Application of beta-Syn protein in preparation of Parkinson's disease medicament

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of beta-Syn protein in preparation of a Parkinson disease medicament. The beta-Syn protein is a protein containing a beta-Syn protein N-terminal structural domain, and the amino acid sequence of the protein containing the beta-Syn protein N-terminal structural domain is as shown in SEQ ID No. 1. In the application, the inventor clones and prepares recombinant proteins of a beta-Syn-N-terminal structural domain and a beta-Syn-C-terminal structural domain respectively, the recombinant proteins act on a PD cell model induced by MPP +, and further Western blot and Tunel dyeing experiments are combined, so that the key structural domain of the beta-Syn protein capable of being used for improving PD diseases is preliminarily determined as the N-terminal structural domain. Based on the results, a good technical foundation can be laid for further development of anti-PD drugs and further modification of related drug protein molecules, so that the method has relatively good technical practical value.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to the application of β-Syn protein in the preparation of Parkinson's disease medicament. Background Art

[0002] Parkinson's disease (PD) is a chronic degenerative disease caused by the loss of dopaminergic neurons projecting from the substantia nigra to the dorsal striatum and dominated by the central nervous system. Its clinical features are mainly progressive deterioration of motor symptoms, including resting tremor, bradykinesia, muscle rigidity, and postural balance disorders. The main pathological feature of PD is the increased expression of α-synuclein (α-Syn) in the substantia nigra region of the brain, which is easy to aggregate to form Lewy bodies (LB). In addition, cell death in the basal ganglia of the brain is also one of the main pathological features.

[0003] Clinically, the first choice of treatment for PD is to take drugs such as levodopa, pramipexole, and amantadine to relieve symptoms. However, there is no treatment that can inhibit the progression of PD or inhibit the potential driving factors of its pathogenesis. Therefore, in-depth exploration of the pathogenesis of PD and protein molecules related to PD progression is of great technical significance for improving the quality of life of PD patients and developing related therapeutic drugs. Summary of the invention

[0004] This application uses MN9D cells (mouse midbrain dopaminergic neuronal cells) as experimental materials, and uses molecular cloning, Western blot, Tunel staining and other experimental techniques to conduct a preliminary analysis of the biologically active domain of β-synuclein (β-Syn), which is highly homologous to α-Syn, in order to lay a certain technical foundation for the further development of polypeptide drugs for PD treatment.

[0005] The technical solution adopted in this application is described in detail as follows.

[0006] Use of β-Syn protein in the preparation of a Parkinson's disease medicament, wherein the β-Syn protein is a protein containing the N-terminal domain of the β-Syn protein, and the amino acid sequence of the protein containing the N-terminal domain of the β-Syn protein (329AA) is shown in SEQ ID No. 1, and is specifically as follows: MGSSHHHHHHSSGLVPRGSHMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN YNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSMDVFMKGLSMAKEGVVAAAEKTKQGVTEAAEKTKEGVLYVGSKTREGVVQGVASVAEKTKLEHHHHHH; The coding nucleotide sequence corresponding to the protein containing the N-terminal domain of β-Syn protein is shown in SEQ ID No. 2, and is as follows: ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGATCCATGGACGTGTTCATGAAGGGCCTGTCCATGGCCAAGGAGGGCGTTGTGGCAGCCGCGGAGAAAACCAAGCAGGGGGTCACCGAGGCGGCGGAGAAGACCAAGGAGGGCGTCCTCTACGTCGGAAGCAAGACCCGAGAAGGTGTGGTACAAGGTGTGGCTTCAGTGGCTGAAAAAACCAAGCTCGAGCACCACCACCACCACCACTGA。

[0007] The application of β-Syn protein in the preparation of Parkinson's disease drugs exerts an anti-Parkinson's disease effect by increasing the expression of the N-terminal domain protein of β-Syn protein; that is, on the one hand, the protein containing the N-terminal domain of β-Syn protein reduces neuronal cell damage by reducing the expression of α-Syn and increasing the expression of TH; on the other hand, it reduces cell apoptosis by down-regulating the expression of cleaved-Caspase3 and Bax and up-regulating the expression of Bcl-2, thereby improving the incidence of PD.

[0008] β-Syn, which is highly homologous to α-Syn, is a protein composed of 134 amino acids encoded by the SNCB gene. It was originally discovered as phospho-neuronal protein (PNP14) in bovine brain. Compared with α-Syn, β-Syn lacks the central NAC domain, so β-Syn is not easy to form amyloid aggregates. Therefore, some studies believe that β-Syn will have an inhibitory effect when α-Syn aggregates, which can reduce the formation of α-Syn fibrils, and can reduce neurotoxicity and slow down the process of neurodegeneration. However, due to the lack of in-depth research on the structure of β-Syn, in-depth analysis of the key domains in β-Syn and targeted development of related protein peptide drugs based on this key domain are of great technical significance for the prevention and treatment of PD (Parkinson's Disease).

[0009] In the present application, the inventors cloned and prepared the recombinant proteins of the β-Syn-N-terminal domain (the front part of the central NAC domain) and the β-Syn-C-terminal domain (the rear part of the central NAC domain), and used them to act on MPP + In the induced PD cell model, combined with further Western blot and Tunel staining experiments, it was preliminarily determined that the key domain of β-Syn protein that can be used to improve PD symptoms is the N-terminal domain. Based on these results, a good technical foundation can be laid for further development of anti-PD drugs and further modification of related drug protein molecules, so it has good technical practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the protein containing β-Syn-N domain and the corresponding recombinant plasmid structure, wherein the upper figure is a schematic diagram of the recombinant plasmid structure containing β-Syn-N terminus, and the lower figure is a schematic diagram of the β-Syn-N terminus recombinant plasmid connection process; Figure 2Schematic diagram of the protein containing β-Syn domain and the corresponding recombinant plasmid structure, wherein the upper figure is a schematic diagram of the recombinant plasmid structure containing β-Syn; the lower figure is a schematic diagram of the β-Syn recombinant plasmid connection process; Figure 3 Schematic diagram of the protein containing the β-Syn-C terminal domain and the corresponding recombinant plasmid structure, wherein the upper figure is a schematic diagram of the β-Syn-C terminal recombinant plasmid structure; the lower figure is a schematic diagram of the β-Syn-C terminal recombinant plasmid connection process; Figure 4 The results of the MTT method used in the construction of the PD cell model and the experimental process are as follows: A shows the cell survival rate when different MPP+ concentrations were used to induce the construction of PD model cells; B shows the changes in cell survival rate after different concentrations of EGFP recombinant protein acted on the PD model; C shows the changes in cell survival rate after different concentrations of β-Syn-N-terminal recombinant protein acted on the PD model; D shows the changes in cell survival rate after different concentrations of β-Syn recombinant protein acted on the PD model; E. Changes in cell survival rate after different concentrations of β-Syn-C-terminal recombinant protein acted on the PD model; Figure 5 Western blot technology was used to detect the expression of α-Syn, TH protein and apoptosis-related proteins; A is the electrophoresis test result; B is the detection and statistical results of α-Syn; C is the detection and statistical results of TH; D is the detection and statistical results of Caspase3; E is the detection and statistical results of Bax; F is the detection and statistical results of Bcl-2; Figure 6 Tunel staining was used to detect the level of cell apoptosis; A is the direct observation result after staining; B is the statistical results after staining. DETAILED DESCRIPTION

[0011] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0012] Biomaterials: MN9D cells are commonly used cell materials in existing PD research and can be obtained through public channels. The inventor is a professional scientific research and teaching staff, and the applicant belongs to a professional teaching and research institution. Therefore, they have purchased and stored relevant experimental cell materials for a long time. Experimental reagents: RIPA lysis buffer, PMSF, 4% paraformaldehyde fixative, immunostaining strong permeabilization solution, endogenous peroxidase blocking solution, Tunel cell apoptosis detection kit (chromogenic method), etc., products of Beyotime Company; Nissl stain, 5×SDS-PAGE Loading Buffer, SDS-PAGE gel preparation kit, TBS buffer, PBS buffer, DMEM cell culture medium, etc., products of Servicebio; Tris, Gly, SDS, trypsin digestion solution, penicillin-streptomycin mixture, DMSO, MTT, Tryptone, Yeast, kanamycin, IPTG, imidazole, urea, etc., products of Solarbio; Protein Marker, a product of Yazyme Corporation; GAPDH, TH, α-Syn primary antibodies, products of HUABIO; Bax primary antibody, Caspase3 primary antibody, Bcl-2 primary antibody, Goat Anti-Rabbit IgG HRP, ECL developer, products of Affinity; MPP + iodide, a product of AbMol; Conventional restriction enzyme BamH1, conventional restriction enzyme Hind Ⅲ, conventional restriction enzyme Xho l, T4 DNA Ligase, products of TaKaRa Company; DH5α competent cells, high-purity plasmid mini-extraction kit, plasmid large-extraction kit, BL21 (DE3) competent cells, His-tag protein purification kit (soluble protein), products of CWBIO; Main instruments and equipment: Multifunctional microplate reader, a product of Biotek; PCR instrument, produced by Eppendorf; Fluorescence microscope, product of Olympus.

[0013] Example 1 Since some studies have shown that β-Syn has certain application prospects in PD treatment, in order to further determine the key domains of β-Syn protein in anti-PD, a certain technical foundation can be laid for further anti-PD drug development. The inventors cloned and prepared the N-terminal and C-terminal gene sequences of β-Syn, and prepared recombinant proteins, respectively, to lay a material foundation for their structural and functional analysis. This embodiment briefly introduces the preparation process of the relevant recombinant protein as follows.

[0014] 1. Determination of the recombinant sequence First of all, it should be noted that, with reference to the existing genome database, the amino acid sequence of the β-Syn protein (395AA) involved in this application is as follows: MGSSHHHHHHSSGLVPRGSHMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQ LADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSMDVFMKGLSMAKEGVVAAAEKTKQGVTEAAEKTKEG VLYVGSKTREGVVQGVASVAEKTKEQASHLGGAVFSGAGNIAAATGLVKREEFPTDLKPEEVAQEAAEEPLIEPLMEPEGESYEDPPQEEYQEYEPEA.

[0015] The corresponding nucleotide sequences are specifically referenced as follows:

[0016] Combined with the analysis of the protein structure, the amino acid sequence of the N-terminal β-Syn-N protein (329AA) finally prepared is shown in SEQ ID No. 1, and the specific design is as follows: MGSSHHHHHHSSGLVPRGSHMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN YNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSMDVFMKGLSMAKEGVVAAAEKTKQGVTEAAEKTKEGVLYVGSKTREGVVQGVASVAEKTKLEHHHHHH; The corresponding nucleotide sequence is shown in SEQ ID No. 2, and the specific reference is as follows: ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGATCCATGGACGTGTTCATGAAGGGCCTGTCCATGGCCAAGGAGGGCGTTGTGGCAGCCGCGGAGAAAACCAAGCAGGGGGTCACCGAGGCGGCGGAGAAGACCAAGGAGGGCGTCCTCTACGTCGGAAGCAAGACCCGAGAAGGTGTGGTACAAGGTGTGGCTTCAGTGGCTGAAAAAACCAAGCTCGAGCACCACCACCACCACCACTGA。

[0017] The amino acid sequence of the prepared C-terminal β-Syn-C protein (324AA) is specifically designed as follows: MGSSHHHHHHSSGLVPRGSHMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKL EYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSKREEFPTDLKPEEVAQEAAEEPLIEPLMEPEGESYEDPPQEEYQEYEPEAKLAAALEHHHHHH.

[0018] The corresponding nucleotide sequences are specifically referenced as follows: ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGATCCAAGAGGGAGGAATTCCCTACTGATCTGAAGCCAGAGGAAGTGGCCCAGGAAGCTGCTGAAGAACCACTGATTGAGCCCCTGATGGAGCCAGAAGGGGAGAGTTATGAGGACCCACCCCAGGAGGAATATCAGGAGTATGAGCCAGAGGCGAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGA。

[0019] After synthesizing the corresponding sequence by chemical synthesis, the corresponding sequence was cloned and recombined into pET28a plasmid according to the conventional molecular biology operation for subsequent application.

[0020] (II) Cloning of corresponding gene fragments For the recombinant plasmid containing the corresponding nucleotide sequence in step (I), the PCR amplification method is used to further clone and amplify, so as to facilitate the subsequent recombination connection with the plasmid. During PCR amplification, the 50μl amplification system is as follows: Template, 0.5 μg; F primer, 2 μL (10 μM); R primer, 2 μL (10 μM); 2×Es Taq MasterMix, 25 μL; RNase-free water, add to 50 μL; The amplification program reference was: pre-denaturation, 95°C, 2 min; denaturation, 94°C, 30 s, annealing, 55°C, 30 s, extension, 72°C, 30 s, 33 cycles; final extension, 72°C, 2 min.

[0021] During PCR amplification, for different target genes, the specific amplification primers are designed as follows: For the gene encoding β-Syn protein, the primers were designed as follows: SNCB-F: 5'-CGCGGATCCATGGACGTGTTCATGAAGGG-3', SNCB-R: 5'-ATAAGAATGCGGCCGCCTACGCCTCTGGCTCATACT-3'; For the gene encoding β-Syn-N protein, the primers were designed as follows: SNCB-NF: 5'-CGCGGATCCATGGACGTGTTCATGAAGGG-3', SNCB-NR: 5'-CCGCTCGAGCTTGGTTTTTTTCAGCCACTG-3'; For the gene encoding β-Syn-C protein, the primers were designed as follows: SNCB-CF: 5'-CGCGGATCCAAGAGGGAGGAATTCCCTAC-3', SNCB-CR: 5'-CCCAAGCTTCGCCTCTGGCTCATACTCCT-3'.

[0022] After PCR amplification, the amplified product is tested by electrophoresis (120V, electrophoresis for 30 minutes), and the amplified product is recovered and purified. When recovering and purifying the amplified product, the specific operation can be referred to as follows: Under ultraviolet light, place the cut agarose gel containing the target gene in a 1.5 mL centrifuge tube, and add an appropriate amount of ADB solution in the centrifuge tube according to the mass ratio of agarose gel: ADB = 1:3; Heat in a 55°C water bath for 8 min. After the agarose gel is completely dissolved into liquid, transfer it to the adsorption column. Centrifuge at 12000 rpm for 1 min and discard the waste liquid in the collection tube; Re-install the adsorption column into the collection tube, add 200 μL of DNA Wash Buffer, centrifuge at room temperature and 12,000 rpm for 1 min, discard the waste liquid, add DNA Wash Buffer again and centrifuge; Place the treated adsorption column into a new labeled 1.5 mL centrifuge tube, add 10 μL of DNAElution Buffer, and centrifuge at 12000 rpm for 1 min. The result is the recovered purified DNA.

[0023] 3. Enzyme digestion and ligation The PCR amplification product and plasmid recovered in step (ii) were double-digested to expose the sticky ends of the empty plasmid and the target gene. When different target gene fragments were digested, the reference design of the 20μL digestion system was as follows: BamH1, 1 μL; NotI, 1 μL; 10×K / M Buffer, 2 μL; Empty vector / target gene (β-Syn), 1 μg; RNase-free water, add to 20 μL; BamH1, 1 μL; Xhol, 1 μL; 10×K / M Buffer, 2 μL; Empty vector / target gene (β-Syn-N terminus), 1 μg; RNase-free water, add to 20 μL; BamH1, 1 μL; HindIII, 1 μL; 10×K / M Buffer, 2 μL; Empty vector / target gene (β-Syn-C terminus), 1 μg; RNase-free water, add to 20 μL; After 2 h of enzyme digestion in a 37°C water bath, the digestion products were detected by electrophoresis and the target products were recovered (refer to step (ii) for gel extraction, recovery and purification).

[0024] The above-mentioned recovered products were connected, and the 20μl connection system was designed as follows: Recover the product (empty plasmid: target gene = 1:4), 3 μL of empty plasmid and 12 μL of target gene; T4 DNA Ligase, 1 μL; 10×T4 DNA Ligase Buffer, 2 μL; RNase-free water, add to 20 μL; Connect at 16°C for 2 h.

[0025] It should be noted that, as a control, the empty plasmid was connected with the coding gene of EGFP, and the amino acid sequence (285AA) corresponding to EGFP is as follows: MGSSHHHHHHSSGLVPRGSHMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR IELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSEFELRRQACGRTRAPPPPPLRSGC.

[0026] (IV) Transformation and screening Add 1 μL of the ligation product from step (III) to 10 μL of thawed DH5α competent cells, mix thoroughly, and place on ice for 30 min. Then, heat shock was placed in a 42°C water bath for 60 seconds, and then immediately returned to the ice bath and allowed to stand for 2 minutes; Then, add 500 μL of LB liquid medium without antibiotics to the above mixture, mix it by pipetting, and culture it at 37°C and 180 rpm for 1 h (to recover the strain); After the culture is completed, 200 μL of the resuscitated bacterial solution was taken and evenly dropped onto LB solid medium (kanamycin resistance, 20 μL of 1 M kanamycin), inverted, and cultured at 37°C overnight; After the culture was completed, a positive monoclonal colony with a full morphology and clear edges was picked and inoculated into 5 mL of LB liquid culture medium. After shaking culture at 37°C and 180 rpm overnight, the plasmid was extracted for sequencing verification to ensure that the plasmid recombination was correct.

[0027] The schematic diagram of the constructed recombinant plasmid structure is shown in Figure 1 , Figure 2 , Figure 3 As shown. Among them: The recombinant plasmid structure containing the β-Syn N-terminal domain is as follows Figure 1 The recombinant plasmid structure containing the β-Syn C-terminal domain is shown in Figure 3 As shown, the recombinant plasmid structure containing β-Syn Figure 2 shown.

[0028] It should be noted that when extracting plasmids, the specific operations can be referred to as follows: Centrifuge the bacterial sample at 13000rpm for 1min, discard the supernatant, add 250μL Buffer P1, and resuspend the bacteria by pipetting and shaking; after resuspending, add 250μL Buffer P2 and turn it upside down to fully lyse the bacteria; Then, add 350 μL of Buffer N3, mix well by inverting, centrifuge at 13,000 rpm for 5 min, transfer the supernatant to a new adsorption column in a collection tube, centrifuge at 13,000 rpm for 30 s, discard the waste liquid, and put it back into the adsorption column; Add 150 μL of Buffer PB to the adsorption column, centrifuge at 13000 rpm for 30 s, and discard the waste liquid; Add 400 μL Buffer PW and centrifuge at 13000 rpm for 1 min. Transfer the adsorption column to a new 1.5mL centrifuge tube, add 50μL of Buffer EB to the center of the adsorption membrane, let it stand at room temperature for 2 minutes, and centrifuge it at 13000rpm for 1 minute. The resulting solution is the plasmid after small extraction, which can be stored at -20℃ or directly subjected to sequencing analysis.

[0029] 5. Transformation and induction of protein expression Refer to the existing routine operations, further extract the recombinant plasmid identified correctly in step (IV), and transform it into BL21 competent cells. The specific transformation operation is as follows: In a 1.5 ml centrifuge tube, add 10 μL of BL21 competent cells and 1 μL of the correctly identified recombinant plasmid in step (iv), mix well, and place on ice for 30 minutes; Then, heat shock was placed in a 42°C water bath for 60 seconds, and then immediately returned to the ice bath and allowed to stand for 2 minutes; Then, add 500 μL of LB liquid medium without antibiotics to the above mixture, mix it by pipetting, and culture it at 37°C and 180 rpm for 1 h (to recover the strain); After the culture is completed, 200 μL of the resuscitated bacterial solution was taken and evenly dropped onto LB solid medium (kanamycin resistance, 20 μL of 1 M kanamycin), inverted, and cultured at 37°C overnight; After the culture was completed, a positive monoclonal colony with a full morphology and clear edges was picked and inoculated into 5 mL of LB liquid medium, and cultured at 37°C and 180 rpm with shaking overnight; The above-mentioned overnight cultured bacterial solution was further transferred to 500 mL of LB liquid medium, and cultured at 37°C and 180 rpm for 3 h. IPTG with a final concentration of 0.2 mM was added and further cultured at 37°C and 180 rpm for 5 h to induce protein expression.

[0030] 6. Protein purification Take 500 mL of the bacterial solution after the induction expression in step (V), centrifuge at 10000 rpm for 2 min, and keep the precipitate; add 30 mL of PBS to resuspend the bacterial precipitate, and lyse it by ultrasonic treatment for 30 min under ice bath (stop lysing when the liquid is no longer turbid); Centrifuge at 4°C and 10,000 rpm for 3 min, collect the supernatant (the target proteins are all soluble proteins), and dilute with an equal volume of Binding Buffer as the sample to be purified; The sample to be purified is added to the pre-equilibrated chromatography column for loading (the chromatography column is filled with Ni-Agarose Resin and pre-equilibrated with 5 column volumes of distilled water and 10 column volumes of Binding Buffer), and 15 column volumes of Soluble Binding Buffer are added to wash the chromatography column to remove non-specifically bound impurities; Subsequently, 300 mM imidazole concentration Soluble Elution Buffer was used for elution. After the elution peak was collected, 10 times column volume of 500 mM imidazole concentration Soluble Elution Buffer was added for full elution; the eluate was collected.

[0031] The eluate was dialyzed in dialysate at 4°C overnight, and the proteins in the dialysis bag (i.e., purified β-Syn, β-Syn-N, and β-Syn-C proteins) were collected and the concentration was measured for later use.

[0032] As a control, the purified EGFP protein was prepared by referring to the above operation.

[0033] Example 2 In this embodiment, the inventors took MN9D cells as an example and constructed a + The induced PD cell model was used to conduct a preliminary test and analysis on the effect of the relevant protein prepared in Example 1. The specific experimental situation is briefly described as follows.

[0034] 1.1 Cell model construction MN9D cells were plated at 5 × 10 3 The amount was inoculated into a 96-well cell culture plate, 100 μL DMEM cell culture medium was added to each well, and after culturing at 37°C for 24 h, MPP+ was added and cultured for another 24 h to induce the formation of PD model cells.

[0035] In order to determine the most suitable MPP+ induction concentration, different concentrations of MPP+ were added during the above culture process, and the most suitable MPP+ concentration was determined based on the cell viability test results of the MTT method.

[0036] When the MTT method is used to detect cell viability, the operation is as follows: Add 150 μL of 0.5 mg / ml MTT working solution to the sample well in the dark, incubate at 37°C for 4 h, discard the supernatant, add 150 μL of DMSO, shake and mix, and measure the absorbance of the sample at 490 nm; Cell survival rate = [(experimental well - blank well) / (negative control - blank well)] × 100%.

[0037] The results are as follows Figure 4 ( Figure 4 As shown in A), it can be seen that the final concentration of MPP+ not less than 0.7mM can effectively induce the formation of the PD model. However, considering the convenience of experimental operation and cell activity, a final concentration of 1mM MPP+ was chosen to induce the construction of the PD model.

[0038] 2. Effect of protein concentration Referring to the experimental operation of "(I) Cell model construction" above, the PD model was constructed with a final concentration of 1mM MPP+. β-Syn-N (β-Syn or β-Syn-C, EGFR as positive) protein working solution was added twice at 12h and 24h after induction of modeling. After continuing to incubate for 24h, the cell survival rate was detected by MTT method as mentioned above to determine the effect of different protein dosages on PD model cells.

[0039] The results are as follows Figure 4 ( Figure 4 B. Figure 4 C. Figure 4 D. Figure 4 E). It can be seen that in the PD model, with the addition of EGFP recombinant protein ( Figure 4B) β-Syn-C-terminal recombinant protein ( Figure 4 E) concentration increased, the cell survival rate did not change significantly; however, with the addition of β-Syn-N-terminal recombinant protein ( Figure 4 C) β-Syn recombinant protein ( Figure 4 D) With the increase of concentration, the cell survival rate increased significantly, while when the concentration of the added recombinant protein increased to 10 μM, the cell survival rate decreased. Therefore, it is more appropriate to add 5 μM recombinant protein concentration to PD model cells at 12h and 24h of culture, respectively, and the data analysis is statistically significant.

[0040] Based on the above experimental results, the inventors further used Western blot and Tunel staining techniques to further analyze the effects of 5 μM recombinant protein on PD cell changes. The specific situation is briefly described as follows.

[0041] (1) Western blot detection RIPA lysis buffer containing PMSF was added to the cells in different treatment groups and incubated on ice for 30 min; After ice bath, centrifuge at 4°C and 12,000 rpm for 10 min, and keep the supernatant (i.e., the total protein extract); after determining the protein concentration in the supernatant, perform SDS-PAGE gel electrophoresis and transfer to a nitrocellulose membrane; The membrane was blocked with 5% skim milk powder at room temperature for 1 hour and then incubated with primary antibodies (GAPDH, TH, α-Syn, Bax, Caspase3, Bcl-2) at 4°C overnight; The next day, the cells were washed three times with TBST, incubated with HRP-labeled rabbit secondary antibody (Goat Anti-Rabbit IgG HRP) at room temperature for 1 hour, washed again with TBST three times (10 minutes each time), and finally, ECL luminescent solution was added and developed using a developer, and the developed images were scanned and analyzed.

[0042] The specific experimental groups include: NC group (group without any treatment), PD group (1mM final concentration MPP+induction group), P+EGFP group (1mM final concentration MPP+induction after adding EGFP protein positive control), P+N group (1mM final concentration MPP+induction after adding β-Syn-N protein experimental group), P+β-Syn group (1mM final concentration MPP+induction after adding β-Syn protein experimental group), P+C group (1mM final concentration MPP+induction after adding β-Syn-C protein experimental group).

[0043] Some existing studies believe that tyrosine hydroxylase (TH) is the rate-limiting enzyme for the synthesis of dopamine (DA), and its reduced expression may be the main cause of PD. In addition, some studies have shown that under the pathological conditions of PD, various apoptosis signaling molecules can trigger the apoptosis pathway to promote the production of free radicals and cause damage to mitochondria and cells. At this time, the expression of apoptosis-related proteins cleaved-Caspase3 and Bax is upregulated, and Bcl-2 is downregulated. Therefore, the above-mentioned Western blot technique was used to detect the changes in the expression levels of α-Syn, TH, cleaved-Caspase3, Bax and Bcl-2 proteins in cells of different experimental groups. The experimental results are as follows Figure 5 As shown. The analysis shows that: As for α-Syn, the protein expression in the PD group was significantly increased compared with the NC group (P<0.01); compared with the PD group, there was no difference in expression in the PD+EGFP group and the PD+C group (P>0.05), while the expression in the PD+N group and the PD+β-Syn group was reduced (P<0.05) ( Figure 5 A. Figure 5 B); That is, the group treated with protein containing the β-Syn N-terminal domain can significantly reduce the content of α-Syn; As for TH, compared with the NC group, the protein expression of PD group was significantly reduced (P<0.001); compared with the PD group, there was no difference in expression in the PD+EGFP group and PD+C group (P>0.05), while the expression in the PD+N group and PD+β-Syn group increased (P<0.05) ( Figure 5 A. Figure 5 C); that is, the group treated with protein containing the β-Syn N-terminal domain can significantly increase the expression of TH; As for Bax, the protein expression in the PD group was significantly increased compared with the NC group (P<0.05); compared with the PD group, there was no difference in the expression of Bax in the PD+EGFP group and the PD+C group (P>0.05), while the expression of Bax in the PD+N group and the PD+β-Syn group was decreased (P<0.05) ( Figure 5 A. Figure 5 E); that is, the group treated with protein containing the β-Syn N-terminal domain can significantly reduce the expression of Bax; As for Caspase3, compared with the NC group, the protein expression in the PD group was significantly increased (P<0.05); compared with the PD group, there was no difference in expression in the PD+EGFP group and the PD+C group (P>0.05), while the expression in the PD+N group and the PD+β-Syn group was reduced (P<0.05) ( Figure 5 A. Figure 5D); That is, the group treated with protein containing the β-Syn N-terminal domain can reduce the expression of Caspase3; As for Bcl-2, compared with the NC group, the protein expression in the PD group was significantly reduced (P<0.01); compared with the PD group, there was no difference in the expression of the PD+EGFP group and the PD+C group (P>0.05), while the expression of the PD+N group and the PD+β-Syn group increased (P<0.05) ( Figure 5 A. Figure 5 F); That is, the group treated with protein containing the β-Syn N-terminal domain can increase the expression of Bcl-2.

[0044] In summary, it can be seen that proteins containing the β-Syn N-terminal domain (β-Syn-N, β-Syn) reduce neuronal damage by reducing the expression of α-Syn and increasing the expression of TH; at the same time, they reduce cell apoptosis by downregulating the expression of cleaved-Caspase3 and Bax and upregulating the expression of Bcl-2, thereby improving the pathogenesis of PD. In other words, the N-terminal domain of β-Syn plays a key role in the anti-PD process.

[0045] (2) Tunel staining Tunel staining operation process is as follows: Place a cell slide in a 24-well plate and inoculate the cells, then add 1 ml of cell culture medium to each well; refer to the above operations to construct a PD cell model and perform related treatments; After the treatment, the medium was discarded and the cells were washed twice with PBS. Then, 500 μL of immunostaining fixative was added to each well to fix the cells for 30 min. After fixation, the cells were washed twice with PBS. Add 500 μL of immunostaining strong permeabilization solution to each well and incubate at room temperature for 5 min. Wash twice with PBS at the end; Add 500 μL of endogenous peroxidase blocking solution to each well and incubate at room temperature for 20 min. Wash twice with PBS after the end; Add 50 μL of pre-prepared biotin labeling solution to each well and incubate at 37°C in the dark for 60 min. Wash once with PBS. Subsequently, 0.2 ml of the reaction stop solution was added dropwise and incubated at room temperature for 10 min. After completion, the cells were washed three times with PBS; Add 50 μL of pre-prepared Streptavidin-HRP working solution to each well and incubate at room temperature for 30 min. Wash three times with PBS after the end; 0.5 ml of DAB color developing solution was added to each well, incubated at room temperature for 20 min, washed 3 times with PBS, and finally the cells were covered with PBS and observed under a fluorescence microscope.

[0046] This experiment reflects the damage or apoptosis of neuronal cells after different protein treatments by the change in the percentage of positive cells after Tunel staining. Figure 6 As shown. It can be seen that: Compared with the NC group, the percentage of Tunel-positive neuronal cells in the PD group was significantly increased (P<0.001). Compared with the PD group, there was no difference in the percentage of Tunel-positive neuronal cells in the PD+EGFP group and the PD+C group (P>0.05), while the percentage of Tunel-positive neuronal cells in the PD+N group (P<0.05) and the PD+β-Syn group (P<0.01) increased ( Figure 6 A. Figure 6 B). In other words, this result once again shows that the group treated with protein containing the β-Syn N-terminal domain can reduce neuronal cell damage.

[0047] Based on the above results, it can be preliminarily concluded that the β-Syn N-terminal domain protein has a neuroprotective effect, which improves the incidence of PD by reducing neuronal cell apoptosis.

Claims

1. The use of β-Syn protein in the preparation of a Parkinson's disease medicament, characterized in that: The β-Syn protein is a protein containing the N-terminal domain of the β-Syn protein, and the amino acid sequence of the protein containing the N-terminal domain of the β-Syn protein is shown in SEQ ID No.

1.

2. The use of β-Syn protein in the preparation of a Parkinson's disease medicament according to claim 1, characterized in that: The coding nucleotide sequence corresponding to the protein containing the N-terminal domain of β-Syn protein is shown in SEQ ID No.

2.

3. The use of β-Syn protein in the preparation of a Parkinson's disease medicament according to claim 1, characterized in that: Proteins containing the N-terminal domain of β-Syn protein reduce neuronal cell damage by reducing the expression of α-Syn and increasing the expression of TH.

4. The use of β-Syn protein in the preparation of a Parkinson's disease medicament according to claim 1, characterized in that: Proteins containing the N-terminal domain of β-Syn protein reduce cell apoptosis by downregulating the expression of cleaved-Caspase3 and Bax, and upregulating the expression pathway of Bcl-2.

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