An anti-alpha synuclein genetically engineered ferritin and a preparation method and application thereof

Abstract

The application discloses genetically engineered ferritin against alpha synuclein, a preparation method and application thereof, and belongs to the technical field of biological medicine and protein engineering. The genetically engineered ferritin is constructed by fusing and expressing NACore polypeptide and human heavy chain ferritin through gene recombination technology, and has alpha synuclein aggregation inhibition activity. The genetically engineered ferritin can specifically combine with the NAC region of alpha synuclein, significantly inhibits the misfolding and aggregation of alpha synuclein, and provides a new drug raw material and treatment strategy for the treatment of Parkinson's disease and other alpha synuclein related neurodegenerative diseases. The genetically engineered ferritin prepared by the application has uniform particle size distribution and stable physicochemical properties. Molecular simulation verifies that the genetically engineered ferritin can efficiently combine with the NAC region of alpha synuclein. ThT fluorescence detection and CCK-8 experiments prove that the genetically engineered ferritin can inhibit the aggregation and fibrosis of alpha synuclein, and lays a solid foundation for the clinical treatment of related diseases.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and protein engineering technology, and in particular to a genetically engineered ferritin that resists α-synuclein, its preparation method, and its applications. Background Technology

[0002] α-synuclein (α-Syn or αS) is a soluble protein primarily expressed on the presynaptic membrane of the central nervous system. Its misfolding and abnormal aggregation are core pathological features of neurodegenerative diseases such as Parkinson's disease (PD). The N-terminal non-amyloid-β component (NAC) region (amino acid sequence 61-95) of α-synuclein is a key region for its misfolding and aggregation. Exposure of hydrophobic fragments in this region can induce α-synuclein to form β-sheet-rich fibrillary aggregates. These aggregates deposit within neurons to form Lewy bodies and Lewy neurites, ultimately leading to neuronal damage and death.

[0003] Currently, treatments for α-synuclein-related neurodegenerative diseases primarily focus on symptom relief, lacking specific drugs that can fundamentally inhibit α-synuclein aggregation and slow disease progression. In recent years, the development of inhibitors targeting the NAC region of α-synuclein has become a research hotspot. By specifically binding to the NAC region, these inhibitors can block the misfolding and aggregation of α-synuclein, thereby reducing its neurotoxicity.

[0004] Ferritin is a cage-like nanoprotein formed by the self-assembly of 24 subunits. It possesses excellent biocompatibility, biodegradability, and structural stability, and is easily genetically engineered, making it an ideal drug carrier and platform for displaying bioactive molecules. Human heavy chain ferritin (FTH1), as an important subtype of ferritin, has unique nanostructures and physicochemical properties. Specific functions can be achieved by fusing exogenous peptides or proteins at its N-terminus or C-terminus using gene recombination technology.

[0005] In the current technology, there are no reports on the fusion of NACore peptides with ferritin to construct genetically engineered ferritin with anti-α-synuclein aggregation activity. Therefore, developing a genetically engineered ferritin that can specifically bind to the NAC region of α-synuclein and efficiently inhibit its aggregation is of great significance for the treatment of α-synuclein-related neurodegenerative diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a genetically engineered ferritin that inhibits α-synuclein, its preparation method, and its applications, thereby addressing the problems existing in the prior art. This invention constructs a protein nanomaterial capable of specifically binding to the α-Syn NAC region and effectively inhibiting its abnormal aggregation by fusing the NACore peptide to the ferritin subunit, thus solving the problems of insufficient targeting and poor stability of existing α-Syn aggregation inhibition methods.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a genetically engineered ferritin that resists α-synuclein, wherein the genetically engineered ferritin consists of a His6 tag, a NACore polypeptide, a flexible linker, and human heavy chain ferritin FTH1 from the N-terminus to the C-terminus. The amino acid sequence of the His6 tag is shown in SEQ ID NO.3; the amino acid sequence of the NACore polypeptide is shown in SEQ ID NO.1; the amino acid sequence of the flexible linker is shown in SEQ ID NO.2; and the accession number of the human heavy chain ferritin FTH1 is NM_002032.4.

[0008] The NACore polypeptide of this invention has the amino acid sequence GAVVTGVTAVA (SEQ ID NO.1) and is capped with thiol groups. This polypeptide can specifically recognize and bind to the NAC region of α-synuclein, thereby blocking the aggregation of α-synuclein. The flexible linker has the amino acid sequence Gly4Ser, which connects the NACore polypeptide and human heavy chain ferritin FTH1, avoiding the influence of steric hindrance on their respective structures and functions. The nucleotide sequence of human heavy chain ferritin FTH1 is obtained from GenBank No. NM_002032.4. Its amino acid sequence is conserved and can self-assemble into a stable cage-like nanostructure. The His6 tag added to the N-terminus facilitates the subsequent purification of the target protein by Ni-NTA affinity chromatography.

[0009] Optionally, the amino acid sequence of the genetically engineered ferritin is shown in SEQ ID NO.9.

[0010] This invention utilizes gene recombination technology to fuse NACore polypeptide with human heavy chain ferritin for expression, constructing a genetically engineered ferritin with α-synuclein aggregation inhibitory activity. This genetically engineered ferritin can specifically bind to the NAC region of α-synuclein, significantly inhibiting the misfolding and aggregation of α-synuclein, providing a new drug raw material and treatment strategy for the treatment of α-synuclein-related neurodegenerative diseases such as Parkinson's disease.

[0011] This invention provides a method for preparing the above-mentioned genetically engineered ferritin, comprising the following steps: A recombinant vector containing the His6 tag encoding gene, the NACore polypeptide encoding gene, the flexible linker encoding gene, and the human heavy chain ferritin FTH1 encoding gene was constructed, followed by prokaryotic expression and protein purification. The nucleotide sequence of the gene encoding the His6 tag is shown in SEQ ID NO.6; the nucleotide sequence of the gene encoding the NACore polypeptide is shown in SEQ ID NO.4; the nucleotide sequence of the gene encoding the flexible linker is shown in SEQ ID NO.7; and the nucleotide sequence of the gene encoding human heavy chain ferritin FTH1 is shown in SEQ ID NO.5.

[0012] More preferably, the recombinant vector is constructed before the design and synthesis of the target gene for fusion; and the protein is purified before the protein is validated.

[0013] A further preferred embodiment is the method for preparing the above-mentioned genetically engineered ferritin against α-synuclein, which includes five steps: design and synthesis of the fusion target gene, construction of the recombinant vector, prokaryotic expression, protein purification, and protein verification, as detailed below: (1) Design and synthesis of the fusion target gene: Based on codon preference, the amino acid sequences of NACore polypeptide (GAVVTGVTAVA, thiol-capped), flexible linker (Gly4Ser) and human heavy chain ferritin FTH1 were codon optimized to design the fusion target gene sequence. A His6 tag coding sequence was added to the N-terminus. NdeⅠ and EcoRⅠ restriction sites were introduced at both ends of the synthesized fusion target gene to facilitate subsequent vector construction. (2) Construction of recombinant vector: The synthesized fusion target gene and pET-30a(+) vector (kanamycin resistance, T7 promoter) were double-digested with NdeI and EcoRI restriction endonucleases, respectively, and digested in a water bath at 37℃ for 4 h. After separation of the digestion products by 1% agarose gel electrophoresis, the target gene fragment and the vector fragment were recovered using a gel recovery kit. The recovered target gene fragment and the vector fragment were mixed at a molar ratio of 3:1, and T4 DNA ligase was added. The ligation was carried out overnight at 16℃. The ligation product was transformed into Escherichia coli DH5α competent cells and plated on LB solid medium containing 50 μg / mL kanamycin. The cells were incubated upside down at 37℃ overnight. Single colonies were picked and inoculated into LB liquid medium containing kanamycin. The cells were cultured at 37℃ and 220 rpm for 12 h with shaking. The plasmid was extracted for enzyme digestion verification and sequencing verification. The recombinant plasmid that was verified to be correct was pET-30a-NACore-FTH1. (3) Prokaryotic expression: The verified recombinant plasmid pET-30a-NACore-FTH1 was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant engineered bacteria; the expression conditions were optimized using orthogonal experiments, with three factors: IPTG concentration (0.1 mM, 0.3 mM, 0.5 mM, 0.7 mM, 1 mM), induction temperature (16℃, 25℃, 37℃), and induction time (4 h, 8 h, 12 h, 16 h, 24 h), each factor set with 5 levels, and the soluble expression level of the target protein was used as the evaluation index to screen the optimal expression conditions; the recombinant engineered bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 220 rpm with shaking until OD. 600 The value is 0.6-0.8. Add IPTG to the optimal final concentration, and culture with shaking at the optimal induction temperature for the optimal time to induce target protein expression. (4) Protein purification: Collect the engineered bacterial cells after induction and centrifuge at 8000 rpm for 10 min, discard the supernatant, and resuspend the bacterial cells in PBS buffer (pH 7.4); place the resuspended solution in an ice bath and sonicate the bacterial cells (300 W power, 30 min total time); after sonication, centrifuge at 12000 rpm and 4℃ for 30 min and collect the supernatant; load the supernatant onto a Ni-NTA affinity chromatography column pre-equilibrated with PBS buffer, wash off impurities with PBS buffer containing 20 mM imidazole, and then elute the target protein with PBS buffer containing 200-300 mM imidazole, and collect the elution peak; load the crude target protein obtained by elution onto a Superdex 200 gel filtration chromatography column, use PBS buffer (pH 7.4) as the elution buffer, flow rate 0.5 mL / min, and collect the main peak, which is the high-purity genetically engineered ferritin; (5) Protein verification: The molecular weight of the target protein was verified by SDS-PAGE electrophoresis. The electrophoresis conditions were: constant voltage 150 V electrophoresis for 50 min, Coomassie brilliant blue R-250 staining for 2 h, and destaining solution until the bands were clear.

[0014] The present invention provides a nucleic acid molecule encoding the above-mentioned genetically engineered ferritin, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0015] The present invention provides a carrier comprising the above-described nucleic acid molecules.

[0016] The present invention provides a recombinant cell comprising at least one of the above-described genetically engineered ferritin, the above-described nucleic acid molecule, and the above-described vector.

[0017] The present invention provides a pharmaceutical composition comprising at least one of the above-described genetically engineered ferritin, the above-described nucleic acid molecule, the above-described vector, and the above-described recombinant cells.

[0018] The present invention provides the use of the above-described genetically engineered ferritin, the above-described nucleic acid molecule, the above-described vector, the above-described recombinant cell, or the above-described pharmaceutical composition in any of the following: (1) Preparation of drugs that inhibit the aggregation of α-synuclein; (2) To prepare drugs for treating α-synuclein-related neurodegenerative diseases; (3) Prepare reagents for detecting α-synuclein.

[0019] Optionally, the α-synuclein-related neurodegenerative diseases include Parkinson's disease.

[0020] The present invention provides a medicine comprising at least one of the above-described genetically engineered ferritin, the above-described nucleic acid molecule, the above-described carrier, the above-described recombinant cell, and the above-described pharmaceutical composition.

[0021] The present invention discloses the following technical effects: This invention, for the first time, fuses NACore peptide with human heavy chain ferritin via a flexible linker to construct a genetically engineered ferritin with α-synuclein aggregation inhibitory activity. The NACore peptide specifically binds to the NAC region of α-synuclein, blocking the misfolding and aggregation of α-synuclein at its source. The cage-like nanostructure of the ferritin not only provides a stable display platform for the NACore peptide but also enhances its biocompatibility and in vivo stability. This solves the problems of poor specificity, low stability, and low bioavailability of existing α-synuclein inhibitors, providing a new drug raw material and treatment strategy for the treatment of α-synuclein-related neurodegenerative diseases such as Parkinson's disease. Furthermore, the results of specific embodiments of this invention show that the genetically engineered ferritin prepared in this invention has a uniform particle size distribution and stable physicochemical properties. Molecular simulations verify its efficient binding to the NAC region of α-synuclein, and ThT fluorescence detection and CCK-8 experiments confirm its ability to inhibit α-synuclein aggregation and fibrosis, laying a solid foundation for the clinical treatment of related diseases. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This document describes the construction and sequence validation process of the recombinant vector pET-30a-NACore-FTH1. Section A outlines the overall construction strategy and experimental procedure, where the target gene was designed, synthesized, and cloned into an expression vector. Section B illustrates the sequencing principle and procedure, with the amplified products validated using Sanger sequencing. Figure 2 This is a schematic diagram of a genetically engineered ferritin that inhibits α-synuclein. A shows the mechanism by which this genetically engineered ferritin inhibits α-synuclein, primarily by binding NACore to the NAC region of synuclein, thereby inhibiting its aggregation. B shows the possible binding site of the simulated NACore polypeptide to α-synuclein. C is a plasmid image of the expression vector encoding NACore-linker-FTH1—the recombinant vector pET-30a-NACore-FTH1. D is a restriction endonuclease map of the recombinant vector pET-30a-NACore-FTH1. Figure 3 The diagram shows the genetically engineered ferritin gene and protein sequence. A and B are Sanger sequencing chromatograms, confirming the successful construction of the designed NACore-HFn sequence. B is a magnified view showing key ferritin nucleotide sites / regions. C is a sequence alignment of NF (NACore-linker-FTH1) and F (FTH1), showing common protein fragments and alignment regions (labeled in the figure). D is a comparison of the N-terminal sequence of N (NACore polypeptide) and NF, highlighting the shared structural motif (GAVVTGVTAVA) (labeled in the figure). Figure 4 The physicochemical properties of NACore-linker-FTH1 are characterized. Among them, A is the absorbance spectrum of NACore-linker-FTH1, which shows that NACore-linker-FTH1 has no obvious characteristic peaks except around 250 nm; B is the SDS-PAGE result, which shows that NACore-linker-FTH1 has a protein band with a molecular weight of about 22 kDa. Figure 5 To analyze the aggregation of α-synaptic protein (αS) in PBS using ThT fluorescence kinetics, including the ThT control, the presence of αS alone, and the co-incubation of αS with NF at specified ratios (1:1, 1:2, 1:5, and 1:10), the fluorescence intensity was monitored over time. Figure 6 This study validates the use of genetically engineered ferritin at the cellular level. A and B represent the assessment of ferritin uptake in SH-SY5Y cells, using flow cytometry to detect intracellular fluorescence signals. C presents the results of the CCK-8 assay. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] 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. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Example 1: Preparation of genetically engineered ferritin against α-synuclein 1. Design and synthesis of the target gene for fusion Based on the amino acid sequences of NACore polypeptide (GAVVTGVTAVA-SH, thiol-capped, SEQ ID NO.1), flexible linker (Gly4Ser, amino acid sequence GGGGS, SEQ ID NO.2), and human heavy chain ferritin FTH1 (GenBank: NM_002032.4), codon optimization was performed using E. coli codon preferences to design the fusion target gene sequence. A His6 tag (amino acid sequence HHHHHH, SEQ ID NO.3) was added to the N-terminus, and NdeⅠ and EcoRI restriction sites were introduced at both ends, respectively. The gene sequences involved are shown in Table 1.

[0030] Table 1 Sequence Information 2. Construction of the recombinant vector pET-30a-NACore-FTH1 The construction and sequence verification process of the recombinant vector pET-30a-NACore-FTH1 is as follows: Figure 1 As shown below: The synthesized fusion target gene NACore-linker-FTH1 (containing a His6 tag, NACore polypeptide, flexible linker, and FTH1 coding sequence, with NdeⅠ / EcoRⅠ restriction sites at both ends) and the pET-30a(+) vector were double-digested. The specific digestion system (50 μL) consisted of: 5 μL 10× restriction endonuclease buffer, 1 μL NdeⅠ (10 U / μL), 1 μL EcoRⅠ (10 U / μL), 10 μg target gene / vector, and sterile deionized water to a final volume of 50 μL. The digestion system was incubated at 37℃ for 4 h. After incubation, 10 μL of the digestion product was subjected to 1% agarose gel electrophoresis for verification. The electrophoresis conditions were 120V constant voltage electrophoresis for 30 min. The digestion effect was observed using a gel imaging system to confirm successful digestion of both the target gene fragment and the vector fragment, which were then recovered.

[0031] Construction of the ligation system (20 μL): 50 ng of the recovered pET-30a(+) vector fragment, 150 ng of the recovered fusion target gene fragment (vector to target gene molar ratio 1:3), 2 μL of 10×T4 DNA ligase buffer, 1 μL of T4 DNA ligase (5 U / μL), and sterile deionized water to a final volume of 20 μL. The ligation system was placed in a 16°C metal bath and ligated overnight (12 h) to obtain the recombinant vector ligation product.

[0032] The ligation product was transformed into *E. coli* DH5α competent cells: 10 μL of the ligation product was added to 100 μL of *E. coli* DH5α competent cells and incubated on ice for 30 min; then, the cells were heat-shocked in a 42°C water bath for 90 s, and quickly transferred to an ice bath to cool for 2 min; subsequently, 800 μL of antibiotic-free LB liquid medium was added, and the cells were cultured at 37°C with shaking at 220 rpm for 1 h to restore the activity of *E. coli* DH5α competent cells and express the resistance gene. 200 μL of the cultured bacterial solution was evenly spread on LB solid medium (containing 1.5% agar) containing 50 μg / mL kanamycin and incubated upside down at 37°C overnight (16 h) to obtain single colonies.

[0033] Three morphologically regular single colonies were selected and inoculated into LB liquid medium containing 50 μg / mL kanamycin. The cultures were incubated at 37°C with shaking at 220 rpm for 12 h. Recombinant plasmids were extracted using a plasmid mini-prep kit. The extracted recombinant plasmids were verified by double enzyme digestion and sequencing. The double enzyme digestion results showed that the recombinant plasmid could yield approximately 627 bp of the target gene fragment and 6000 bp of the vector fragment, consistent with expectations, indicating that the recombinant vector pET-30a-NACore-FTH1 was successfully constructed.

[0034] 3. Prokaryotic expression and condition optimization The validated recombinant vector pET-30a-NACore-FTH1 was transformed into *E. coli* BL21(DE3) competent cells, following the same transformation procedure as described above for transforming DH5α competent cells with the recombinant vector, to obtain recombinant engineered bacteria. Positive single colonies were picked and inoculated into LB broth containing 50 μg / mL kanamycin, and cultured at 37°C with shaking at 220 rpm for 12 h to obtain a seed culture. The seed culture was then inoculated at a 1:100 ratio into fresh LB broth containing 50 μg / mL kanamycin, and cultured at 37°C with shaking at 220 rpm until OD (dose elapsed). 600 When the value is 0.6-0.8, the expression condition optimization stage begins.

[0035] Using L 16 (4) 3An orthogonal experimental design was used to optimize expression conditions. Three influencing factors were selected: IPTG concentration (Factor A: 0.1 mM, 0.3 mM, 0.5 mM, 1 mM), induction temperature (Factor B: 16℃, 25℃, 37℃), and induction time (Factor C: 4 h, 8 h, 12 h, 24 h). Each factor had four levels, and the soluble expression level of the target protein (engineered ferritin) was used as the evaluation index. A total of 16 experimental groups were set up in the orthogonal experiment, with three replicates in each group. Subsequent large-scale expression was conducted under these optimal conditions: the recombinant engineered bacterial seed culture was inoculated into a 5L fermenter (containing 2L LB liquid medium and 50 μg / mL kanamycin) at a volume ratio of 1:100, and cultured at 37℃ with shaking at 220 rpm until OD... 600 The value was 0.7, and IPTG was added to a final concentration of 0.5 mM for induction culture. The induction culture conditions were: 25℃, 200 rpm shaking culture for 12 h to induce the expression of the target protein.

[0036] 4. Protein purification (1) Collection and disruption of bacterial cells: After the induction of expression, the fermentation broth was placed in a centrifuge bottle and centrifuged at 8000 rpm and 4℃ for 10 min. The supernatant was discarded and the bacterial cell pellet was collected. The bacterial cell pellet was resuspended in pre-cooled PBS buffer (pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4) (10 mL of PBS buffer was added per gram of bacterial cells) and placed in an ice bath. The bacterial cells were disrupted using an ultrasonic disruptor with the following parameters: power 300 W and total disruption time 30 min, to ensure complete disruption of the bacterial cells. After disruption, the bacterial solution was placed in a centrifuge tube and centrifuged at 12000 rpm and 4℃ for 30 min. The supernatant (containing soluble target protein) was collected and the pellet (containing insoluble protein and bacterial cell fragments) was discarded.

[0037] (2) Ni-NTA affinity chromatography purification: The Ni-NTA affinity chromatography column was fixed on the chromatography rack, and the column was equilibrated with 10 column volumes of PBS buffer at a flow rate of 0.5 mL / min. The collected supernatant was slowly loaded into the chromatography column at a flow rate of 0.3 mL / min to ensure that the target protein (containing the His6 tag) in the supernatant was compatible with Ni. 2+After loading the sample, wash the column with 10 column volumes of washing buffer (PBS buffer + 20 mM imidazole, pH 7.4) at a flow rate of 0.5 mL / min to remove unbound contaminants. Then, perform gradient elution with elution buffer (PBS buffer + 200-300 mM imidazole, pH 7.4), gradually increasing the imidazole concentration by 5 mM / tube (200 mM → 205 mM → 210 mM → … → 300 mM), for a total elution volume of 20 column volumes (20 mL). Collect 10 tubes (2 mL per tube). Then, 5 column volumes (5 mL) of PBS buffer containing 300 mM imidazole were eluted, and the remaining eluent (2-3 tubes) was collected until the A280 absorbance returned to baseline (the target genetically engineered ferritin was completely eluted). The protein concentration of each elution tube was detected using a BCA protein quantification kit, and SDS-PAGE electrophoresis was performed to verify the concentration. Elution tubes with high target protein content and high purity were collected, combined, and placed in a dialysis bag. The tubes were dialyzed overnight at 4°C with PBS buffer to remove imidazole, yielding the genetically engineered ferritin against α-synuclein.

[0038] 5. Protein Validation (1) SDS-PAGE electrophoresis verification: Prepare stacking gel and separating gel, mix the sample with loading buffer at a volume ratio of 5:1, heat at 95℃ for 5 min to denature the protein; load the denatured sample into the gel wells, and simultaneously load the protein marker; electrophoresis conditions: 150V constant voltage electrophoresis for 50 min; after electrophoresis, place the gel in Coomassie Brilliant Blue R-250 staining solution and shake at room temperature for 2 h; then transfer the gel to destaining solution (methanol:acetic acid:water = 4:1:5, volume ratio), shake at room temperature until the protein bands are clear, take pictures and analyze using a gel imaging system, the results are as follows. Figure 4 As shown in B in the figure. The results showed that the purified sample showed a single protein band at approximately 22 kDa, consistent with the theoretical molecular weight (22 kDa) of the genetically engineered ferritin monomer, and no obvious contaminating protein bands.

[0039] 6. The principle, binding sites, and map of genetically engineered ferritin that inhibits α-synuclein. The principle behind genetically engineered ferritin that inhibits α-synuclein is as follows: Figure 2 As shown in A, this engineered ferritin that inhibits α-synuclein mainly relies on NACore binding to the NAC region of synuclein to suppress its aggregation; the possible binding sites of the NACore polypeptide simulated by AlphaFold3 with α-synuclein are as follows. Figure 2 As shown in B; the plasmid diagram and restriction endonuclease map of the recombinant vector pET-30a-NACore-FTH1 are shown in Figure B. Figure 2 As shown in C and D.

[0040] The amino acid sequence of genetically engineered ferritin that resists α-synuclein and the nucleotide sequence of its encoding gene are as follows: Figure 3 As shown. The amino acid sequence of this engineered ferritin against α-synuclein, from N-terminus to C-terminus, is as follows: His6 tag, NACore polypeptide, flexible linker, and human heavy chain ferritin FTH1.

[0041] Example 2: Verification of the structure and function of genetically engineered ferritin 1. Absorption spectroscopy detection of genetically engineered ferritin The purified anti-α-synuclein genetically engineered ferritin from Example 1 was diluted to 0.1 mg / mL with PBS buffer. Using PBS buffer as a blank control, the sample was scanned in the 200-600 nm wavelength range using a UV-Vis spectrophotometer with a scan interval of 10 nm. The results are as follows. Figure 4 As shown in A in the figure. The results show that the genetically engineered ferritin against α-synuclein folds correctly, shows no obvious degradation, and has stable physicochemical properties; at the same time, it can be seen that the genetically engineered ferritin against α-synuclein has no obvious characteristic peak except around 250 nm.

[0042] 2. Detection of particle size and potential of genetically engineered ferritin The purified anti-α-synuclein genetically engineered ferritin from Example 1 was diluted to 0.2 mg / mL with PBS buffer and placed in a sample cell. Particle size distribution and Zeta potential were determined using dynamic light scattering (DLS) three times. The results showed that the hydrated particle size of the genetically engineered ferritin was 22.3 ± 0.4 nm, and the particle size distribution index (PDI) was 0.18 ± 0.02, indicating uniform protein particle dispersion without significant aggregation. The Zeta potential was -10.2 ± 0.5 mV, indicating good stability of the protein in aqueous solution and resistance to precipitation.

[0043] 3. ThT fluorescence detection to inhibit αS aggregation (1) Experimental grouping: A control group and an experimental group were set up, with 3 parallel samples in each group. Control group: αS monomer (final concentration 10 μM) + PBS buffer (denoted as αS), PBS (PBS buffer (denoted as PBS)), ThT (thioflavin T, ThT (denoted as ThT)); Experimental group: αS monomer (final concentration 20 μM) + genetically engineered ferritin against α-synuclein prepared in Example 1 (final concentrations 1:1, 1:2, 1:5, 1:10, denoted as NF1:1, NF1:2, NF1:5 and NF1:10 respectively), each with a volume of 100 μL, placed in a 96-well fluorescent plate.

[0044] (2) Incubation and detection: Add 100 μL of 40 μM THT to each well and incubate at 37°C for 48 h. Detect the fluorescence intensity every 2 h using a multi-functional microplate reader.

[0045] (3) Results analysis: The results are as follows Figure 5 As shown in the figure. The results showed that αS monomers in the αS group began to aggregate significantly after 2 hours of incubation, and the ThT fluorescence intensity gradually increased, reaching its maximum value after 48 hours of incubation. In the experimental group, the increase in ThT fluorescence intensity decreased significantly with the increase in the concentration of genetically engineered ferritin against α-synuclein, showing a concentration-dependent inhibition of αS aggregation.

[0046] Example 3: Cellular Experimental Validation of Genetically Engineered Ferritin 1. Flow cytometry confirms that ferritin can be phagocytosed by cells. Remove SH-SY5Y cells cultured to the logarithmic growth phase, discard the old culture medium, and wash the cells twice with PBS buffer. Add 2 mL of 0.25% trypsin solution and incubate at 37°C for 3 min to digest the cells. When the cells become rounded and detach, add 4 mL of complete culture medium (prepared with DMEM high-glucose medium, 10% serum, and 1% penicillin antibody; DMEM high-glucose medium manufacturer: Solarbio, catalog number: No. 11995; serum manufacturer: Pronos, catalog number: 164210-50; penicillin antibody manufacturer: Pronos, catalog number: PB180120) to stop digestion. Gently pipette the cell suspension to disperse the cells evenly. Take a small amount of the cell suspension and count the cells using a hemocytometer to adjust the cell concentration to 5 × 10⁶ cells / mL. 4 Cells / mL; Add 500 μL of adjusted cell suspension (5 × 10⁶ cells / mL) to each well of a 6-well cell culture plate. 3 Add 500 μL of PBS buffer to each well (to avoid edge effects); place the 6-well plate in a 37°C, 5% CO2 cell culture incubator and incubate for 24 h to allow the cells to adhere and grow.

[0047] The engineered ferritin against α-synuclein prepared in Example 1 was coupled with CY5.5-NHS ester stock solution (manufacturer: DuoFluor, catalog number: D10013) to prepare an engineered ferritin-labeled solution against α-synuclein. The specific method is as follows: Coupling reaction: Add 200 μL of genetically engineered ferritin against α-synuclein and 295.85 μL of PBS to a 1.5 mL light-protected centrifuge tube and gently pipette to mix. Quickly add 4.15 μL of 1 mM CY5.5-NHS ester stock solution and vortex at low speed for 10 s (≤1000 rpm) to avoid destroying the cage-like structure of ferritin. Shake slowly on a horizontal shaker at room temperature for 1 h (60 rpm) to ensure sufficient coupling and no protein precipitation.

[0048] To terminate the reaction: Add 1M Tris-HCl (pH 8.0) to a final concentration of 50mM (approximately 27.8μL), gently invert to mix, and incubate at room temperature in the dark for 15 minutes to block unreacted NHS ester groups (the total volume of the reaction solution becomes 527.8μL).

[0049] Dialysis purification: Transfer 527.8 μL of the terminated reaction solution to a pretreated dialysis bag, clamp both ends with dialysis clamps to ensure no leakage. Take a 500 mL beaker, add 400 mL of pre-cooled pH 7.4 PBS buffer (dialysis external solution), and place a magnetic stir bar in it; completely immerse the dialysis bag containing the reaction solution in PBS, wrap the beaker with aluminum foil to protect it from light, and place it on a magnetic stirrer at 4℃. Change the solution every 4 hours and dialyze for 12 hours with stirring.

[0050] Group setup, 3 replicates per group, operation must be conducted in the dark throughout: Blank control group (hereinafter referred to as Control): Only SH-SY5Y cells were added, without any drug treatment; Negative control group (denoted as Blank): SH-SY5Y cells were added + an equal volume of serum-free complete culture medium (prepared by using DMEM high-glucose medium, 10% serum, and 1% penicillin antibiotics; DMEM high-glucose medium manufacturer: Solarbio, catalog number: No. 11995; serum manufacturer: Pronos, catalog number: 164210-50; penicillin antibiotic manufacturer: Pronos, catalog number: PB180120) + 1 mM CY5.5-NHS ester stock solution; Experimental group (denoted as NF): SH-SY5 cells were added with a genetically engineered ferritin-labeled solution that was anti-α-synuclein.

[0051] After incubation for 4 hours, single-cell suspensions were prepared according to requirements and analyzed by flow cytometry.

[0052] Combining quantitative and qualitative analysis data from flow cytometry ( Figure 6In the studies (A and B in the original text), SH-SY5Y cells showed significantly higher uptake efficiency of genetically engineered ferritin labeled with CY5.5 fluorescent dye against α-synuclein than that of free CY5.5 dye. Quantitative fluorescence intensity results showed that the fluorescence signal in the experimental group was more than an order of magnitude higher than that in the negative control group, with a statistically significant difference. The cell population distribution in the scatter plot also clearly showed that the experimental group cells generally shifted towards the high-fluorescence region, while only a small number of cells in the negative control group showed weak fluorescence signals. This phenomenon indicates that, compared to free dyes which can only be taken up by cells through inefficient methods such as passive diffusion, genetically engineered ferritin against α-synuclein can efficiently enter cells due to its unique structural features and cell interaction mechanisms (such as receptor-mediated endocytosis). This result not only directly verifies the excellent cell internalization ability of genetically engineered ferritin against α-synuclein but also reveals its outstanding advantages as a delivery carrier in enhancing transmembrane transport. From the perspective of application value in subsequent research, this highly efficient cell internalization characteristic has multiple key implications.

[0053] 2. CCK-8 assays confirm that genetically engineered ferritin can inhibit αS aggregation. Remove SH-SY5Y cells cultured to the logarithmic growth phase, discard the old culture medium, and wash the cells twice with PBS buffer. Add 2 mL of 0.25% trypsin solution and incubate at 37°C for 3 min to digest the cells. When the cells become rounded and detach, add 4 mL of complete culture medium to stop the digestion. Gently pipette the cell suspension to disperse the cells evenly. Take a small amount of the cell suspension and count the cells using a hemocytometer to adjust the cell concentration to 5 × 10⁶ cells / mL. 4 Cells / mL; In a 6-well cell culture plate, add 100 μL of cell suspension of adjusted concentration (5 × 10³ cells per well) to each well, and add 100 μL of PBS buffer to the edge wells (to avoid edge effect); Place the 24-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 24 h to allow the cells to adhere and grow.

[0054] Except for the blank group, 4 μL of 1M αS (final concentration 20 μM) and 196 μL of genetically engineered ferritin with different concentrations of anti-α synuclein (final concentration 2, 4, 8, 16, 32 or 64 μg / mL) were added to each well. After incubation for 10 h, working solution was added according to the CCK-8 instructions. At the same time, the treatment with only 200 μL of 20 μM αS was used as a control. After incubation at 37 °C for 30 min, the results were detected at 450 nm using a microplate reader.

[0055] The results are as follows Figure 6As shown in C. The results showed that without αS, the cell survival rate was around 30%. After adding the genetically engineered ferritin that inhibits α-synuclein, the cell survival rate reached around 80%, and the cell viability was significantly improved, further demonstrating that the genetically engineered ferritin can inhibit αS aggregation.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A genetically engineered ferritin that resists α-synuclein, characterized in that, The genetically engineered ferritin consists of a His6 tag, NACore polypeptide, flexible linker, and human heavy chain ferritin FTH1, from N-terminus to C-terminus. The amino acid sequence of the His6 tag is shown in SEQ ID NO.3; the amino acid sequence of the NACore polypeptide is shown in SEQ ID NO.1; the amino acid sequence of the flexible linker is shown in SEQ ID NO.2; and the accession number of the human heavy chain ferritin FTH1 is NM_002032.

4.

2. The genetically engineered ferritin according to claim 1, characterized in that, The amino acid sequence of the genetically engineered ferritin is shown in SEQ ID NO.

9.

3. The method for preparing the genetically engineered ferritin according to claim 1, characterized in that, Includes the following steps: A recombinant vector containing the His6 tag encoding gene, the NACore polypeptide encoding gene, the flexible linker encoding gene, and the human heavy chain ferritin FTH1 encoding gene was constructed, followed by prokaryotic expression and protein purification. The nucleotide sequence of the gene encoding the His6 tag is shown in SEQ ID NO.6; the nucleotide sequence of the gene encoding the NACore polypeptide is shown in SEQ ID NO.4; the nucleotide sequence of the gene encoding the flexible linker is shown in SEQ ID NO.7; and the nucleotide sequence of the gene encoding human heavy chain ferritin FTH1 is shown in SEQ ID NO.

5.

4. A nucleic acid molecule encoding the genetically engineered ferritin of claim 1 or 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

8.

5. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 4.

6. A recombinant cell, characterized in that, The recombinant cells comprise at least one of the genetically engineered ferritin of claim 1 or 2, the nucleic acid molecule of claim 4, and the vector of claim 5.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the following: the genetically engineered ferritin of claim 1 or 2, the nucleic acid molecule of claim 4, the vector of claim 5, and the recombinant cell of claim 6.

8. The use of the genetically engineered ferritin of claim 1 or 2, the nucleic acid molecule of claim 4, the vector of claim 5, the recombinant cell of claim 6, or the pharmaceutical composition of claim 7 in any of the following: (1) Preparation of drugs that inhibit the aggregation of α-synuclein; (2) To prepare drugs for treating α-synuclein-related neurodegenerative diseases; (3) Prepare reagents for detecting α-synuclein.

9. The application according to claim 8, characterized in that, The α-synuclein-related neurodegenerative diseases include Parkinson's disease.

10. A medicine, characterized in that, The drug comprises at least one of the following: the genetically engineered ferritin of claim 1 or 2, the nucleic acid molecule of claim 4, the vector of claim 5, the recombinant cell of claim 6, and the pharmaceutical composition of claim 7.

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