A recombinant alpha-synuclein protein with low self-aggregation tendency and its use in the preparation of a neurodegenerative disease alpha-syn-rt-quic detection system

Abstract

The application belongs to the technical field of molecular diagnosis and protein engineering, and discloses a recombinant alpha-synuclein protein with low self-aggregation tendency and application thereof in preparation of an alpha-syn-RT-QuIC detection system for neurodegenerative diseases. The recombinant alpha-synuclein protein is composed of alpha-synuclein and a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 inserted after the M site of the first amino acid of the wild-type alpha-synuclein protein. The application aims to overcome the defects of the existing wild-type alpha-synuclein protein monomer, such as high background noise, false positive and decreased detection specificity caused by spontaneous aggregation in the alpha-syn-RT-QuIC detection. In the detection, the recombinant alpha-synuclein protein can significantly reduce the non-specific aggregation signal, improve the detection sensitivity and specificity of the pathological alpha-synuclein seed activity, and thus provide a more reliable tool for early diagnosis and biomarker detection of neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostics and protein engineering technology, specifically relating to a recombinant α-synuclein with low self-aggregation tendency and its application in the preparation of an α-syn-RT-QuIC (Real-Time Quaking-Induced Conversion) detection system for neurodegenerative diseases (especially synucleinopathies, such as Parkinson's disease and Lewy body dementia). Background Technology

[0002] α-Synuclein (α-syn) is a naturally occurring disordered protein highly expressed at the presynaptic terminals of the central nervous system. Pathological α-synuclein aggregates, formed by its misfolding and abnormal aggregation, are a common pathological marker of a range of synucleinogenic disorders, including Parkinson's Disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA). These insoluble aggregates, particularly their oligomeric intermediates and fibrillary forms, are considered neurotoxic and can spread and replicate between cells via a prion-like "seeding" mechanism, driving disease progression.

[0003] Real-time vibration-induced transformation (α-syn) of α-syn protein (α-syn-RT-QuIC) technology can amplify misfolded α-syn. α-syn-RT-QuIC detection utilizes a seed nucleation mechanism, amplifying α-syn aggregates (α-syn seeds) present in pathological α-synuclein from patients. These seeds can induce the polymerization of α-synuclein (monomers) to form amyloid-β aggregates. The α-syn-RT-QuIC assay uses the fluorescent dye thioflavin T (ThT), which binds to the amyloid-β structures of aggregated α-syn, assessing the differential levels of pathological α-syn in terms of fluorescence signal intensity. α-syn-RT-QuIC detection of α-synuclein seeds in cerebrospinal fluid (CSF) has shown extremely high diagnostic sensitivity and specificity, making it a promising biomarker detection platform.

[0004] However, the performance of α-syn-RT-QuIC detection is highly dependent on the biological properties of the α-synuclein monomer substrate used. Wild-type α-synuclein monomers, due to their inherent amphiphilicity, especially their N-terminal domain (approximately amino acids 1-60), tend to undergo intermolecular interactions in solution. These properties are associated with the aggregation tendency of α-Syn (Reference: Bertoncini CW, et al. Release of long-range tertiary interactions potentials aggregation of natively unstructured alpha-synuclein. Proc Natl Acad Sci USA. 2005;102(5):1430-1435).Furthermore, a few α-synuclein monomers unfold, exposing previously hidden hydrophobic regions or β-sheet-prone regions (Reference: Mathias Jucker, et al. Self-propagation of pathogenic protein aggregates in neurodegenerative diseases. Nature. 501, 45-51(2013)) and the NACore region (amino acids 68-78: GAVVTGVTAVA, a highly hydrophobic core segment with a strong tendency to self-aggregate and a key step in the entire aggregation cascade) (Reference: Benoit I. Giasson, et al. A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. The Journal of Biological Chemistry). 2001;276(4):2380-2386;Israeli, Eitan.Structure of the toxic core of α-synuclein from invisible crystals. Israel Medical Association Journal, 2015,17(10):659), which then forms unstable oligomers or fibrils through random collisions. This tendency to self-aggregate will generate background fluorescence signals during long-term α-syn-RT-QuIC incubation, leading to false positive results, which severely limits the specificity and repeatability of the detection, especially when detecting samples with extremely low seed concentrations (such as blood) or when a high sensitivity threshold is required.

[0005] To address these issues, existing technologies primarily focus on: optimizing protein purification processes to reduce pre-existing aggregates, such as employing optimized expression systems to avoid amino acid misinsertion (see patent: US11970520B2, α-synuclein substrate and its preparation and use method); or using single or a few point mutations (such as K23E, K23Q, etc.) to attempt to reduce aggregation tendency (see patent: US12105101B2, detection method for detecting α-synuclein inoculation activity associated with synucleinosis); or designing inhibitory peptides that bind to α-synuclein molecules, such as the structure-based peptide inhibitor of α-synuclein aggregation disclosed in patent CN109641028A, which inhibits amyloid aggregation, cytotoxicity, and spread by binding to residues 68-78 of α-synuclein, and describes methods for preparing and using the inhibitory peptide (e.g., for the treatment of Parkinson's disease, Lewy body dementia, or MSA).

[0006] However, the above methods all have significant limitations: optimization of the purification process cannot fundamentally change the inherent aggregation characteristics of the protein; point mutation strategies are scattered across different positions in the sequence, with complex mechanisms and potential unintended effects on protein function; and repressive peptide strategies target non-N-terminal regions (residues 68-78), directly interfering with the core seed recognition interface (NACore region) upon which α-syn-RT-QuIC detection relies, thus preventing the repressive peptide from being used as a detection substrate simultaneously. In other words, there is a fundamental contradiction between inhibiting aggregation and preserving seed-induced aggregation activity, making such repressive peptides unsuitable for direct substrate modification in α-syn-RT-QuIC detection systems. More importantly, current technologies have not fully explored the potential of the N-terminal region as an "engineering platform." Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant α-synuclein with low self-aggregation tendency and its application in the preparation of α-syn-RT-QuIC detection products for neurodegenerative diseases. By molecularly designing a short peptide sequence fused to its N-terminus, this recombinant α-synuclein serves as a detection substrate, aiming to overcome the shortcomings of existing wild-type α-synuclein monomers in RT-QuIC detection, which generate high background noise, leading to false positives and decreased detection specificity due to spontaneous aggregation. During detection, this recombinant α-synuclein significantly reduces non-specific aggregation signals, improving the detection sensitivity and specificity for pathological α-synuclein seed activity, thereby providing a more reliable tool for the early diagnosis and biomarker detection of neurodegenerative diseases (synucleinopathy).

[0008] This invention is achieved through the following technical solution: a recombinant α-synuclein with low self-aggregation tendency, wherein the recombinant α-synuclein is composed of α-synuclein and a polypeptide fused to the N-terminus of the α-synuclein. The polypeptide is inserted after the M site of the first amino acid of wild-type α-synuclein, and the amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

[0009] Preferably, the amino acid sequence of the recombinant α-synuclein is shown in SEQ ID NO:2.

[0010] Preferably, the recombinant α-synuclein is obtained by inserting a nucleic acid sequence encoding the polypeptide between the original start codon ATG of the nucleotide sequence encoding α-synuclein and the subsequent coding sequence (the coding sequence of α-synuclein).

[0011] Preferably, the nucleic acid sequence encoding the recombinant α-synuclein is shown in SEQ ID NO:3.

[0012] Another technical solution of the present invention is to provide the application of the above-mentioned recombinant α-synuclein in the preparation of an α-syn-RT-QuIC detection system for neurodegenerative diseases. Specifically, the recombinant α-synuclein is used as a substrate in the α-syn-RT-QuIC detection reaction.

[0013] Preferably, the α-syn-RT-QuIC detection system includes recombinant α-synuclein, reaction buffer, ThT, and the sample to be tested.

[0014] Preferably, the neurodegenerative diseases include Parkinson's disease, Lewy body dementia, and multiple system atrophy.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention is the first to fuse a short peptide sequence (EDPGEDPGED) at the N-terminus of α-synuclein. By introducing charge repulsion (E and D residues) and conformational restriction (P and G residues), it effectively inhibits the spontaneous aggregation and misfolding of monomer molecules from a dual mechanism, thereby solving the problem of wild-type α-synuclein easily forming background aggregates in solution and during incubation from the source.

[0016] (2) The present invention uses N-terminal extension modification for molecular design, which does not involve the core functional region of α-synuclein (especially the NACore region, amino acids 68-78) and does not change its bulk sequence. Therefore, the recombinant α-synuclein can still be recruited normally by pathological α-synuclein aggregates (seeds) and undergo conformational change, and completely retains the substrate reactivity required for α-syn-RT-QuIC detection.

[0017] (3) In the α-syn-RT-QuIC detection, the recombinant α-synuclein described in this invention is used as the α-syn-RT-QuIC substrate. During the long-term oscillation incubation, the background fluorescence signal is extremely low, which significantly reduces the risk of false positives and improves the signal-to-noise ratio, repeatability and threshold discrimination of the detection. It is especially suitable for sample types with extremely low seed concentration (such as cerebrospinal fluid, blood, etc.), making ultrasensitive detection possible.

[0018] (4) This invention can be achieved by inserting a nucleic acid sequence encoding a short peptide at the N-terminus through genetic engineering. It does not rely on complex purification optimization or multi-point mutation. It is easy to obtain soluble recombinant protein in high yield in expression systems such as E. coli. The preparation process is simple and low cost, making it suitable for industrial production and standardized reagent kit development.

[0019] (5) The α-syn-RT-QuIC detection system provided by the present invention can be effectively applied to the detection of various α-synuclein diseases such as Parkinson's disease, Lewy body dementia, and multiple system atrophy, and has a good range of disease coverage and application prospects. Attached Figure Description

[0020] Figure 1 This is a predicted diagram of the recombinant α-synuclein and wild-type α-synuclein structures described in this invention.

[0021] Figure 2 This is a schematic diagram of the α-syn-RT-QuIC detection results for a positive sample.

[0022] Figure 3 This is a schematic diagram of the α-syn-RT-QuIC test results for a negative sample. Detailed Implementation

[0023] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] This invention aims to address the shortcomings of existing wild-type α-synuclein monomers in α-syn-RT-QuIC detection, which generate high background noise, lead to false positives, and reduce detection specificity due to spontaneous aggregation. Existing techniques for addressing these issues do not consider using the N-terminal region as an "engineering platform," failing to solve the self-aggregation problem at its source. Therefore, this invention proposes for the first time a modification strategy based on the N-terminal extension and fusion of a specific polypeptide with α-synuclein. By introducing a dual synergistic mechanism of charge repulsion and conformational restriction, this strategy significantly reduces the tendency for spontaneous protein aggregation while fully preserving its ability to recognize and amplify pathological α-synuclein aggregates.

[0026] The technical solution of this invention can be summarized as follows: A recombinant α-synuclein variant (with the amino acid sequence EDPGEDPGED (as shown in SEQ ID NO:1) is constructed by fusing a polypeptide with the amino acid sequence EDPGEDPGED (as shown in SEQ ID NO:1) to the N-terminus of wild-type α-synuclein. This variant is then used as a substrate in the α-syn-RT-QuIC detection reaction and applied to an α-syn-RT-QuIC detection system for neurodegenerative diseases such as Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA). The α-syn-RT-QuIC detection system preferably comprises recombinant α-synuclein, a reaction buffer, ThT, and the sample to be tested.

[0027] The molecular design of this invention (N-terminal fusion of a specific polypeptide) is based on the understanding of the self-aggregation initiation mechanism of α-synuclein: the initial steps of self-aggregation are mainly driven by intermolecular interactions between hydrophobic residues within the N-terminal domain. This invention employs an N-terminal extension strategy, fusing a 10-amino acid sequence to the N-terminus of the wild-type sequence: Glu-Asp-Pro-Gly-Glu-Asp-Pro-Gly-Glu-Asp (EDPGEDPGED). See the molecular structure below. Figure 1 , Figure 1 In the diagram, A represents the conformation of wild-type human α-synuclein, B represents the conformation of recombinant α-synuclein, and C is a comparison between the two.

[0028] This extended sequence collaboratively suppresses self-aggregation through the following mechanism: (1) Charge repulsion mechanism (E and D residues): Glutamic acid (E) and aspartic acid (D) are both negatively charged at physiological pH. The six negative charges form a high-density charge cluster at the N-terminus, generating long-range electrostatic repulsion, which greatly increases the energy barrier for non-specific approach between monomers. The electrostatic barrier covers the hydrophobic region at the N-terminus, preventing random collisions between hydrophobic regions.

[0029] (2) Conformation restriction mechanism (P and G residues): The rigid pyrrole ring of proline (P) restricts the conformation of the main chain, disrupts the regularity of the α-helix, and introduces molecular kinks at the N-terminus; glycine (G) has no side chain, providing maximum conformational flexibility. As a molecular hinge, it allows the extended sequence to move relatively independently, avoiding the influence on the function of the main structure.

[0030] (3) Synergistic effect: The alternating arrangement of E, D, P and G forms a dual barrier unit of charge and conformation; 10 amino acids form a sufficiently long physical barrier (about 35-40 Å), and the dual mechanism ensures that the spontaneous aggregation process is suppressed.

[0031] Furthermore, the design of this invention ensures that the aforementioned N-terminal extension variants remain highly sensitive to aggregation induced by pathological seeds. This design follows the principle of suppressing spontaneous interactions without affecting template-guided binding: the extension sequence is added only to the N-terminus, 50 amino acids away from the NACore region (amino acids 68-78) responsible for seed recognition. This flexible connection ensures complete accessibility to the NACore region, unhindered by the spatial obstruction of the N-terminal extension; the regular β-sheet edges exposed on the surface of the aggregate seed provide monomers with high-affinity, high-specificity binding sites. Therefore, when encountering a seed, the complete NACore region of the variants of this invention can efficiently recognize and bind to the seed template, initiating rapid aggregation.

[0032] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0033] Example 1: Design, gene synthesis, and vector construction of recombinant α-synuclein This embodiment provides a recombinant α-synuclein variant with low self-aggregation tendency (named EDPGEDPGED-α-Syn). The design strategy for this variant is as follows: a polypeptide with the amino acid sequence shown in SEQ ID NO:1 is directly inserted between the original starting amino acid and subsequent amino acid sequences of wild-type human α-synuclein (amino acid sequence as shown in SEQ ID NO:4, encoding gene NCBI reference sequence NM_000345.4). After insertion, the polypeptide is attached to the N-terminus of the α-synuclein. To facilitate subsequent purification, a 6×His tag is retained at the C-terminus.

[0034] SEQ ID NO:1: EDPGEDPGED.

[0035] SEQ ID NO:4: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA.

[0036] The amino acid sequence of the recombinant α-synuclein variant (containing a C-terminal 6×His tag, totaling 156 amino acids) is as follows (SEQ ID NO:2): MEDPGEDPGEDDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEAHHHHHH.

[0037] Based on the codon usage frequency table of E. coli, the nucleotide sequence encoding the above-mentioned recombinant α-synuclein was optimized. The optimized nucleic acid sequence encoding the recombinant α-synuclein variant is as follows (SEQ ID NO:3): ATG GAG GAT CCG GGT GAG GAT CCG GGG GAA GAT GAC GTT TTT ATG AAG GGACTG AGT AAA GCG AAG GAG GGT GTG GTT GCA GCG GCA GAG AAA ACC AAG CAG GGC GTGGCT GAA GCC GCA GGC AAA ACA AAA GAA GGC GTT CTG TAT GTG GGT TCA AAA ACC AAAGAA GGT GTG GTA CAC GGA GTC GCA ACC GTA GCC GAG AAA ACC AAA GAG CAG GTG ACTAAC GTG GGC GGC GCT GTA GTC ACT GGC GTG ACT GCC GTG GCG CAG AAA ACA GTC GAAGGT GCT GGG AGC ATT GCA GCG GCG ACC GGT TTT GTA AAA AAA GAT CAG TTA GGG AAGAAT GAA GAA GGA GCG CCG CAG GAA GGA ATA CTT GAA GAT ATG CCG GTA GAC CCG GATAAT GAA GCA TAT GAA ATG CCA TCT GAA GAA GGT TAT CAA GAT TAC GAA CCG GAG GCACAT CAC CAT CAT CAC CAT TAA. The nucleic acid sequence was cloned into the prokaryotic expression vector pET-28a(+) between the Nde I and Xho I restriction sites, placing the target gene under the control of the T7 strong promoter. The recombinant expression plasmid was constructed and named pET28a-EDPGEDPGED-α-Syn-His.

[0038] Example 2: Expression and purification of recombinant α-synuclein The recombinant expression plasmid pET28a-EDPGEDPGED-α-Syn-His obtained in Example 1 was heat-shocked and transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C with shaking at 220 rpm as seed culture. The seed culture was transferred to fresh medium at a ratio of 1:100 and cultured until the OD600 reached 0.6 to 0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM, and the culture temperature was lowered to 25°C to induce expression for 16 to 18 hours.

[0039] The protein was purified using the following steps: (1) Cell lysis: Collect bacterial cells by centrifugation at 4°C. Resuspend the bacterial cells in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 1 mM PMSF, pH 8.0). Disrupt the cells using a probe sonicator on ice.

[0040] (2) Metal chelate affinity chromatography: The filtered sample was loaded onto a Ni-NTA agarose gel column pre-equilibrated with binding buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0). The sample was washed thoroughly with wash buffers containing 20 mM and 50 mM imidazole to remove non-specifically bound proteins. Finally, the target protein was eluted with elution buffer containing 250 mM imidazole, and the elution peak was collected.

[0041] (3) Dialysis and concentration: The eluent was dialyzed thoroughly with phosphate-buffered saline (PBS, pH 7.4) at 4°C to remove imidazole and salt.

[0042] (4) Quantification and storage: Protein concentration was determined using the BCA method and protein purity was verified by SDS-PAGE analysis. The concentration was adjusted to storage concentration (1 mg / mL) with PBS. After aliquoting, the protein was stored at -80°C for later use.

[0043] Example 3: Establishment of the α-syn-RT-QuIC detection method (1) Preparation of the reaction system Prepare the master mixture in sterile low-adsorption centrifuge tubes to the following final concentrations: 100 mM PIPES buffer (pH 6.5); 170 mM NaCl; 10 μM ThT; 0.1 mg / mL EDPGEDPGED-α-Syn variant (prepared in Example 2).

[0044] After vortexing the master mixture, briefly centrifuge. Aliquot 95 μL of the master mixture into each RT-QuIC reaction well. Then add 5 μL of the test sample (cerebrospinal fluid stock solution or serum IP product). Set up at least 4 replicate wells for each sample.

[0045] (2) Reaction initiation and monitoring Using a black transparent-bottomed 96-well plate, two 1.0 mm diameter silica beads were pre-added to each well. The above reaction mixture was added to the wells and sealed with an optical sealing film. A BMG FLUOstar Omega microplate reader (Germany) was used at 600 rpm, dual-track, 440 nm excitation and 480 nm emission; bottom readout, oscillation for 14 min, pause for 1 min, and recording every 30 min.

[0046] (3) Criteria for determining a positive result Positive result criteria: The test sample shows a peak in 3 / 4 replicates within 80 hours, meaning the time-to-threshold (TTT) is reached before 80 hours, with the threshold defined as 30,000 RFU. In this embodiment, the detection results of positive samples are as follows: Figure 2 As shown.

[0047] (4) Criteria for determining negative results Negative test criteria: Test time (TTT) > 80 hours for 3 / 4 replicates of the sample. In this embodiment, the test results for negative samples are as follows: Figure 3 As shown.

[0048] Comparative Example 1: Recombinant protein variant fused with the C-terminus EDPGEDPGED peptide To verify the position specificity of the N-terminal fusion polypeptide EDPGEDPGED (amino acid sequence as shown in SEQ ID NO: 1) in this invention, a recombinant protein variant fused with the same polypeptide at the C-terminus of α-synuclein was constructed in this comparative example.

[0049] The nucleic acid sequence encoding EDPGEDPGED was inserted before the stop codon of the wild-type α-synuclein coding sequence, fusing the polypeptide to the C-terminus of the α-synuclein while retaining the C-terminal 6×His tag. Recombinant expression and purification were performed using the same expression vector and E. coli expression system as in Example 1 to obtain the C-terminal fusion variant (α-Syn-EDPGEDPGED-His). The amino acid sequence of this recombinant protein is as follows (SEQ ID NO:5): MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEAEDPGEDPGEDHHHHHH.

[0050] Comparative Example 2: Recombinant protein variants with N-terminal fusion of other sequence peptides To verify the synergistic effect of the dual mechanism of "charge repulsion + conformational restriction" in the polypeptide EDPGEDPGED described in this invention, two recombinant protein variants with other sequences fused to the N-terminus were constructed in this comparative example, namely: (1) Only charge-repellent polypeptides are provided, the amino acid sequence of which is shown in SEQ ID NO:6: EEDEEDDEE.

[0051] (2) Only conformationally restricted polypeptides are provided, with the amino acid sequence shown in SEQ ID NO:7: APGSAPGSAP.

[0052] Using the same N-terminal fusion strategy as in Example 1, the nucleic acid sequences encoding the above-mentioned polypeptides (SEQ ID NO:6) and (SEQ ID NO:7) were inserted after the start codon ATG and before the rest of the coding sequence of the α-synuclein protein, respectively, with a 6×His tag retained at the C-terminus. Two variants (EEDDEEDDEE-α-Syn-His; APGSAPGSAP-α-Syn-His) were obtained after expression and purification.

[0053] The amino acid sequences of the two variants are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0054] SEQ ID NO:8: MEEDDEEDDEEDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEAHHHHHH.

[0055] SEQ ID NO:9: MAPGSAPGSAPDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEAHHHHHH. Experimental Example 1: Comparison of sensitivity detection of positive seeds by variant and wild-type monomeric substrates The recombinant α-synuclein fibrils standard (seeds) of known concentrations was serially diluted 10-fold. The α-synuclein seeds at different dilutions were subjected to α-syn-RT-QuIC assays (using the same method as in Example 3) with the EDPGEDPGED-α-Syn-His variant and wild-type substrate of this invention to determine the detection limit. The results are shown in Table 1.

[0056] Table 1. Results of α-syn-RT-QuIC assays on different seed concentrations using variant and wild-type substrates.

[0057] As shown in Table 1, the 100% detection concentration of the two substrates for seeds was the same, 100 pg / mL. For seeds at 100 pg / mL, the mean time to temperature (TTT) was 24.8 ± 1.9 hours for the EDPGEDPGED-α-Syn-His variant and 26.8 ± 3.0 hours for the wild-type group, with no statistically significant difference between the two groups (p > 0.05). Statistical differences in this patent were all determined using t-tests.

[0058] The above results demonstrate that the seed recognition and amplification efficiency of the EDPGEDPGED-α-Syn-His variant of this invention is comparable to that of the wild type, and the N-terminal fusion strategy does not affect its core function as an α-syn-RT-QuIC substrate. Furthermore, at a seed concentration of 100 pg / mL, both substrates achieved 100% detection, and the TTT values ​​were concentrated (variant CV = 7.7%, wild type CV = 11.2%), indicating that this concentration represents a reliable detection limit. When the seed concentration decreased to 10 pg / mL, the detection rate significantly decreased (variant 25%, wild type 17%), suggesting that this concentration is close to the methodological detection limit. Notably, in the negative control group (no seeds), the wild type substrate exhibited spontaneous aggregation in 2 wells (50%), while the variant substrate did not spontaneously aggregate, preliminarily suggesting that the variant maintains seed recognition sensitivity while having lower background noise.

[0059] Experimental Example 2: Assessment of the self-aggregation tendency of variant, wild-type, and comparative substrates (negative sample specificity) Using the EDPGEDPGED-α-Syn-His variant of this invention, commercially available wild-type human α-synuclein, the C-terminal fusion variant of Comparative Example 1, and two N-terminal fusion variants of Comparative Example 2 as substrates, cerebrospinal fluid samples (n=112) of patients not clinically diagnosed with α-synucleinosis were mixed for α-syn-RT-QuIC detection (detection method as in Example 3). The specificity of negative samples was statistically analyzed, and the results are shown in Table 2.

[0060] Table 2. Detection specificity of each substrate for non-α-synucleinosis samples (n=112)

[0061] As shown in Table 2, in the detection of cerebrospinal fluid samples from 112 non-α-synucleinosis patients, the negative specificity of the EDPGEDPGED-α-Syn-His variant of this invention as a substrate reached 95.5%, significantly better than the wild-type monomeric substrate (79.5%, p<0.001). In Comparative Example 1, the C-terminal fusion of the EDPGEDPGED variant (specificity 81.2%) showed no significant difference from the wild type (p>0.05), proving that C-terminal fusion cannot achieve the technical effect of N-terminal fusion. In Comparative Example 2, the specificity of the charge-repulsive variant (specificity 87.5%) and the conformation-restricted variant (specificity 85.7%) was better than that of the wild type (p<0.05), but significantly lower than that of the dual-mechanism variant of this invention (p<0.01), proving that although a single mechanism has a certain effect, only the synergistic effect of "charge repulsion + conformation restriction" can achieve the optimal self-aggregation inhibition effect.

[0062] The above results demonstrate that the dual-mechanism design of N-terminal fusion EDPGEDPGED of this invention has significant technical advantages in reducing background noise and improving negative specificity in α-syn-RT-QuIC detection.

[0063] Experimental Example 3: Sensitivity of Variant, Wild-type, and Comparative Substrates for Detecting Clinical Parkinson's Disease Samples Using the EDPGEDPGED-α-Syn-His variant of this invention, commercially available wild-type human α-synuclein, the C-terminal fusion variant of Comparative Example 1, and two N-terminal fusion variants of Comparative Example 2 as substrates, cerebrospinal fluid samples (n=46) from patients clinically diagnosed with Parkinson's disease (PD) were mixed and α-syn-RT-QuIC detection was performed (detection method is the same as in Example 3). The positive detection rate was calculated, and the results are shown in Table 3.

[0064] Table 3. Detection sensitivity of each substrate for Parkinson's disease samples (n=46)

[0065] As shown in Table 3, the recognition and amplification capabilities of the EDPGEDPGED-α-Syn-His variant (93.5%) and the comparative variants of this invention for positive seeds were not significantly different from those of the wild-type monomer (91.3%) (p>0.05), further verifying that the N-terminal fusion modification did not affect its sensitivity to pathological aggregates.

[0066] Based on the verification results of the above comparative examples and experimental cases, the recombinant α-synuclein variant (EDPGEDPGED-α-Syn-His) with N-terminal fusion of the EDPGEDPGED polypeptide constructed in this invention, while fully retaining seed recognition and amplification capabilities comparable to the wild-type substrate (same detection limit, no significant difference in positive rate of clinical PD samples), significantly inhibits spontaneous aggregation background through a dual synergistic mechanism of N-terminal specific fusion and charge repulsion-conformation restriction (in 112 cerebrospinal fluid samples of non-α-synucleinopathy, the negative specificity of the variant substrate reached 95.5%, far superior to the 79.5% of the wild-type substrate). Meanwhile, Comparative Examples 1 and 2 also demonstrate that other sequences with C-terminal or N-terminal fusion (providing only charge repulsion / only conformation restriction) cannot achieve this effect. Therefore, the variant of this invention effectively overcomes the defects of wild-type α-synuclein in α-syn-RT-QuIC detection, which leads to false positives and decreased specificity due to self-aggregation, and provides a higher quality substrate for the ultrasensitive and highly specific diagnosis of synucleinosis.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A recombinant α-synuclein with low self-aggregation tendency, characterized in that: The amino acid sequence of the recombinant α-synuclein is shown in SEQ ID NO:

2.

2. A nucleic acid encoding the recombinant α-synuclein of claim 1, characterized in that: The sequence of the nucleic acid is shown in SEQ ID NO:

3.

3. The application of the recombinant α-synuclein as described in claim 1 in the preparation of a Parkinson's disease α-syn-RT-QuIC detection system, characterized in that: The recombinant α-synuclein was used as a substrate in the α-syn-RT-QuIC detection reaction.

4. The application according to claim 3, characterized in that: The α-syn-RT-QuIC detection system includes recombinant α-synuclein, reaction buffer, thioflavin T, and the sample to be tested.

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