oxidized forms of dopamine also impair the CMA pathway leading to upregulation of autophagy. Functional clearance disorder of alpha-synuclein and the damage of protein quality control system caused by the functional clearance disorder, especially autophagy, become a significant pathogenic mechanism of neurodegeneration.

Other studies have shown that chaperonin-mediated autophagy, also known as giant autophagy, plays an important role in the degradation of α -synuclein polymers. Based on these studies, it was found that wild-type α -synuclein can be cleared by the CMA system, whereas polymeric α -synuclein can only be cleared by giant autophagy. However, it is also contemplated that interference with the autophagy system itself, as seen in a degenerative cell stress environment, may also contribute to the toxicity of alpha-synuclein.

4. Therapeutic anti-alpha-synuclein antibodies

In fact, the presence of aggregates is usually detected during the intervention, and autoantibodies do not completely eliminate aggregates, even if they improve symptoms at this time, which is not statistically different when comparing PD patients with normal autoantibodies on a large scale. With the intensive research on the clearance mechanism of alpha-synuclein, the clearance mediated by antibodies aiming at alpha-synuclein becomes the breakthrough point for treating alpha-synuclein diseases. In animal experiments, the antibody combined with the C terminal of the alpha-synuclein can reduce the aggregation of the alpha-synuclein in the brain of a PD model mouse and relieve pathological symptoms. While lysosomal inhibitors can reduce antibody protection, suggesting that autophagy-mediated clearance mechanisms are involved in the degradation of α -synuclein aggregates.

In addition, the ability to effectively inhibit the aggregation of alpha-synuclein has also become one of the breakthrough for the treatment of alpha-synuclein disease. The antibody is used for inhibiting the aggregation of the alpha-synuclein, so that the toxicity of the alpha-synuclein polymer to neurons can be effectively reduced, the damage of the neurons can be reduced, and meanwhile, the reduction of the polymer can promote the clearance of an UPS (uninterrupted Power supply) and CMA (CMA) system in vivo to the alpha-synuclein and play a role in protecting neuron cells. Alleviate and slow down the symptoms of the alpha-synuclein disease, and play a good role in treatment.

The prior art studies of antibodies against alpha-synuclein have focused primarily on distinguishing differences in affinity for alpha-synuclein monomers and polymers, binding to different forms and epitopes of alpha-synuclein, and the source of the antibody (e.g., hybridoma pathways and autoantibody sources). Furthermore, most antibodies exhibit their function only by binding to α -synuclein monomers or polymers (e.g., using ELISA, immunohistochemical, immunoblotting, etc.), but the efficacy of antibodies in pharmaceutical applications is not determined, and only a small amount of antibodies demonstrate efficacy in animals, but are still in the initial stages of the study. However, in the development process of antibody pharmaceuticals, the intervention of in vitro cytology is indispensable, can make up the deficiency of the research on the cytological activity in animals, and provides theoretical and practical basis for the screening of new drug molecules.

According to the pathological mechanism of alpha-synuclein, the aggregation of alpha-synuclein monomers and the toxicity of polymers are the causes for triggering a series of diseases, so that the development of a high-efficiency anti-alpha-synuclein antibody, effective prevention of the aggregation of the alpha-synuclein monomers and elimination of the polymers are the basis of the treatment of the alpha-synuclein diseases, and the method has important significance.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide an anti-alpha-synuclein monoclonal antibody which has high and proper affinity, high specificity and good biological activity to alpha-synuclein monomers and polymers and an application thereof.

In order to realize the purpose, the technical scheme of the invention is as follows: the monoclonal antibody can be combined with alpha-synuclein monomers of human, mice and monkeys and alpha-synuclein polymers of human, has higher affinity for the alpha-synuclein monomers and the polymers of the human, can inhibit the polymerization of the alpha-synuclein monomers in a dose-dependent manner and promote the phagocytosis of microglia on the alpha-synuclein polymers, and simultaneously reduces the deposition of the alpha-synuclein polymers by inhibiting and eliminating two mechanisms to relieve the neurotoxic effect caused by the polymerization of the alpha-synuclein.

Specifically, first, the present invention provides an anti- α -synuclein monoclonal antibody or fragment thereof capable of specifically binding to α -synuclein monomer and α -synuclein aggregate, the monoclonal antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, the CDR region sequences of the heavy chain variable region being as follows:

(1) CDR1: has any sequence shown in SEQ ID NO.1-3 or has an amino acid sequence of protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 1-3;

(2) CDR2: has any sequence shown in SEQ ID NO.4-6 or has an amino acid sequence of protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 4-6;

(3) CDR3: has any sequence shown in SEQ ID NO.7-9 or has the amino acid sequence of the protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 7-9.

The CDR region sequence of the light chain variable region of the anti-alpha-synuclein monoclonal antibody or the fragment thereof is as follows:

(1) CDR1: has any sequence shown in SEQ ID NO.10-12 or has an amino acid sequence of the protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 10-12;

(2) CDR2: has any sequence shown in SEQ ID NO.13-15 or has an amino acid sequence of the protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 13-15;

(3) CDR3: has any sequence shown in SEQ ID NO.16-18 or has an amino acid sequence of the protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 16-18.

The CDR region of the monoclonal antibody determines the affinity and specificity of the antibody for combining with antigen to a great extent, and the CDR region with the sequence is favorable for improving the binding affinity of the monoclonal antibody and alpha-synuclein, reducing the binding affinity with beta-synuclein and gamma-synuclein and improving the specificity.

Further, the sequences of the FR regions of the heavy chain variable regions of the anti-alpha-synuclein monoclonal antibodies or fragments thereof are as follows:

(1) FR1: has any sequence shown in SEQ ID NO.19-21 or has an amino acid sequence of the protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 19-21;

(2) FR2: has any sequence shown in SEQ ID NO.22-24 or has an amino acid sequence of protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 22-24;

(3) FR3: has any sequence shown in SEQ ID NO.25-27 or has an amino acid sequence of the protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 25-27;

(4) FR4: has any sequence shown in SEQ ID NO.28-30 or has the amino acid sequence of the protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 28-30.

Further, the sequences of the FR regions of the light chain variable regions of the anti-alpha-synuclein monoclonal antibodies or fragments thereof are as follows:

(1) FR1: has any sequence shown in SEQ ID NO.31-33 or has an amino acid sequence of protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 31-33;

(2) FR2: has any sequence shown in SEQ ID NO.34-36 or has an amino acid sequence of the protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 34-36.

(3) FR3: has any sequence shown in SEQ ID NO.37-39 or has an amino acid sequence of protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 37-39;

(4) FR4: has any sequence shown in SEQ ID NO.40-42 or has the amino acid sequence of the protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any sequence shown in SEQ ID NO. 40-42.

Preferably, the heavy chain variable region of the anti-alpha-synuclein monoclonal antibody or fragment thereof of the present invention has any one of the sequences shown in SEQ ID Nos. 43-45 or has an amino acid sequence of a protein having the same function obtained by deleting, replacing or inserting one or more amino acids in any one of the sequences shown in SEQ ID Nos. 43-45.

Preferably, the variable region of the light chain of the monoclonal antibody or the fragment thereof against alpha-synuclein has any one sequence shown in SEQ ID NO.46-48 or an amino acid sequence of a protein with the same function obtained by deleting, replacing or inserting one or more amino acids in any one sequence shown in SEQ ID NO. 46-48.

In the present invention, the above-mentioned "amino acid sequence of a protein having the same function by deletion, substitution, or insertion of one or more amino acids" refers to a sequence which differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule, and may be a "conservatively modified variant" or modified by "conservative amino acid substitution". "conservatively modified variants" or "conservative amino acid substitutions" refer to amino acid substitutions known to those of skill in the art that are made without typically altering the biological activity of the resulting molecule. In general, one skilled in the art recognizes that a single amino acid substitution in a non-essential region of a monoclonal antibody function does not substantially alter biological activity. Exemplary substitutions are preferably made according to the substitutions shown below:

TABLE 1 exemplary conservative amino acid substitutions

In the invention, the "plurality" of the amino acid sequences of the proteins with the same function obtained by deleting, replacing or inserting one or more amino acids "is more than or equal to 2 and less than or equal to 30.

In a preferred embodiment of the present invention, the anti- α -synuclein monoclonal antibody or fragment thereof comprises a combination of a light chain variable region and a heavy chain variable region selected from any one of the following:

(1) The sequence of the light chain variable region is shown as SEQ ID NO.46 or is a sequence which has at least 90 percent of homology with the sequence shown as SEQ ID NO.46, and the sequence of the heavy chain variable region is shown as SEQ ID NO.43 or is a sequence which has at least 90 percent of homology with the sequence shown as SEQ ID NO. 43; preferably, the homology is at least 95%; more preferably at least 98%;

(2) The sequence of the light chain variable region is shown as SEQ ID NO.47 or is a sequence which has at least 90 percent of homology with the sequence shown as SEQ ID NO.47, and the sequence of the heavy chain variable region is shown as SEQ ID NO.44 or is a sequence which has at least 90 percent of homology with the sequence shown as SEQ ID NO. 44; preferably, the homology is at least 95%; more preferably at least 98%;

(3) The light chain variable region has a sequence shown in SEQ ID NO.48 or a sequence having at least 90% homology with the sequence shown in SEQ ID NO.48, and the heavy chain variable region has a sequence shown in SEQ ID NO.45 or a sequence having at least 90% homology with the sequence shown in SEQ ID NO. 45; preferably, the homology is at least 95%; more preferably at least 98%.

The above sequence having at least 90% homology or at least 95% or at least 98% homology means homology of not less than 90%, 95% or 98% obtained by homology alignment with the sequence of the antibody and having the same function as the antibody, i.e., being capable of binding to α -synuclein monomer and α -synuclein aggregate.

On the basis of the light chain variable region and the heavy chain variable region of the monoclonal antibody or the fragment thereof against alpha-synuclein, one skilled in the art can construct a full-length monoclonal antibody molecule by using the conventional technical means in the art as required.

Preferably, the full-length monoclonal antibody molecule is of the IgG or IgM class. The IgG type comprises one of IgG1, igG2, igG3 and IgG 4.

Preferably, the light chain of the monoclonal antibody is of the kappa type.

Further, the present invention provides a hybridoma cell producing the anti-a-synuclein monoclonal antibody or fragment thereof.

Further, the invention also provides an antigen which has an amino acid sequence shown as SEQ ID NO.61, SEQ ID NO.62 or SEQ ID NO. 63.

Further, the present invention also provides a nucleic acid encoding the anti-a-synuclein monoclonal antibody or fragment thereof.

Based on the amino acid sequences of the heavy chain and the light chain, one skilled in the art can design different nucleotide sequences capable of encoding the heavy chain and the light chain according to the codon preference of an expression host, and all nucleic acids capable of encoding the light chain and the heavy chain provided by the present invention are within the protection scope of the present invention.

Preferably, the nucleic acid encoding the heavy chain variable region and the light chain variable region of the anti- α -synuclein monoclonal antibody or fragment thereof has any one of the following nucleotide sequences:

(1) The nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO.49-51 or a complementary sequence thereof; the nucleic acid encoding the light chain variable region is shown in SEQ ID NO.52-54 or is the complementary sequence thereof;

wherein the complementary sequences shown as SEQ ID NO.49-51 are shown as SEQ ID NO.55-57, and the complementary sequences shown as SEQ ID NO.52-54 are shown as SEQ ID NO. 58-60.

(2) A sequence which encodes the same monoclonal antibody or a fragment thereof as the nucleotide sequence of (1) but differs from the nucleotide sequence of (1) due to the degeneracy of the genetic code

(3) A sequence having at least 80% homology to the sequence of (1) or (2).

(4) And (2) a nucleotide sequence which is obtained by replacing, deleting or adding one or more nucleotides in the nucleotide sequence shown in (1), (2) or (3) and has the same or similar functions with the nucleotide sequence shown in (1), (2) or (3).

In the invention, the number of the nucleotides is more than or equal to 2 and less than or equal to 30 in the 'one or more nucleotides are replaced, deleted or added'.

The invention also provides biological materials containing the nucleic acid, wherein the biological materials comprise expression cassettes, vectors, transposons, host cells, engineering bacteria or transgenic cell lines.

The vector includes but is not limited to cloning vector, expression vector, plasmid vector, and all vectors containing the nucleic acid encoding the anti-alpha-synuclein monoclonal antibody or fragment thereof are within the scope of the present invention.

The host cell or transgenic cell line may be a cell or cell line derived from a microorganism, a plant or an animal, and all host cells or transgenic cell lines containing the nucleic acid encoding the anti-alpha-synuclein monoclonal antibody or fragment thereof or the vector comprising the nucleic acid are within the scope of the present invention.

Further, the present invention provides a complex or conjugate comprising or prepared from said anti-a-synuclein monoclonal antibody or fragment thereof.

Preferably, the complex is obtained by chemical labeling or biological labeling of the anti-alpha-synuclein monoclonal antibody or fragment thereof.

The chemical labels include, but are not limited to, isotopes, immunotoxins, and/or chemical drugs.

The biomarkers include, but are not limited to, biotin, avidin, or enzyme markers.

Preferably, the conjugate is obtained by coupling the anti-alpha-synuclein monoclonal antibody or fragment thereof or the chemically or biologically labeled complex with a solid or semi-solid medium.

The solid medium or non-solid medium includes, but is not limited to, colloidal gold, polystyrene plates or beads.

Further, the present invention provides any one of the following uses of the anti-a-synuclein monoclonal antibody or fragment thereof or the nucleic acid encoding the anti-a-synuclein monoclonal antibody or fragment thereof or the biological material containing the nucleic acid or the complex or conjugate:

(1) The use in the manufacture of a medicament for the prevention or treatment of an alpha-synucleinopathy;

(2) The use in the manufacture of a product for alleviating the polymer toxicity of α -synuclein or controlling the development of α -synucleinopathies;

(3) Use in the manufacture of a product for inhibiting polymerization of alpha-synuclein monomers or formation of oligomers, fibrils, cellulosomes, lewy bodies of alpha-synuclein;

(4) Use in the preparation of a product for reducing the accumulation of alpha-synuclein aggregates;

(5) Use in the manufacture of a product for promoting the phagocytosis and clearance of α -synuclein polymers by microglia;

(6) The application in preparing alpha-synuclein, alpha-synuclein polymer and alpha-synuclein antibody detection reagents or kits.

Further, the invention provides a medicament which comprises the monoclonal antibody or the fragment thereof for resisting alpha-synuclein or is prepared by the monoclonal antibody or the fragment thereof for resisting alpha-synuclein.

The medicine has any one of the following functions:

(1) For the prevention or treatment of alpha-synucleinopathies;

(2) For alleviating polymer toxicity or controlling development of alpha-synuclein disease;

(3) For inhibiting polymerization of alpha-synuclein monomers or formation of oligomers, fibrils, fibroids, lewy bodies of alpha-synuclein;

(4) For reducing the accumulation of alpha-synuclein aggregates, fibrils, fibroids, lewy bodies.

(5) Is used for promoting the phagocytosis and the clearance of alpha-synuclein polymers by microglia.

Preferably, the IC50 value of the antibody concentration of the anti-alpha-synuclein monoclonal antibody or the fragment thereof for blocking alpha-synuclein monomer polymerization is 2.693-3.678 mu g/ml.

Preferably, the dosage of the anti-alpha-synuclein monoclonal antibody or the fragment thereof mediating the phagocytosis of alpha-synuclein polymers by microglia is 2 to 5 mu g/mL in terms of antibody concentration.

In the present invention, the alpha-synucleinopathies include, but are not limited to, parkinson's disease, lewy body dementia, combined Alzheimer's and Parkinson's disease, pure autonomic failure, multiple system atrophy.

Preferably, the medicament is in the form of injection or powder injection.

In one embodiment, the concentration of the anti- α -synuclein monoclonal antibody or fragment thereof in the injection is 5 μ g/mL.

Proved by experimental verification, the monoclonal antibody or the fragment thereof for resisting alpha-synuclein provided by the invention can be combined with alpha-synuclein monomers and alpha-synuclein polymers, so that the monoclonal antibody can be used for detecting the alpha-synuclein monomers or the alpha-synuclein polymers. In addition, because the expression of the alpha-synuclein in cerebrospinal fluid, blood and intestinal tracts of patients with synuclein is abnormal, the monoclonal antibody or the fragment thereof for resisting the alpha-synuclein provided by the invention can be used for preparing a kit for detecting the alpha-synuclein marker.

The detection can be carried out by antigen or antibody detection methods conventional in the art, including but not limited to ELISA.

Further, the present invention provides a detection reagent or kit for detecting a-synuclein monomer or polymer thereof, comprising the anti-a-synuclein monoclonal antibody or fragment thereof, or comprising the complex or conjugate.

In order to facilitate the detection of the alpha-synuclein, the detection kit further comprises one or more of coating buffer solution, washing solution, confining solution and developing solution.

The coating buffer solution, the washing solution, the blocking solution and the developing solution can adopt the formula commonly used in the field, such as: the coating buffer solution is carbonate buffer solution; the washing liquid contains PBS, tween, sodium chloride, potassium chloride, disodium hydrogen phosphate and dipotassium hydrogen phosphate; the blocking solution contains PBS and BSA; the color development liquid contains TMB solution, substrate buffer solution and stop solution; the substrate buffer solution contains citric acid and disodium hydrogen phosphate; the stop solution is aqueous hydrogen peroxide solution.

When used to detect cells expressing α -synuclein on their surface, the assay kit also included PBS, goat anti-mouse IgG Fc, and a TITC secondary antibody.

Furthermore, the invention also provides a preparation method of the anti-alpha-synuclein monoclonal antibody or fragment thereof, which comprises the steps of introducing nucleic acid encoding the anti-alpha-synuclein monoclonal antibody or fragment thereof into a host cell, expressing the anti-alpha-synuclein monoclonal antibody or fragment thereof, and separating and purifying to obtain the product.

The invention also provides a method for diagnosing diseases, which uses the detection kit provided by the invention to detect the expression and accumulation of alpha-synuclein, and judges whether the diseases related to the alpha-synuclein exist according to the amount of the alpha-synuclein in cerebrospinal fluid and plasma and by referring to normal controls (1.57 +/-0.6 ng/ml (cerebrospinal fluid) and 59.54 +/-35.1 ng/ml (plasma)).

The present invention also provides a method for preventing or treating a disease, comprising: the medicaments of the present invention are administered.

Such diseases include, but are not limited to, parkinson's disease (PD, including idiopathic parkinson's disease), dementia with lewy bodies (DLB), combined alzheimer's and parkinson's disease, pure autonomic failure and Multiple System Atrophy (MSA).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in Molecular Biology (Ausubel).

Wherein the abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

An "antibody" refers to a protein composed of one or more polypeptides that specifically bind to an antigen. One form of antibody constitutes the basic building block of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains are joined together and are responsible for binding to antigen, while the constant regions are responsible for the effector functions of the antibody.

The "variable region" of an antibody heavy or light chain is the N-terminal mature region of the chain. The types of antibodies currently known include kappa and lambda light chains, as well as alpha, gamma (IgG 1, igG2, igG3, igG 4), delta, epsilon and mu heavy chains or other class equivalents thereof. Full-length immunoglobulin "light chains" (about 25kDa or about 214 amino acids) contain a variable region of about 110 amino acids at the NH 2-terminus, and a kappa or lambda constant region at the COOH-terminus. The full-length immunoglobulin "heavy chain" (about 50kDa or about 446 amino acids) likewise comprises a variable region (about 116 amino acids), and one of the heavy chain constant regions, e.g., gamma (about 330 amino acids).

"antibody" includes any isotype of antibody or immunoglobulin, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like. The antibodies can also be bound to a solid support including, but not limited to, polystyrene plates or beads, and the like.

The term "monoclonal antibody" refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The medicament contains at least one functional component and also comprises a medicinal carrier. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffered solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.

As used herein, "CDR region" or "CDR" refers to the hypervariable regions of the heavy and light chains of an immunoglobulin, as defined by Kabat et al (Kabat et al, sequences of proteins of immunological interest,5th Ed., U.S. department of Health and Human Services, NIH,1991, and later). There are three heavy chain CDRs and three light chain CDRs. As used herein, the term CDR or CDRs is intended to indicate one of these regions, or several or even all of these regions, which comprise the majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope, as the case may be.

The invention has the following beneficial effects:

the anti-alpha-synuclein monoclonal antibody provided by the invention can specifically bind to monomers and polymers of alpha-synuclein, has higher affinity to the monomers and polymers of the alpha-synuclein (the highest affinity to human alpha-synuclein monomers is 15.6nM; the highest affinity to human alpha-synuclein polymers is 8.9 nM), and has no binding force to beta-synuclein and gamma-synuclein; can effectively inhibit the polymerization of alpha-synuclein monomers (the inhibition rate is up to 98.78 percent, and the IC50 is 2.693 mu g/ml), promote the clearance of microglia to the formed alpha-synuclein polymers (the phagocytic rate of BV2 microglia to the human alpha-synuclein polymers is up to 89.55 percent, and the IC50 is 1.97 mu g/ml), protect neuron cells from the toxicity of the alpha-synuclein polymers, and can be used for preventing, treating and diagnosing alpha-synuclein related diseases and symptoms, such as Parkinson's disease, lewy body dementia, combined Alzheimer's disease and Parkinson's disease, pure autonomic nerve failure, multi-system atrophy and the like.

The antibodies of the present invention exhibit superior biological activity in addition to the beneficial effects described above, relative to prior art anti-a-synuclein monoclonal antibodies. When the biological activity is verified, the antibody-mediated clearance function is verified at a cellular level through a cytological activity detection method, and the function of the antibody in the aspect of antibody-mediated microglia phagocytosis is completely verified. Accords with the pathological pathogenesis of alpha-synuclein related diseases and can be used as the basis and the foundation for screening patent medicine molecules. In the aspect of biological activity, the anti-alpha-synuclein monoclonal antibody plays a role of treating both principal and secondary aspects of diseases for the accumulation of alpha-synuclein polymers, inhibits the polymerization of monomers on the basis of the principle, eliminates the polymers on the basis of the principle, and activates the intrinsic UPS and CMA clearance systems in a living body, so that the in-vivo clearance systems gradually restore balance, and toxic and side effects caused by continuous or excessive administration are avoided. Based on the requirement of a drug-forming molecule on the affinity of the antibody, the antibody has higher affinity to both a monomer and a polymer, can ensure effective combination with an alpha-synuclein monomer and a polymer, but still has difference, and can increase the difference of the monomer and the polymer through affinity maturation again, thereby ensuring that the combination of the monomer and the polymer is achieved, the monomer is prevented from polymerizing, the combination of the polymer and the microglia in the brain is promoted to phagocytize and remove the polymer. Meanwhile, the affinity of the antibody is moderate, and the high affinity cannot be generated, so that on one hand, the continuous elimination of peripheral alpha-synuclein caused by the high affinity of the monomer is avoided, and on the other hand, the side effects of intracerebral inflammation and the like caused by the continuous combination of the antibody and intracerebral alpha-synuclein polymers and the excessive activation of intracerebral macrophages are also avoided. Therefore, the moderate affinity of the antibody of the invention can ensure that the polymerization of monomers is prevented, and polymers are properly cleared, so that the brain inflammation is not caused, and the alpha-synuclein polymers can be cleared through the mediation of the antibody on one hand and through the UPS and CMA systems in the body on the other hand by activating a clearing mechanism, so that the alpha-synuclein polymers are maximally cleared, and the toxic and side effects caused by the antibody are minimized.

Drawings

FIG. 1 shows the detection of purified human, monkey and murine α -synuclein-linker-7 his fusion proteins by non-reducing SDS-PAGE electrophoresis in example 1 of the present invention; marker: protein molecular weight markers; lane1: human α -synuclein-linker-His; lane2 monkey alpha-synuclein-linker-His; lane3 murine α -synuclein-linker-His.

FIG. 2 shows the detection of purified monoclonal antibodies 2A1, 2B1 and 2C1 by non-reducing SDS-PAGE in example 3 of the present invention; marker, protein molecular weight Marker; lane1:2A1 purified antibody; lane2:2B1 purified antibody; lane3:2C1 purified antibody.

FIG. 3 shows the total RNA electrophoresis detection in example 4 of the present invention; m: DL2000 molecular weight markers, lane1-3, are electrophoretic bands for 2A1, 2B1 and 2C1 total RNA, respectively.

FIG. 4 shows the results of agarose electrophoresis detection of the heavy chain variable region and the light chain variable region of the antibodies 2A1, 2B1 and 2C1 amplified by PCR in example 4 of the present invention; a: amplifying agarose electrophoresis detection results of the heavy chain variable regions of the candidate antibodies by PCR; m: DL2000 molecular weight marker, lane1-3 for 2A1, 2B1, 2C1 heavy chain variable region strip; b: amplifying the agarose electrophoresis detection result of the candidate antibody light chain variable region by PCR; m: DL2000 molecular weight marker, lane1-3 is 2A1, 2B1, 2C1 light chain variable region band.

FIG. 5 is a graph showing the effect of the antibodies 2A1, 2B1, 2C1 on inhibiting the polymerization of human alpha-synuclein monomers in example 9 of the present invention.

FIG. 6 is a graph showing the effect of antibodies 2A1, 2B1, 2C1 on microglial phagocytosis of human α -synuclein polymers in example 10.

Detailed Description

The invention provides an anti-alpha-synuclein monoclonal antibody and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The heavy chain constant region of the anti-alpha-synuclein monoclonal antibodies 2A1, 2B1 and 2C1 provided by the invention is mouse IgG1, and the light chain constant region is the constant region of a mouse kappa chain, wherein the amino acid sequence of the heavy chain variable region of the antibody 2A1 is shown as SEQ ID No.43, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 46; the amino acid sequence of the heavy chain variable region of the antibody 2B1 is shown as SEQ ID NO.44, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 47; the amino acid sequence of the heavy chain variable region of the antibody 2C1 is shown as SEQ ID NO.45, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 48.

The anti-alpha-synuclein monoclonal antibodies 2A1, 2B1 and 2C1 provided by the invention can be combined with human alpha-synuclein monomers and polymers, the detection of the effect is carried out by an ELISA method, and the result is obtained by OD ₄₅₀ Values represent, the invention provides ODs of antibodies binding to human alpha-synuclein monomers and polymers ₄₅₀ The value is not less than 0.5.

The anti-alpha-synuclein monoclonal antibodies 2A1, 2B1 and 2C1 provided by the invention specifically bind to alpha-synuclein and do not bind to beta-synuclein and gamma-synuclein of synuclein family, the effect is measured by an ELISA method, and the result is measured by OD ₄₅₀ The values are represented.

The anti-alpha-synuclein monoclonal antibodies 2A1, 2B1 and 2C1 provided by the invention have species cross property, can be combined with human, monkey and mouse alpha-synuclein, the detection of the effect is carried out by an ELISA method, and the result is obtained by OD ₄₅₀ The values are expressed.

The affinity between the anti-alpha-synuclein monoclonal antibodies 2A1, 2B1 and 2C1 and the alpha-synuclein provided by the invention is characterized by KD (equilibrium dissociation constant), and the KD value of the anti-alpha-synuclein monoclonal antibody provided by the invention is not higher than 30nM.

The anti-alpha-synuclein monoclonal antibodies 2A1, 2B1 and 2C1 provided by the invention can block the polymerization of alpha-synuclein monomers in a dose-dependent manner, and the blocking effect is expressed by the percentage of inhibition rateThe antibody inhibition rate is not less than 50%, and the dosage effect IC is ₅₀ The value was not higher than 2.693. Mu.g/ml.

The anti-alpha-synuclein monoclonal antibodies 2A1, 2B1 and 2C1 provided by the invention can mediate the phagocytosis of alpha-synuclein polymers by microglia in a dose-dependent manner, and the result is expressed by the percentage of phagocytosis rate. The phagocytosis rate of the antibody mediated microglia to alpha-synuclein polymers is not lower than 50%, and the agent effect IC50 value is not higher than 1.97 mu g/ml.

The anti-alpha-synuclein monoclonal antibodies 2A1, 2B1 and 2C1 provided by the invention can effectively inhibit the polymerization of alpha-synuclein monomers to polymers; and the formed alpha-synuclein polymer can promote the phagocytosis and the elimination of the alpha-synuclein polymer by microglia through the antibody-mediated action, and relieve or slow down the toxic action of the alpha-synuclein polymer on neuron cells. Therefore, the alpha-synuclein monoclonal antibody provided by the invention not only can play a role in treatment, but also can effectively prevent alpha-synucleinopathy.

Alpha-synucleinopathies described herein include, but are not limited to, parkinson's disease (PD, including idiopathic Parkinson's disease), lewy body Dementia (DLB), combined Alzheimer's and Parkinson's disease, pure autonomic failure and Multiple System Atrophy (MSA).

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of human, monkey, murine alpha-synuclein protein

1. Human, monkey and mouse alpha-synuclein gene synthesis and expression vector construction

The amino acid sequences of human, monkey and mouse alpha-synuclein full-length proteins are respectively fused with a connecting peptide-7 his amino acid sequence, and the amino acid sequences of human, monkey and mouse alpha-synuclein are respectively shown as SEQ ID NO.61, SEQ ID NO.62 and SEQ ID NO. 63. Designing a coding nucleotide sequence corresponding to human, monkey and mouse alpha-synuclein full-length fusion protein (alpha-synuclein-connecting peptide-7 his), adding HindIII enzyme cutting sites and Kozak sequence GCCGCCACC at the 5 'end, adding termination codons TAG and EcoRI enzyme cutting sites at the 3' end, amplifying coding DNA of the alpha-synuclein fusion protein by PCR, and cloning into a pUC57simple vector to obtain human, monkey and mouse pUC57 simple-alpha-synuclein-connecting peptide-7 his plasmid.

Carrying out enzyme digestion (HindIII and EcoRI) on plasmid pUC57 simple-alpha-synuclein-connecting peptide-7 his of human, monkey and mouse and vector pcDNA3.1, carrying out electrophoretic recovery on the obtained fusion gene fragment alpha-synuclein-connecting peptide-7 his, carrying out ligation reaction and recombination with vector pcDNA3.1 to construct an expression plasmid of human, monkey and mouse alpha-synuclein full-length fusion protein (alpha-synuclein-connecting peptide-7 his), which comprises the following specific steps:

pcDNA3.1-human α -synuclein-linker-7 his;

pcDNA3.1-monkey α -synuclein-linker-7 his;

pcDNA3.1-murine α -synuclein-linker-7 his.

2. Instantaneous expression

Transient expression was performed on pcDNA3.1-human α -synuclein-linker-7 his, pcDNA3.1-monkey α -synuclein-linker-7 his, and pcDNA3.1-murine α -synuclein-linker-7 his.

Using FreeStyle ^TM 293F cells were transiently expressed in Freestyle medium. 24 hours before transfection, 6X 10 were inoculated in 125ml Erlenmeyer flasks ⁵ 293F cells per ml, 5% CO at 37 ℃ ₂ The incubator was incubated with a shaker (120 rpm/min). When in transfection, 60 mu l of 293fectin is firstly added into 1ml of OPTIMEM, and after being fully and uniformly mixed, the mixture is incubated for 5 minutes at room temperature; the recombinant light chain plasmid and the recombinant heavy chain plasmid were simultaneously mixed at a ratio of 1. Then, DNA and 293fectin were mixed well to a total volume of 2ml, incubated at room temperature for 15 minutes, and then the mixture was mixedAdding the total composition into cell culture well, mixing, and adding CO at 37 deg.C 5% ₂ Shaking and culturing at 130rpm in incubator for 7 days. And (4) centrifuging the culture solution at a high speed, and performing vacuum filtration by using a microporous filter membrane.

3. Protein purification

Purifying by nickel column (protein purification liquid chromatography system/AKTA Purifier 10, GE) according to the operation method provided by the manufacturer to obtain purified human, monkey and mouse alpha-synuclein-connecting peptide-7 his fusion protein. The purity of the purified fusion protein was determined by SDS-PAGE as shown in FIG. 1.

EXAMPLE 2 preparation of hybridoma cells

1. Immunization of mice

The human alpha-synuclein-connecting peptide-7 his fusion protein prepared in example 1 was used as an antigen, and the antigen and an adjuvant were mixed at a volume ratio of 1. The first immunization was carried out by emulsifying the antigen with Freund's complete adjuvant, and after 2 weeks, the second immunization was carried out by emulsifying the antigen with Freund's incomplete adjuvant and intraperitoneally injecting the same in an amount of 150. Mu.g per mouse and a volume of 200. Mu.L per injection.

3 days after the third immunization, the mice are subjected to orbital blood collection, a small amount of blood samples are taken for serum titer detection, and the mice are subjected to boosting immunization after the serum titer reaches 1.

2. Preparation of cells for fusion (myeloma cells)

Myeloma cells P3X63Ag8.653 for fusion were recovered three weeks in advance, and cultured for two weeks in a DMEM medium containing 1X 8-azaguanine and 10% fetal bovine serum, and then cultured in DMEM medium containing 10% fetal bovine serum one week before fusion, maintaining the density of P3X63Ag8.653 at 70% -80% until the day of fusion.

3. Cell fusion and HA screening:

acquisition and preparation of splenocytes: 2 mice after boosting were taken, immune serum was collected, and then the neck was pulled to be killed and soaked in 75% alcohol for 2-3 minutes. The skin and peritoneum were cut from the ventral side of the immunized mice, exposing the spleen. Removing peripheral tissues by using a tip to obtain a spleen, grinding by using a grinding rod, and filtering by using a cell screen to prepare a single cell suspension.

Cell fusion pretreatment: P3X63Ag8.653 in the culture flask was collected, centrifuged at 1000rpm/5min and the supernatant discarded, resuspended and then viable cells counted. Spleen cell suspensions were centrifuged at 2000rpm/5min, and the supernatants were discarded and resuspended and viable cells counted. The number of p3x63ag8.653 viable cells, the number of splenocytes viable cells were recorded.

Cell fusion: according to the ratio of splenocytes: p3x63ag8.653=2, the cells were mixed at a ratio of p3x63ag8.653= 1, centrifuged at 2000rpm/5min, the supernatant was decanted, the cells were sedimented, 1mL of a preheated 50% peg1500 solution was slowly dropped into a 37 ℃ water bath, the bottom of the tube was shaken in 37 ℃ water with a circling motion, the above operation time was controlled to about 1min, the reaction was left to stand for 30s, a 37 ℃ preheated DMEM medium was added into the tube from slow to fast, and the reaction was terminated. And (4) centrifuging the cell suspension after the reaction is stopped at 800rpm for 3min, discarding the supernatant, and gently shaking and scattering the cell precipitate.

HAT medium screening: HAT selection medium was prepared containing 1 XHAT, 1 XHE-streptomycin, 15% fetal bovine serum and 85% DMEM medium. The rat hybridoma cells and feeder cells were resuspended in HAT screening medium as described above and mixed. The cell suspension was added to 20 96-well cell culture plates at 300. Mu.l/well and cultured in a 37 ℃ cell culture chamber. After 1 week of culture, carrying out first liquid change by using an HT culture medium, and culturing in a cell culture box at 37 ℃; after 3 days of culture, a second change was made with HT medium.

HAT medium screening: HAT selection medium was prepared containing 1 XHAT, 1 XHam-streptomycin, 15% fetal bovine serum and 85% DMEM medium. The rat hybridoma cells and feeder cells were resuspended in HAT screening medium as described above and mixed. The cell suspension was added to 20 96-well cell culture plates at 300. Mu.l/well and incubated in a 37 ℃ cell incubator. After 1 week of culture, carrying out first liquid change by using an HT culture medium, and culturing in a cell culture box at 37 ℃; after 3 days of culture, a second change was made with HT medium.

4. Screening of Positive cell lines

2 weeks after fusion, taking cell supernatant to perform ELISA experiment, detecting the combination condition of the cell supernatant and human alpha-synuclein protein monomer and polymer, screening out cells with positive combination of the alpha-synuclein protein monomer and polymer and the cell supernatant, and performing expanded culture and subcloning on the double-positive cells.

5. Expanding culture

Transferring the cell strain with positive ELISA monomer and polymer detection into 24-hole from 96-hole for culture, and transferring into 25cm after full growth ² Culturing in a culture flask.

6. Subcloning by limiting dilution method

And blowing and mixing the positive cell strains uniformly, and sucking a small amount of the positive cell strains to count the living cells. About 200 cells were pipetted into 80ml of complete medium and mixed well and plated into 4 plates. About 400 additional cells were pipetted into 80ml of complete medium and mixed well and plated in 4 plates. About 1000 additional cells were pipetted into 20mL of complete medium and mixed well and plated in 1 block. In total, 9 blocks were plated at 3 different cell densities, 0.5, 1, and 10 cells/well, respectively. Placing 96 well plates at 37 ℃ 5% CO ₂ Culturing in an incubator.

7. Clone detection and expanded culture

And (3) taking the supernatant of the monoclonal cell hole to carry out ELISA alpha-synuclein monomer and polymer combination detection, and respectively detecting the combination condition of the cell clone antibody and the human alpha-synuclein monomer and polymer. Transferring the cell strain with double positive ELISA detection into 24-hole from 96-hole for culture, and transferring into 25cm after full growth ² Culturing in a culture flask.

8. Identification of subclasses

Coating goat anti-mouse IgG1, igG2a, igG2b, igG2c, igG3, igM and IgGA,50ng/100 μ l/well, overnight at 4 ℃, blocking 3% BSA at room temperature, adding cell supernatant to be tested, incubating at room temperature for 2 hours, adding enzyme-labeled secondary goat anti-mouse IgG, κ, λ, developing, terminating, reading at 450 nm. The subclass of the cell strain to be detected is IgG1 and kappa.

9. Cell cryopreservation

Preparing a frozen stock solution: 90% fetal bovine serum, 10% DMSO.

Resuspending the cells in the flask, counting the cells, centrifuging at 1000rpm/min for 5min, discarding the supernatant, pipetting the suspension with 10% DMSO-containing fetal bovine serum,at 1 × 10 ⁷ Cells/tubes were cryopreserved in a cryopreservation box at-80 ℃ overnight, and transferred into liquid nitrogen the next day.

10. Monoclonal hybridoma gene preservation

Collecting positive monoclonal cell strain, adding Trizol to crack cell and extract RNA, reverse transcription to cDNA, and storing at-80 deg.c.

Example 3 monoclonal antibody preparation and characterization

1. Antibody preparation by in vitro culture method

The hybridoma cell line prepared in example 2 was recovered by recovering a DMEM medium containing 10% fetal bovine serum and 1% streptomycin and cultured in a small vial, and after the degree of cell confluence was about 90%, subculture was performed until the cell culture supernatant amounted to about 200mL.

2. Antibody purification

Cell supernatants cultured for about 7 days were collected, volume was measured (about 200 mL), naCl was added to the supernatant to 2.5M, and the supernatant was vacuum filtered through a 0.22 μ M mixed cellulose microporous membrane, stored at 4 ℃ and subjected to antibody purification by Protein A affinity chromatography. Sampling: filtering the cell culture supernatant containing 2.5M NaCl through a 0.22 mu M filter membrane, concentrating to 30ml, and directly loading; flow washing: washing with PBS (pH 7.4.2.5M) until the UV280 base line is 0; and (3) elution: pH3.5.0.1M citric acid solution, each section of 2ml collection eluent, each tube is added with 100 u l 1M Tris solution; the collected solution was concentrated and eluted with PBS until the initial fraction was less than 0.1%. SDS-PAGE assays verified the purity of the purified antibodies, as shown in FIG. 2.

EXAMPLE 4 monoclonal antibody screening and Gene sequencing

After mouse immunization, hybridoma cell construction and monoclonality, three monoclonal antibody cell strains with high cytological activity are finally obtained based on antibody combination Experiments (ELISA), family proteins (beta-synuclein and gamma-synuclein) and species proteins (human, mouse and monkey alpha-synuclein) cross Experiments (ELISA), experiments of affinity determination and antibody inhibition alpha-synuclein monomer polymerization and results of antibody-mediated phagocytosis experiments of BV2 microglia on alpha-synuclein polymers (phagocytosis rate > 50%), which are respectively named as 2A1, 2B1 and 2C1 monoclonal antibody cell strains, total RNA extraction is carried out on the three cell strains, the three cell strains are subjected to reverse transcription to form cDNA, and then heavy chain variable regions and light chain variable regions of the antibodies are amplified by PCR by taking the cDNA as a template.

Three monoclonal antibody total RNAs were obtained from Invitrogen corporation

The reagent kit (15596-026) is extracted according to the instruction, and the extraction result is shown in FIG. 3.

Then, 5' RACE FULL kit (D315) from Takara was used, and a random primer in the kit was reverse-transcribed into first strand cDNA using total RNA as a template, and then a heavy chain was PCR-amplified using a constant region design primer (mVH-R) and an adaptor primer in the kit, and a light chain was PCR-amplified using a constant region design primer (mVL-R) and an adaptor primer in the kit. The sequences of mVH-R and mVL-R are as follows:

mVH-R：CTCAGGGAARTARCCYTTGAC

mVL-R：TCACTGCCATCAATCTTCCAC

the heavy chain variable region and the light chain variable region of the three monoclonal antibody cell strains are subjected to PCR amplification and then electrophoresis detection, and the detection is shown in a figure 4.

The agarose gel recovery kit recovered the PCR fragment and sent the sample to hong news science and technology for sequencing. Finally, determining that the heavy chain variable region sequences of the monoclonal antibodies 2A1, 2B1 and 2C1 are respectively shown as SEQ ID NO.1-3, and the light chain variable region sequences are respectively shown as SEQ ID NO. 4-6; the nucleotide sequences of the heavy chain variable regions are respectively shown in SEQ ID NO.49-51, and the nucleotide sequences of the light chain variable regions are respectively shown in SEQ ID NO.52-54 (Table 2).

TABLE 2 sequences of monoclonal antibodies 2A1, 2B1 and 2C1

EXAMPLE 5 binding assay (ELISA) of anti-alpha-synuclein monoclonal antibody and human alpha-synuclein protein monomer and Polymer

Human alpha-synuclein monomer protein and human alpha-synuclein polymer protein were diluted with 0.01M PBS buffer, pH7.2 (ab 218819,abcam) and coated with 100ng protein/100 μ l/well in 96-well plates overnight at 4 ℃. After washing the plate 3 times with 250. Mu.l/well PBST (PBS +1% Tween 20), 2% milk-PBS was added at 250. Mu.l/well and blocked at 37 ℃ for 2 hours. After washing the plate three times, the cell culture supernatant of the candidate monoclonal antibody or a negative antibody control (100. Mu.l/well) was added and incubated at room temperature for 1 hour. The plate was then washed 6 times, and a 10000-fold dilution of goat anti-mouse IgG Fc-HRP secondary antibody (100. Mu.l/well) in 2% mil-PBS was added and incubated at room temperature for 1 hour. After washing the plate 4 times, the plate was developed with a TMB color development kit (100. Mu.l/well), and developed under dark conditions at room temperature for 5 minutes, and then developed with 2M H ₂ SO ₄ (100. Mu.l/well) the color development was stopped. Immediately after the color development was terminated, the OD value was measured at a wavelength of 450nm with a microplate reader. Experimental data As shown in Table 3, all the tested alpha-synuclein monoclonal antibodies were well bound to human alpha-synuclein protein monomers and polymers.

TABLE 3 ELISA detection of monoclonal antibodies 2A1, 2B1 and 2C1 binding to human alpha-synuclein monomers and polymers

Example 6 family Cross-over assay (ELISA) of anti-alpha-synuclein monoclonal antibodies with human alpha, beta, gamma-synuclein proteins

Human α -synuclein protein (ab 218818, abcam), human β -synuclein protein (ab 82630, abcam) and human γ -synuclein protein (ab 48712, abcam) were each diluted in 0.01M PBS buffer, pH7.2, and 96-well plates were coated with 100ng of protein/100 μ l/well and overnight at 4 ℃. After washing the plate 3 times with 250. Mu.l/well PBST (PBS +1% Tween 20), 2% milk-PBS was added at 250. Mu.l/well and blocked at 37 ℃ for 2 hours. After washing the plate three times, monoclonal purified antibody or negative antibody control (100. Mu.l/well) was added and incubated for 1 hour at room temperature. The plate was then washed 6 times, and a 10000-fold dilution of goat anti-mouse IgG Fc-HRP secondary antibody (100. Mu. In 2% mil-PBS) was addedl/well) was incubated at room temperature for 1 hour. After washing the plate 4 times, the plate was developed with a TMB color development kit (100. Mu.l/well), and developed under dark conditions at room temperature for 5 minutes, and then developed with 2M H ₂ SO ₄ (100. Mu.l/well) the color development was stopped. Immediately after the color development was terminated, the OD value was measured at a wavelength of 450nm with a microplate reader. Experimental data As shown in Table 4, all the tested alpha-synuclein monoclonal antibodies were able to bind well to human alpha-synuclein protein, but not to human beta-synuclein protein and human gamma-synuclein protein. The experimental result shows that the detected alpha-synuclein monoclonal antibody is specifically combined with the alpha-synuclein protein.

TABLE 4 ELISA detection of the binding of monoclonal antibodies 2A1, 2B1 and 2C1 to human alpha, beta, gamma-synuclein proteins

Example 7 species crossover Experiments (ELISA) of anti-alpha-synuclein monoclonal antibodies with human, monkey, murine alpha-synuclein proteins

Human, monkey, and murine α -synuclein proteins were each diluted with 0.01M PBS buffer, pH7.2, and 96-well plates were coated with 100ng of protein/100. Mu.l/well and overnight at 4 ℃. The plate was washed 3 times with 250. Mu.l/well PBST (PBS +1% Tween 20), then 250. Mu.l/well of 2% milk-PBS was added, and blocked at 37 ℃ for 2 hours. After washing the plate three times, monoclonal purified antibody or negative antibody control (100. Mu.l/well) was added and incubated for 1 hour at room temperature. The plate was then washed 6 times, and a 10000-fold dilution of goat anti-mouse IgG Fc-HRP secondary antibody (100. Mu.l/well) in 2% mil-PBS was added and incubated at room temperature for 1 hour. After washing the plate 4 times, the plate was developed with a TMB color development kit (100. Mu.l/well), and developed under dark conditions at room temperature for 5 minutes, and then developed with 2M H ₂ SO ₄ (100. Mu.l/well) the color development was stopped. Immediately after the color development was terminated, the OD value was measured at a wavelength of 450nm with a microplate reader. Experimental data As shown in Table 5, all the tested alpha-synuclein monoclonal antibodies were well bound to human, monkey, and murine alpha-synuclein proteins. The experimental result shows that the detected alpha-synuclein monoclonal antibody can be used for carrying out pharmacodynamic drug-induced experiments in monkey and mouse bodies.

TABLE 5 ELISA detection of the binding of monoclonal antibodies 2A1, 2B1 and 2C1 to human, monkey, murine alpha-synuclein proteins

Example 8 affinity assay of anti-alpha-synuclein monoclonal antibodies with human alpha-synuclein protein monomers and polymers (Biacore)

A Biacore T200 instrument was used to detect the affinity constant of the anti-alpha-synuclein monoclonal antibody, an anti-mouse Fc antibody (GE Healthcare, BR-1008-38) was coupled to a CM5 biosensing chip (GE Healthcare) by amino covalent binding, the anti-mouse Fc antibody on the chip captured the candidate monoclonal antibody, different concentrations of human alpha-synuclein protein monomer or human alpha-synuclein protein polymer were flowed through the candidate monoclonal purified antibody on the chip at a flow rate of 30. Mu.l/min, the human alpha-synuclein protein monomer or polymer was bound to the candidate monoclonal purified antibody for 120 seconds and the dissociation time was 300 seconds, kinetic fitting was performed with BIAevaluation software (GE Healthcare), the affinity constant results are shown in Table 6, where 2B1 had the highest affinity for binding to human alpha-synuclein protein monomer and 2A1 had the highest affinity for binding to human alpha-synuclein protein polymer.

TABLE 6 affinity constants for binding of monoclonal antibodies 2A1, 2B1 and 2C1 to human alpha-synuclein protein monomers or polymers

Example 9 anti-alpha-synuclein monoclonal antibody inhibits the polymerization of human alpha-synuclein monomers

In order to detect whether the anti-alpha-synuclein monoclonal antibody to be detected can inhibit the polymerization of the human alpha-synuclein monomer so as to reduce the generation of alpha-synuclein polymer with neurotoxicity, in the embodiment, a small amount of human alpha-synuclein polymer is used as seeds to induce the spontaneous polymerization of the human alpha-synuclein monomer, and the generation of the polymer is inhibited by co-incubation with the anti-alpha-synuclein monoclonal antibody to be detected, a thioflavin T fluorescence detection method is used to compare the fluorescence intensity of an antibody group to be detected and a negative control group, and the inhibition rate of the antibody to be detected on the polymerization of the alpha-synuclein monomer is calculated.

Preparing a thioflavin T (ThT) solution: 3.19mg of thioflavin T (ab 120751, abcam) was dissolved in 10ml of deionized water to prepare a 1mM aqueous solution of thioflavin T, which was sterilized by filtration through a 0.2 μm filter.

Freshly prepared human α -synuclein polymers (ab 218819, abcam) at final concentrations of 10nM, human α -synuclein monomers (ab 218818, abcam) at 100 μ M, thioflavin T at 25 μ M, and either the test antibody or isotype control antibody (murine IgG 1) or blank control (PBS buffer) at 50 μ g/ml were mixed in PBS (pH 7.4) buffer, applied to a 96-well microplate (3603, corning) at 100 μ l/well (3 replicates), sealed and placed on a shaker, and incubated at room temperature for 24 hours at 600 rpm/min. Fluorescence values were then read on a microplate reader (excitation light 450 nm/emission light 485 nm) and the inhibition rate of the antibody on the polymerization of α -synuclein monomer was calculated by the following formula.

Inhibition rate = [ (ThT) _blank -ThT _ab )/(ThT _blank -ThT _{isotype control} )]*100％

The results are shown in Table 7, where antibody 2A1 had the best anti-polymerization effect, followed by 2B1 and 2C1.

TABLE 7 inhibition rate of monoclonal antibodies 2A1, 2B1 and 2C1 on human alpha-synuclein monomer polymerization

Name of antibody	Inhibition ratio (%)
		2A1	98.78
2B1	93.20
		2C1	86.46
Negative isotype control antibody	1.61
		Blank control	0

To examine whether 2A1, 2B1 and 2C1 were able to inhibit the polymerization of human α -synuclein monomer in a dose-dependent manner and further compare the polymerization inhibition ability of three antibodies, 2A1, 2B1 and 2C1, the initial final concentration of 2A1, 2B1 and 2C1 antibody was increased to 200 μ g/ml and a dose-response curve was prepared by diluting 10 concentration points twice, the specific experimental procedure was as follows:

freshly prepared human α -synuclein polymers (ab 218819, abcam) at final concentrations of 10nM, human α -synuclein monomers (ab 218818, abcam) at 100 μ M and thioflavin T at 25 μ M were mixed well, and the initial concentration of 200 μ g/ml, 2-fold diluted 10 concentration points of the test antibody or isotype control antibody (murine IgG 1) or blank control (PBS buffer) were added to each well in PBS (pH 7.4) buffer and added to a 96-well microplate (3603, corning) at 100 μ l/well (3-well). The plate was sealed and placed on a shaker and incubated at 600rpm/min for 24 hours at room temperature. Fluorescence values were then read on a microplate reader (excitation light 450 nm/emission light 485 nm) and the inhibition rate of the antibody on the polymerization of α -synuclein monomer was calculated by the following formula.

The antibody concentration was plotted on the abscissa and the thioflavin T fluorescence on the ordinate as the dose-response curve using Prism6 software, and the IC50 values were generated and are shown in table 8. The IC50 value of 2A1 is the lowest, and 2B1 and 2C1 are the second, which shows that 2A1 has the best capability of inhibiting the polymerization of alpha-synuclein monomers, is better than 2B1 and 2C1, and accords with the experimental result of single-point detection in Table 7. In addition, all three antibodies tested have complete dose-response curves, as shown in FIG. 5, which indicates that the effect of the antibody in inhibiting the polymerization of alpha-synuclein monomer is dose-dependent, and the IC50 range of the antibody concentration is 2.693. Mu.g/ml-3.678. Mu.g/ml.

TABLE 8 IC50 values for the inhibition of the polymerization of human alpha-synuclein monomers by monoclonal antibodies 2A1, 2B1 and 2C1

Name of antibody	IC50(μg/ml)
		2A1	2.693
2B1	2.948
		2C1	3.678

Example 10 anti-alpha-synuclein monoclonal antibody mediated phagocytosis of alpha-synuclein polymers by microglia (BV 2)

In order to detect whether the anti-alpha-synuclein monoclonal antibody to be detected can mediate microglia (BV 2) to phagocytize human alpha-synuclein polymers so as to accelerate the removal of the human alpha-synuclein polymers, the embodiment compares the fluorescence intensity of an antibody group to be detected and a negative control group by co-incubating the BV2 microglia, the antibody to be detected and the human alpha-synuclein polymers by using a thioflavin T fluorescence detection method, and calculates the phagocytosis rate of the microglia mediated by the antibody to be detected on the human alpha-synuclein polymers.

Preparation of BV2 mouse microglia: resuscitating BV2 cells and 5% CO at 37% ₂ Expanding the cells to the desired number in DMEM medium (DMEM +10% FBS +1% PS) under conditions, removing the medium, washing the cells twice with DPBS followed by trypsinization, centrifuging the cells at 1000rpm for 3 minutes, discarding the supernatant, resuspending the cells in 2ml DMEM medium and counting, diluting the cells to 2x10 after counting ⁵ Perml, 100. Mu.l of the cell suspension was applied to a 96-well microplate (3606, corning) to give a 2X10 cell count per well ⁴ Per 100. Mu.l (3 wells) at 37 ℃ 5% ₂ Incubate in incubator for 16 hours.

After incubation, the BV2 cells were added with the antibody to be tested or the negative control antibody (murine IgG 1) or the blank control (DMEM medium) at a final concentration of 50. Mu.g/ml and the alpha-synuclein polymer at 30. Mu.g/ml, respectively, to make the final volume 200. Mu.l. At 37 ℃,5% CO ₂ After 5 hours incubation in the incubator, 50. Mu.l of supernatant per well was transferred to a new 96-well microplate (3606, corning), and mixed by adding thioflavin T at a final concentration of 25. Mu.M to achieve a final volume of 100. Mu.l per well. The microplate is placed in a microplate reader (excitation light 450 nm/emission light 485 nm) to read the fluorescence value, and the phagocytosis rate of the BV2 microglia mediated by the antibody to be detected on the alpha-synuclein polymer is calculated by the following formula.

Phagocytosis rate = [ (ThT) _blank -ThT _ab )/(ThT _blank -ThT _{isotype control} )]*100％

The results are shown in table 9, where antibody 2A1 mediated BV2 microglia have the highest rate of phagocytosis of human α -synuclein aggregates, followed by 2C1 and 2B1.

TABLE 9 phagocytosis rates of human alpha-synuclein polymers by BV2 microglia mediated by monoclonal antibodies 2A1, 2B1 and 2C1

Name of antibody	Phagocytosis ratio (%)
		2A1	89.55
2B1	78.61
		2C1	85.79
Negative isotype control antibody	1.39
		Blank control	0

In order to detect the dose-effect relationship of antibody-mediated phagocytosis of BV2 microglia on alpha-synuclein polymers and further compare the phagocytosis abilities of microglia mediated by three antibodies, namely 2A1, 2B1 and 2C1, the initial final concentration of the 2A1, 2B1 and 2C1 antibodies is increased to 200 mu g/ml, and 10 concentration points are diluted twice to make a dose-effect curve, the specific experimental process is as follows:

BV2 microglia at 37 ℃,5% ₂ Expanding the cells to the desired number in DMEM medium (DMEM +10% FBS +1% PS) under conditions, removing the medium, washing the cells twice with DPBS followed by trypsinization, centrifuging the cells at 1000rpm for 3 minutes, discarding the supernatant, resuspending the cells in 2ml DMEM medium and counting, diluting the cells to 2x10 after counting ⁵ Perml, 100. Mu.l of the cell suspension was applied to a 96-well microplate (3606, corning) to give a 2X10 cell count per well ⁴ Per 100. Mu.l (3 wells), 5% CO at 37 ℃ ₂ Incubate in incubator for 16 hours.

Respectively adding an antibody to be detected or a negative control antibody (mouse IgG 1) or a blank control (DMEM culture medium) which is diluted twice to 10 concentration points with the concentration of 200 mu g/ml all the time into the incubated BV2 cells, and adding alpha-synuclein polymer with the final concentration of 30 mu g/ml into each concentration point to ensure that the final volume of each hole reaches200. Mu.l. At 37 ℃,5% CO ₂ After 5 hours incubation in the incubator, 50. Mu.l of supernatant per well was transferred to a new 96-well microplate (3606, corning), and mixed with thioflavin T at a final concentration of 25. Mu.M to achieve a final volume of 100. Mu.l per well. The microplate is placed in a microplate reader (excitation light 450 nm/emission light 485 nm) to read the fluorescence value, and the phagocytosis rate of the BV2 microglia mediated by the antibody to be detected on the alpha-synuclein polymer is calculated by the following formula.

The concentration of the antibody is used as the abscissa and the fluorescence value of thioflavin T is used as the ordinate to be used as an agent effect curve by Prism6 software, and an IC50 value is generated, and the experimental result is shown in a table 10. Among these, the IC50 value for 2A1 was the lowest, indicating that 2A1 mediated microglia phagocytosis was the best, followed by 2C1 and 2B1. The results of this experiment are consistent with the results of single-point detection in table 7. In addition, the 3 antibodies tested were dose-dependent in mediating the effect of microglia phagocytosis of α -synuclein aggregates, as shown in fig. 6, with an antibody effect concentration IC50 ranging from 1.97 μ g/ml to 3.43 μ g/ml.

TABLE 10 monoclonal antibodies 2A1, 2B1 and 2C1 mediated phagocytosis of human alpha-synuclein polymers by BV2 microglia IC50 values

Name of antibody	IC50(μg/ml)
		2A1	1.97
2B1	3.43
		2C1	2.59

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Sequence listing

<110> Changchun university of industry

<120> monoclonal antibody against alpha-synuclein and use thereof

<130> KHP191111330.0

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Gly Asn Lys Asn Thr Arg Thr Asn Gly Ser Thr Tyr Asp Gln Glu Phe

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Leu Glu Ile Asn Asn Leu Lys Ser Glu Asp Thr Ala Thr Tyr Phe Cys

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Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

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Asp Ile Leu Met Thr Gln Thr Pro Ser Ser Met Ser Val Ser Leu Gly

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Ser Arg Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

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Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Thr Ser Phe Pro Trp

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Thr Phe Gly Ala Gly Thr Lys Leu Glu Met Lys

100 105

<210> 49

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

cagatccagt tggtgcagtc tggacctgaa ctgaagaagc ttggagagac agtcaagatc 60

tcctgcaagg cttctgggta taccttcaca gactatggaa tgaactgggt gaagcaggct 120

ccaggaaagg gtttaaagtg gatgggctgg aaaaacacca ggtctggaga gtcaacatat 180

gatgaagagt tcaagggacg gtttgccttt tcttcggaaa catctgccag cactgcctat 240

ttgcagatca acaacctcaa gagtgaggac acggctacat atttctgtgc aagggctctt 300

gactactggg gccaaggaac cactctcaca gtctcctca 339

<210> 50

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gagatccagc tggtgcagcc cggccccgag ctgaagaagc tgggcgagac catgaagatg 60

agctgcaagg ccagcggcca caccttcacc agctacggca tgcactgggt gaagcagagc 120

cacggcaaga acctgaagtg gatgggcaac aagaacacca ggaccaacgg cagcacctac 180

gaccaggagt tcaaggacag gttcgccttc agcgtggaga ccagcgccac caccgcctac 240

ctggagatca acaacctgaa gagcgaggac accgccacct acttctgcgc caagtggctg 300

gactactggg gccaggccac cagcctgacc gtgagcgcc 339

<210> 51

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<400> 51

gagatccagg tggtgcagag cggcgccgag ctgaagaagc tgggcgagac catgaggatc 60

agctgcaagg ccagcggcta cagcttcacc aactactgga tgaactggat caagcagaag 120

cccgagaagg gcctgaagtg gatgggctgg aaggacacca ggaacagcga cagcaccagc 180

gaccaggagt tcaggggcag gttcctgttc agcctggaga ccagcgccag caccgccttc 240

ctgcagatca acaacctgaa gagcgaggac accgccacct acttctgcgg caggagcctg 300

gcctactggg gccagggcac caccctgacc gtgagcagc 339

<210> 52

<211> 315

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<400> 52

gacatcctga tgacccaatc tccatcctcc atgtctgtat ctctgggaga cacagtcagc 60

atcacttgcc atgcaagtca gggcattagc aggaatatag ggtggttgca gcagaaacca 120

gggaaatcat ttaagggcct gatttatcat ggaaccaact tggaagatgg agttccatca 180

aggttcagtg gcagtggatc tggagcagat tattctctca ccatcagcag cctggaatct 240

gaagattttg cagactatta ctgtgtacag tatactcagt ttccgctcac gttcggtgct 300

gggaccaagc tggag 315

<210> 53

<211> 315

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt atttacttaa gctggtttca gcagaaacca 120

gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccgaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg cagactatta ctgtctacaa tatgctagtt atccgtggac gttcggtgga 300

ggcaccaagc tggaa 315

<210> 54

<211> 321

<212> DNA

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<400> 54

gacatcctga tgacccagac ccccagcagc atgagcgtga gcctgggcga cagggtgagc 60

atcacctgca gggccagcca gggcctgaac aggtacctgg gctggctgca gcagaagccc 120

ggcaagacct tcaagggcct gatctacgcc ggcagcaacc tggacagcgg cgtgcccagc 180

aggttcagcg gcagcaggag cggcgccgac tacagcctga ccatcagcag cctggagagc 240

gaggacttcg ccgactacta ctgcctgcag tacaccagct tcccctggac cttcggcgcc 300

ggcaccaagc tggagatgaa g 321

<210> 55

<211> 339

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<400> 55

tgaggagact gtgagagtgg ttccttggcc ccagtagtca agagcccttg cacagaaata 60

tgtagccgtg tcctcactct tgaggttgtt gatctgcaaa taggcagtgc tggcagatgt 120

ttccgaagaa aaggcaaacc gtcccttgaa ctcttcatca tatgttgact ctccagacct 180

ggtgtttttc cagcccatcc actttaaacc ctttcctgga gcctgcttca cccagttcat 240

tccatagtct gtgaaggtat acccagaagc cttgcaggag atcttgactg tctctccaag 300

cttcttcagt tcaggtccag actgcaccaa ctggatctg 339

<210> 56

<211> 339

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<400> 56

ggcgctcacg gtcaggctgg tggcctggcc ccagtagtcc agccacttgg cgcagaagta 60

ggtggcggtg tcctcgctct tcaggttgtt gatctccagg taggcggtgg tggcgctggt 120

ctccacgctg aaggcgaacc tgtccttgaa ctcctggtcg taggtgctgc cgttggtcct 180

ggtgttcttg ttgcccatcc acttcaggtt cttgccgtgg ctctgcttca cccagtgcat 240

gccgtagctg gtgaaggtgt ggccgctggc cttgcagctc atcttcatgg tctcgcccag 300

cttcttcagc tcggggccgg gctgcaccag ctggatctc 339

<210> 57

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

gctgctcacg gtcagggtgg tgccctggcc ccagtaggcc aggctcctgc cgcagaagta 60

ggtggcggtg tcctcgctct tcaggttgtt gatctgcagg aaggcggtgc tggcgctggt 120

ctccaggctg aacaggaacc tgcccctgaa ctcctggtcg ctggtgctgt cgctgttcct 180

ggtgtccttc cagcccatcc acttcaggcc cttctcgggc ttctgcttga tccagttcat 240

ccagtagttg gtgaagctgt agccgctggc cttgcagctg atcctcatgg tctcgcccag 300

cttcttcagc tcggcgccgc tctgcaccac ctggatctc 339

<210> 58

<211> 315

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

ctccagcttg gtcccagcac cgaacgtgag cggaaactga gtatactgta cacagtaata 60

gtctgcaaaa tcttcagatt ccaggctgct gatggtgaga gaataatctg ctccagatcc 120

actgccactg aaccttgatg gaactccatc ttccaagttg gttccatgat aaatcaggcc 180

cttaaatgat ttccctggtt tctgctgcaa ccaccctata ttcctgctaa tgccctgact 240

tgcatggcaa gtgatgctga ctgtgtctcc cagagataca gacatggagg atggagattg 300

ggtcatcagg atgtc 315

<210> 59

<211> 315

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

ttccagcttg gtgcctccac cgaacgtcca cggataacta gcatattgta gacagtaata 60

gtctgcaaaa tcttcagact caaggctgct gatggtgaga gaataatctg acccagacct 120

actgccactg aaccttttcg ggacaccaga atctaaagtg gatgcggcgt agatcaggcg 180

tttaatagtt ccatctggtt tctgctgaaa ccagcttaag taaatactaa tttcctgact 240

tgcccgacaa gtgagactga ctctttctcc cagagaggca gataaggagg atggagactg 300

ggtcatctgg atgtc 315

<210> 60

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

cttcatctcc agcttggtgc cggcgccgaa ggtccagggg aagctggtgt actgcaggca 60

gtagtagtcg gcgaagtcct cgctctccag gctgctgatg gtcaggctgt agtcggcgcc 120

gctcctgctg ccgctgaacc tgctgggcac gccgctgtcc aggttgctgc cggcgtagat 180

caggcccttg aaggtcttgc cgggcttctg ctgcagccag cccaggtacc tgttcaggcc 240

ctggctggcc ctgcaggtga tgctcaccct gtcgcccagg ctcacgctca tgctgctggg 300

ggtctgggtc atcaggatgt c 321

<210> 61

<211> 140

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 61

Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val

1 5 10 15

Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys

20 25 30

Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val

35 40 45

Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr

50 55 60

Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys

65 70 75 80

Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys

85 90 95

Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile

100 105 110

Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro

115 120 125

Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

130 135 140

<210> 62

<211> 140

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 62

Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val

1 5 10 15

Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys

20 25 30

Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val

35 40 45

Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr

50 55 60

Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys

65 70 75 80

Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Ile Lys

85 90 95

Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile

100 105 110

Leu Gln Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro

115 120 125

Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

130 135 140

<210> 63

<211> 140

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 63

Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val

1 5 10 15

Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys

20 25 30

Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val

35 40 45

Val His Gly Val Thr Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr

50 55 60

Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys

65 70 75 80

Thr Val Glu Gly Ala Gly Asn Ile Ala Ala Ala Thr Gly Phe Val Lys

85 90 95

Lys Asp Gln Met Gly Lys Gly Glu Glu Gly Tyr Pro Gln Glu Gly Ile

100 105 110

Leu Glu Asp Met Pro Val Asp Pro Gly Ser Glu Ala Tyr Glu Met Pro

115 120 125

Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

130 135 140

Claims

1. An anti- α -synuclein monoclonal antibody or fragment thereof, wherein the monoclonal antibody or fragment thereof is capable of specifically binding to α -synuclein monomers and α -synuclein aggregates; the monoclonal antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.43, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 46;

or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.44, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 47;

or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.45, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 48.

2. The anti- α -synuclein monoclonal antibody or fragment thereof, as claimed in claim 1, characterized in that the monoclonal antibody is of the IgG1 type.

3. A nucleic acid encoding the anti-a-synuclein monoclonal antibody or fragment thereof of claim 1 or 2.

4. The nucleic acid of claim 3, wherein the nucleic acid sequence encoding the heavy chain variable region of the anti- α -synuclein monoclonal antibody or fragment thereof is set forth in SEQ ID No.49, and the nucleic acid sequence encoding the light chain variable region of the anti- α -synuclein monoclonal antibody or fragment thereof is set forth in SEQ ID No. 52;

or, the nucleic acid sequence of the heavy chain variable region of the monoclonal antibody or the fragment thereof for coding the anti-alpha-synuclein is shown as SEQ ID NO.50, and the nucleic acid sequence of the light chain variable region of the monoclonal antibody or the fragment thereof for coding the anti-alpha-synuclein is shown as SEQ ID NO. 53;

or the nucleic acid sequence of the heavy chain variable region of the monoclonal antibody or the fragment thereof for coding the alpha-synuclein is shown in SEQ ID NO.51, and the nucleic acid sequence of the light chain variable region of the monoclonal antibody or the fragment thereof for coding the alpha-synuclein is shown in SEQ ID NO. 54.

5. Biological material comprising nucleic acids according to claim 3 or 4, wherein the biological material is an expression cassette, a vector, a transposon or a host cell.

6. A complex or conjugate comprising or produced from the monoclonal antibody or fragment thereof of claim 1 or 2.

7. The complex or conjugate of claim 6, wherein the complex is chemically or biologically labeled with the monoclonal antibody or fragment thereof of claim 1 or 2;

the conjugate is obtained by conjugating the monoclonal antibody or the fragment thereof or the complex of claim 1 or 2 with a solid or semisolid medium.

8. Use of the anti-alpha-synuclein monoclonal antibody or fragment thereof of claim 1 or 2 or the nucleic acid of claim 3 or 4 or the biological material of claim 5 or the complex or conjugate of claim 6 or 7 for any of the following:

(3) Use in the manufacture of a product for inhibiting polymerization of alpha-synuclein monomers or formation of oligomers, fibrils, fibrids, lewy bodies of alpha-synuclein;

9. Use according to claim 8, wherein the α -synucleinopathies include Parkinson's disease, lewy body dementia, combined Alzheimer's and Parkinson's disease, pure autonomic failure, multiple system atrophy.

10. A medicament comprising or produced from the monoclonal antibody or fragment thereof of claim 1 or 2;

the medicine has any one of the following functions:

(1) For the prevention or treatment of alpha-synucleinopathies;

(3) For inhibiting polymerization of alpha-synuclein monomers or formation of oligomers, fibrils, cellulosomes, lewy bodies of alpha-synuclein;

(4) For reducing the accumulation of alpha-synuclein aggregates, fibrils, fibroids, lewy bodies;

11. The medicament of claim 10, wherein the α -synucleinopathies include parkinson's disease, lewy body dementia, combined alzheimer's and parkinson's disease, pure autonomic failure, multiple system atrophy.

12. An assay reagent or kit for detecting a-synuclein monomer or polymer thereof, comprising the monoclonal antibody or fragment thereof of claim 1 or 2, or comprising the complex or conjugate of claim 6 or 7.