A polypeptide that reverses pathological aggregation of human crystallin gamma c-g129c

Abstract

The application discloses a polypeptide for reversing pathological aggregation of human crystallin gamma C-G129C. The application screens an optimal polypeptide for targeting human crystallin gamma C-G129C through a self-made polypeptide chip microarray. The polypeptide screened by the application can significantly reverse the aggregation of human crystallin gamma C-G129C in vivo and in vitro, and indicates that the polypeptide provided by the application has a potential effect of treating cataract and has a wide application prospect in the clinic.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a polypeptide that reverses the pathological aggregation of human lens protein γC-G129C. Background Technology

[0002] Pathological aggregation of lens proteins is closely related to various eye diseases, including but not limited to age-related cataracts, congenital cataracts, secondary cataracts, and traumatic cataracts. Cataract treatment typically involves surgical removal of the cloudy lens and implantation of an artificial lens. Targeted therapies such as hormones, antioxidants, and lanosterol are used, but their effectiveness is limited and can only slow cataract progression; currently, there are no effective targeted therapies. Since abnormal aggregation of lens proteins is one of the key mechanisms of cataract formation, developing drugs targeting pathological aggregation of lens proteins is of great significance. The γCG129C mutation in lens protein is a point mutation closely related to cataracts; this mutation exacerbates protein aggregation, leading to cataracts. The γCG129C mutation also has a familial genetic component; children carrying the mutated gene may develop lens opacity within months of birth, and severe cases require early surgical intervention. However, surgical methods are not readily available for young children, and drugs that delay the onset of the disease are not applicable to individuals who have already developed the disease and formed abnormal protein aggregations. Currently, there are no drugs specifically targeting pathological protein aggregations of γCG129C in the lens.

[0003] Research on the treatment of pathological aggregation of lens proteins has reported that lanosterol plays a key role in inhibiting lens protein aggregation and reducing cataract formation. However, lanosterol has poor solubility, stability, and ocular penetration, making it difficult to achieve ideal therapeutic concentrations within the lens and thus preventing a complete cure for cataracts. Current research attempts to optimize the chemical structure of lanosterol, but the high cost of raw materials and the expensive modification process have hindered subsequent drug development. Peptide drugs offer advantages such as lower cost, simple structure, easy metabolism, good biocompatibility, easy penetration of human tissue and cell barriers, and ease of structural modification and industrial production. Therefore, screening for potential peptide drugs that can target cataracts caused by γCG129C mutations is crucial. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the purpose of this invention is to develop potential drugs that can regulate and reverse the pathological aggregation of γC-G129C by targeting small polypeptide molecules that target pathological aggregates of lens proteins.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a polypeptide that targets the human lens protein γC-G129C.

[0007] Furthermore, the polypeptide comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having one or more amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the amino acid sequence shown in SEQ ID NO:2.

[0008] Furthermore, the N-terminus of the polypeptide is modified with biotin.

[0009] In this invention, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds having amino acid residues covalently linked by a peptide bond.

[0010] In this invention, the terms "homology" and "identity" are used interchangeably. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0011] In some embodiments, the polypeptide of the present invention further includes a polypeptide formed by substituting, deleting or adding one or more (2, 3, 4, 5 or 6) amino acid residues of the polypeptide shown in SEQ ID NO:2, and having a specific binding function to human lens protein γC-G129C.

[0012] A second aspect of the present invention provides a biomaterial.

[0013] Furthermore, the biomaterial includes:

[0014] 1) A nucleic acid molecule encoding the polypeptide described in the first aspect of the present invention;

[0015] 2) A vector comprising the nucleic acid molecule described in 1);

[0016] 3) A host cell containing the nucleic acid molecule described in 1) or the vector described in 2).

[0017] Furthermore, the vector includes plasmids and viral vectors.

[0018] Furthermore, the viral vector includes lentivirus, adenovirus, and adeno-associated virus vector.

[0019] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0020] In this invention, the terms "polynucleotide," "nucleic acid molecule," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, namely deoxyribonucleotides or ribonucleotides or their analogues. Polynucleotides may be further modified after polymerization, for example by conjugation with labeled components. The term also refers to double-stranded and single-stranded molecules.

[0021] In this invention, the term "vector" refers to a nonchromosomal nucleic acid containing a complete replicon, such that the vector can be replicated when placed within a permitted cell, for example, through a transformation process. A vector can replicate in one cell type (e.g., bacteria) but has limited ability to replicate in another cell type (e.g., mammalian cells). Vectors can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; and DNA, alone or in combination with a cationic polymer, in a cationic lipid complex; anionic and cationic liposomes; DNA-protein complexes; and particles containing DNA condensed with cationic polymers (such as heteropolymers of polylysine, fixed-length oligopeptides, and polyethyleneimine), and in some cases, also contained in liposomes.

[0022] In some embodiments, the vectors described in this invention include plasmids (expression plasmids, cloning vectors, small loops, microvectors, double microchromosomes), lentiviral vectors, adenovirus vectors, or adeno-associated virus vectors.

[0023] Furthermore, the carrier also includes one or more control elements.

[0024] Furthermore, the regulatory element includes a promoter, an enhancer, a ribosome binding site for translation initiation, a terminator, a polyadenylate sequence, and a selection marker gene.

[0025] Furthermore, the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

[0026] In some embodiments, the host cells described in this invention include one or more of Escherichia coli, Streptomyces, Agrobacterium, yeast cells, plant cells, animal cells, or viruses.

[0027] A third aspect of the present invention provides a polypeptide derivative.

[0028] Furthermore, the polypeptide derivative is a complex obtained by modifying the polypeptide described in the first aspect of the present invention; the modification includes conjugation modification using a detectable marker, conjugation modification using a therapeutic agent, and conjugation modification using an imaging agent.

[0029] Furthermore, the detectable markers include fluorescent dyes, avidin, paramagnetic atoms, radioactive isotopes, and enzyme markers.

[0030] Furthermore, the fluorescent pigments include fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, and polydinoflavin-chlorophyll protein.

[0031] Furthermore, the avidin includes biotin, egg white avidin, streptavidin, egg yolk avidin, and avidin-like substances.

[0032] Furthermore, the radioactive isotopes include radioactive iodine, radioactive cesium, radioactive iridium, and radioactive cobalt.

[0033] Furthermore, the enzyme markers include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme, and malate dehydrogenase.

[0034] Furthermore, the therapeutic agents include cytotoxic agents, hormonal preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of co-stimulatory molecules, and inhibitors of inhibitory molecules.

[0035] A fourth aspect of the present invention provides a pharmaceutical composition for treating cataracts.

[0036] Furthermore, the pharmaceutical composition includes the polypeptide described in the first aspect of the present invention.

[0037] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

[0038] Furthermore, the pharmaceutically acceptable carriers and / or excipients include diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0039] In some implementations, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, water, etc.

[0040] In some embodiments, the adhesive includes, but is not limited to: starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginate, xanthan gum, hydroxypropylcellulose and hydroxypropylmethylcellulose, etc.

[0041] In some embodiments, the surfactant includes, but is not limited to: polyethylene oxide sorbitan fatty acid ester, sodium lauryl sulfate, glyceryl monostearate, hexadecyl alcohol, etc.

[0042] In some embodiments, the humectant includes, but is not limited to, glycerin, starch, etc.

[0043] In some embodiments, the adsorbent carrier includes, but is not limited to, starch, lactose, bentonite, silica gel, kaolin, and soap clay.

[0044] In some embodiments, the lubricant includes, but is not limited to: zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.

[0045] In some embodiments, the filler includes, but is not limited to: mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, and sodium bicarbonate, etc.

[0046] In some embodiments, the disintegrant includes, but is not limited to: crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides, etc.

[0047] In some embodiments, the pharmaceutically acceptable carrier and / or excipients may additionally contain liquids such as water, saline, glycerin, and ethanol. The dosage forms of the pharmaceutical compositions include, but are not limited to: tablets, pills, powders, granules, capsules, lozenges, syrups, solutions, emulsions, suspensions, controlled-release formulations, aerosols, films, injections, intravenous infusions, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories, nasal preparations, pulmonary preparations, eye drops, etc., for patient ingestion.

[0048] In some embodiments, suitable routes of administration of the pharmaceutical composition include any of a variety of methods and delivery systems known to those skilled in the art to physically introduce the pharmaceutical composition of the present invention into a subject, including but not limited to: oral administration, non-gastrointestinal administration, administration via inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration, or administration via an implanted storage device. When the pharmaceutical composition is intended for injection, it may be in the form of a suspension, solution, or emulsion in an oily or aqueous medium and may contain formulation agents such as suspending agents, preservatives, stabilizers, and / or dispersants.

[0049] In some embodiments, the pharmaceutical composition or active ingredient (the polypeptide of the present invention) of the present invention may also be used in combination with other drugs for treating cataract diseases. The combination includes simultaneous or sequential use, such that a therapeutically effective amount of the pharmaceutical composition or active ingredient of the present invention and one or more other drugs for treating cataract diseases are present in the patient's body.

[0050] The fifth aspect of the present invention provides a method for preparing the polypeptide described in the first aspect of the present invention.

[0051] Furthermore, the method includes the following steps: culturing the host cells described in the second aspect of the present invention to obtain a culture product, and separating and purifying the polypeptide described in the first aspect of the present invention from the culture product.

[0052] The sixth aspect of the present invention provides a method for inhibiting and / or reversing the pathological aggregation of human lens protein γC-G129C in vitro for non-therapeutic purposes.

[0053] Furthermore, the method includes incubating the polypeptide described in the first aspect of the present invention with human lens protein γC-G129C.

[0054] Furthermore, the concentration ratio of the polypeptide to human lens protein γC-G129C is 0.03:1-10:1.

[0055] The seventh aspect of the present invention provides any of the following applications, the applications including:

[0056] 1) The application of the polypeptide described in the first aspect of this invention in the preparation of polypeptide derivatives, nucleic acid molecules, and carriers;

[0057] 2) The use of the polypeptides described in the first aspect of the present invention, the biomaterials described in the second aspect of the present invention, and the polypeptide derivatives described in the third aspect of the present invention in the preparation of products that inhibit and / or reverse the pathological aggregation of human lens protein γC-G129C;

[0058] 3) The use of the polypeptide described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, and the polypeptide derivative described in the third aspect of the present invention in the preparation of products for treating cataracts.

[0059] Advantages and beneficial effects of the present invention:

[0060] This invention utilizes a self-made peptide chip microarray to screen for an optimal peptide targeting the human lens protein γC-G129C. The peptide screened in this invention significantly reverses the aggregation of human lens protein γC-G129C both in vivo and in vitro, indicating that the peptide provided by this invention has potential therapeutic effects on cataracts and has broad clinical application prospects. Attached Figure Description

[0061] Figure 1 The graph shows the trend of absorbance at 350 nm of hydrogen peroxide solution of different concentrations and γC-WT / G129C over time.

[0062] Figure 2 This is a graph showing the morphological changes of hydrogen peroxide solutions of different concentrations and γC-WT / G129C over time under a differential interference microscope.

[0063] Figure 3 The graph shows the trend of absorbance at 350 nm over time for different concentrations of γC-WT / G129C and 0.1% hydrogen peroxide solution.

[0064] Figure 4 Figure 1 shows the results of fluorescence bleaching recovery experiment of γC-WT and γC-G129C aggregates (left: γC-G129C, right: γC-WT);

[0065] Figure 5 The fusion trend of γC-WT and γC-G129C aggregates over time is shown in the figure.

[0066] Figure 6 The proportion of the secondary structure of γC-G129C aggregates (left: 0.1% H2O2, right: 1% H2O2);

[0067] Figure 7 The figure shows the effect of DTT and 1,6-hexanediol on γC-G129C aggregates.

[0068] Figure 8 A schematic diagram for screening peptides that specifically bind to γC-G129C using a microarray chip (top: original image; bottom: model image);

[0069] Figure 9 The graph shows the trend of absorbance at 350 nm over time after mixing the peptide candidate with γC-G129C aggregates.

[0070] Figure 10 Optical microscopy image of the endpoint of incubation of the polypeptide candidate with γC-G129C aggregate;

[0071] Figure 11 The figure shows the concentration-dependent experimental results of peptide No.2 reversing the γC-G129C aggregate;

[0072] Figure 12 The graph shows the affinity determination results of γC-G129C, γC-WT and No.2 peptide;

[0073] Figure 13 This diagram shows the endogenous and exogenous expression of γC-WT and γC-G129C in HeLa cells.

[0074] Figure 14 Fluorescence microscopy imaging and schematic diagram of HeLa cells expressing γC-G129C intracellularly;

[0075] Figure 15 The image shows fluorescence microscopy imaging results of HeLa cells with γC-WT / G129C under exogenous stimulation.

[0076] Figure 16 Figure 1 shows the effect of exogenous oxidants and reducing agents on the intracellular aggregation rate of γC-G129C.

[0077] Figure 17 The graph shows the cytotoxicity results of peptide No.2 as detected by CCK-8 assay.

[0078] Figure 18 Image of cell fluorescence microscopy after incubation of peptide No.2 for 24 hours;

[0079] Figure 19 Real-time imaging results of live cells after 24 hours of incubation of peptide No.2;

[0080] Figure 20 The graph shows the effect of concentration gradient of peptide No. 2 on intracellular aggregation rate. Detailed Implementation

[0081] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0082] The experimental materials used in the following examples were sourced from the following sources:

[0083] 1. Chemical reagents: Sodium chloride (NaCl), glucose, calcium chloride (CaCl2), and magnesium sulfate (MgSO4) were purchased from Tianjin Damao Chemical Reagent Factory; anhydrous ethanol, ammonium chloride (NH4Cl), glacial acetic acid, and ZnSO4·7H2O were purchased from Sinopharm Chemical Reagent Co., Ltd.; disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and potassium dihydrogen phosphate (KH2PO4) were purchased from Xilong Scientific Co., Ltd.; pantothenic acid, folic acid, inositol, nicotinamide, ferrous sulfate heptahydrate (FeSO4·7H2O), manganese sulfate tetrahydrate (MnSO4·4H2O), and copper sulfate pentahydrate (CuSO4·5H2O) were purchased from Shanghai Aladdin Biotechnology Co., Ltd.; boric acid (H3BO3) and sodium dodecyl sulfate (Sodium dodecyl sulfate) were purchased from Shanghai Aladdin Biotechnology Co., Ltd. Sulfate (SDS) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich; cobalt chloride hexahydrate (CoCl2·6H2O), choline chloride, biotin, thiamine, riboflavin, and (NH4)6Mo7O were also present. 244H2O was purchased from Shanghai Maclean Biochemical Reagent Co., Ltd.; peroxide disinfectant (3% v / v, purchased from Haisheng Hainuo); 1,1,1,3,3,3-hexafluoroisopropanol was purchased from China Innocare Technology Co., Ltd.; guanidine hydrochloride was purchased from ACROS, Belgium; 24-well, 48-well, and 96-well cell culture plates were purchased from Corning, USA; MEM cell culture medium, streptomycin-penicillin antibiotics (PS, 100×), dithiothreitol (DTT), 2-morpholinoethanesulfonic acid (MES), trometamol (Tris), glutathione, and pyridoxal were purchased from Beijing Lanbolide Trading Co., Ltd.; fetal bovine serum (FBS, Gibco), Opti-MEM, and Protein Labeling Kits (Alexa Fluor 488, A10235 Invitrogen) were purchased from Thermo. Fisher Scientific; phosphate-buffered saline (PBS) and enzyme-free sterile water were purchased from Wuhan Sewell Biotechnology Co., Ltd.; kanamycin (kana) was purchased from Tiangen Biotech Co., Ltd.; yeast extract and tryptone were purchased from Thermo Fisher Scientific; isopropyl-β-D-thiogalactoside (IPTG) was purchased from Beijing TransGen Biotech Co., Ltd.; Escherichia coli BL21(DE3)pLysS was purchased from Beijing TransGen Biotech Co., Ltd.; ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) was purchased from Beijing Innocare Technology Co., Ltd.; Coomassie brilliant blue staining solution (self-made); peptide synthesis was performed by Guoping Pharmaceutical Co., Ltd.

[0084] 2. Consumables and Equipment: Cation exchange chromatography column and packing material purchased from Topfan Ltd.; nickel column packing material and CCK-8 purchased from Beijing Lamborghini Trading Co., Ltd.; 10kDa concentration tube purchased from Millipore; electric thermostatic incubator (XMTD HH.B11-600); eight-well coverslip (Hangzhou Xinyou Biotechnology Co., Ltd.); vertical pressure steam sterilizer (Shanghai Boxun, China); thermostatic bacterial incubator (Shanghai Boxun, China); benchtop centrifuge (Eppendorf, Centrifuge 5415D); electric thermostatic water bath (SHH W21600); micro-volume UV spectrophotometer (NanoDrop 2000); electronic balance (Sartorius 2000S); optical inverted microscope (XDS-1B); micropipette (Eppendorf Research). (plus); -80℃ ultra-low temperature freezer (SANYO, MDF-382E); high-speed refrigerated centrifuge (Beckman, Germany); pH meter (Thermo Orion 868); magnetic stirrer (IKA RH-KT / C); ultrasonic breaker; Pure 25 protein purification system (Superdex 75, GE Healthcare); SDS-PAGE electrophoresis system (Bio-Rad); gel imaging system (Tanon); 2mL disposable sterile syringe (purchased from BD Medical Devices Co., Ltd.); electrophoresis apparatus (Beijing Longfang Technology Co., Ltd., China); polyacrylamide gel electrophoresis precast gel (Beijing Qingke Biotechnology Co., Ltd., China); differential interference difference microscope (DMI8, Leica GmbH, Germany); Fortibio OctetRed96 molecular interaction detection system (ForteBio); laser scanning confocal microscope Leica Stellar Leica Stellaris 5 Confocal Microscope (Leica GmbH, Germany); Incucyte S3 live cell long-term analyzer (Sartorius); shaker (Harbin Donglian Electronic Technology Development Co., Ltd.); miniature micro-oscillator, miniature vortex shaker (Axygen, USA); DNA- and RNase-free centrifuge tubes (15mL, 50mL) (Corning, USA); RNase-free EP tubes (Axygen, USA); pipettes of various sizes (Eppendorf, Germany); streptavidin (SA) biosensor (Sartorius, Sartorius, Germany); CO2 cell incubator (Thermo, USA); biosafety cabinet (Haier, China); fume hood (Harbin Donglian Harbin, China); circulating water multi-purpose vacuum pump (Zhengzhou Great Wall, China); 0.22μm filter membrane (Merck Millipore, USA); high-speed refrigerated centrifuge (Eppendorf, Germany); 4× protein loading buffer (Beijing Lamborghini, China); PerkinElmer Spectrum One FTIR spectrometer (Waltham, Massachusetts, USA); Bruker Avance III 600 NMR spectrometer (Bruker Corporation, Germany); Multifunctional microplate reader (BioTek, USA).

[0085] Example 1: Expression and purification of γC-WT and γC-G129C proteins

[0086] 1. After codon optimization, the DNA encoding the γC-WT protein sequence and the γC-G129C protein sequence was cloned into the pET-28a(+) vector plasmid through NcoI / XhoI double restriction sites to construct the pET-28a(+)-γC-WT and pET-28a(+)-γC-G129C vectors.

[0087] After codon optimization, the DNA encoding the γC-WT and γC-G129C protein sequences was cloned into the peGFP-N1 vector plasmid via XhoI / BamH1 double restriction sites to construct the peGFP-N1-γC-WT and peGFP-N1-γC-G129C vectors.

[0088] The sequence of the γC-WT protein is as follows:

[0089] MGKITFYEDRAFQGRSYETTTDCPNLQPYFSRCNSIRVESGCWMLYERPNYQGQQYLLRRGEYPDYQQWMGLSDSIRSCCLIPQTVSHRLRLYEREDHKGLMMELSEDCPSIQDRFHLSEIRSLHVLEGCWVLYELPNYRGRQYLLRPQEYRRCQDWGAMDAKAGSLRRVVDLY(SEQ ID NO:7); The sequence of γC-G129C protein is: MGKITFYEDRAFQGRSYETTTDCPNLQPYFSRCNSIRVESGCWMLYERPNYQGQQYLLRGEYPDYQQWMGLSDSIRS CCLIPQTVSHRLRLYEREDHKGLMMELSEDCPSIQDRFHLSEIRSLHVLECCWVLYELPNYRGRQYLLRPQEYRRCQDWGAMDAKAGSLRRVVDLY(SEQ ID NO:8).

[0090] 2. Transformation:

[0091] 1) Melt BL21(DE3)pLysS competent cells with γC-WT and γC-G129C recombinant plasmids on ice;

[0092] 2) Add 0.5 μL (0.2 μg / μL) of γC-WT and γC-G129C recombinant plasmids to BL21(DE3)pLysS competent cells and incubate on ice for 15 min;

[0093] 3) 42℃ water bath heat shock for 90 seconds;

[0094] 4) Quickly place on ice and in an ice bath for 5 minutes;

[0095] 5) Add 500 μL of antibiotic-free LB and incubate at 37℃ for 40-60 min;

[0096] 6) Take the bacterial culture, γC-WT and γC-G129C are inoculated on LB solid medium containing Kanamycin (25 μg / mL) and incubated overnight at 37°C.

[0097] 3. Bacterial culture:

[0098] Using Kanamycin (25 μg / mL) as a selection marker, positive monoclonal clones BL21(DE3)pLysSγC-WT and γC-G129C from the solid medium in the previous step were picked and inoculated into 20 mL of LB liquid medium containing Kanamycin resistance, and cultured overnight at 37°C and 210 rpm with shaking. The bacterial suspensions were then inoculated into 1 L of LB liquid medium containing Kanamycin resistance at a ratio of 1:100 and cultured at 37°C and 210 rpm with shaking.

[0099] 4. Induction and acquisition of the target protein:

[0100] 1) For γC-WT and γC-G129C, when the optical density (OD) of E. coli at 600 nm... 600 When the concentration of the target protein was 0.8-1.2, IPTG was added to a final concentration of 0.5 mM, and the mixture was cultured at 37℃ and shaking at 210 r / min for 4 h to induce the expression of the target protein.

[0101] 2) Enrich the expressed bacterial culture, centrifuge at 6000g for 20 min at 4°C, and discard the supernatant. Resuspend in 20mM MES (pH 6.0, containing 1mM EDTA) (γC-WT, γC-G129C) lysis buffer; collect after resuspending, and add PMSF and DTT to a final concentration of 1mM to prevent degradation or aggregation of the target protein;

[0102] 3) Ultrasonic disruption of bacteria: Ice bath ultrasound, power 30%, working time 25 min, ultrasound on time 3 s, ultrasound off time 9 s;

[0103] 4) Protein collection: Centrifuge at 12000g for 30 minutes at 4℃, collect the supernatant, and the soluble target protein is present in the supernatant.

[0104] 5. Purification of the target protein:

[0105] 5.1 γC-WT and γC-G129C proteins were purified by cation exchange chromatography:

[0106] 1) Equilibrate the cation exchange column: After activating the cation exchange column with 2M NaCl, pass ultrapure water through the column for 2-3 column volumes, and then pass the corresponding Lysis Buffer out of the cation exchange column for about 2-3 column volumes before loading the sample.

[0107] 2) Load the supernatant obtained from centrifugation onto the cation exchange column 2-3 times;

[0108] 3) Wash the column with Lysis Buffer (without NaCl) to elute contaminating proteins;

[0109] 4) Wash the column with Lysis Buffer containing 100mM NaCl and 1mM DTT to debind the target protein from the cation exchange column;

[0110] 5) Concentration: Place the target protein collected by elution in step 4) into a 10kDa concentration tube, centrifuge at 4000×g for 50 min at 4℃. Add a small amount of DTT during the concentration process to prevent the target protein from agglomerating and precipitating.

[0111] 5.2 Pure 25 protein purification:

[0112] 1) Cleaning the chromatography column: Pre-clean the chromatography column with 20% ethanol solution, approximately one column volume. Then place the pump head in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution and clean one column volume, approximately 24 mL.

[0113] 2) Initial parameter settings: System flow: 0.5 mL / min; Column position: 1; Alarm deltacolumn pressure enabled: 3.0; Alarm pre column pressure enabled: 5.0; Collect 100% of the target component;

[0114] 3) Sample loading: First, rinse the sample loading loop 2-3 times with 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution, then inject about 2mL of concentrated sample into the sample loading loop, set the collection tube, and set the parameter inject valve:inject;

[0115] 4) Collection: When the UV 280 marker line, which represents protein content, rises near the target size, collect the target protein using an automated sample collector, setting the collection volume to 0.5 mL / EP;

[0116] 5) Cleaning the chromatography column: After collecting the sample, place the pump head in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution and continue running until 20mL has passed; then replace with sterile water for cleaning, running 10mL; then replace with 20% ethanol solution and run one column volume, then turn off the machine.

[0117] 6) All proteins are extracted fresh for immediate use. Liquid nitrogen freezing may affect their properties. They can be stored briefly in a 4°C freezer.

[0118] 6. SDS-PAGE identification of the expressed product:

[0119] 1) Sample preparation: After whole bacteria are sonicated and the sample is retained, the supernatant sample is centrifuged, the protein supernatant sample is passed through the chromatography column, the sample is washed with Lysis Buffer, the sample is passed through, and the protein sample is collected at three points: before the molecular sieve peak, at the peak tip, and after the peak.

[0120] 2) Gel formulation: 5-20% gradient stacking gel, 15% separating gel;

[0121] 3) Sample preparation: Mix 4× Loading Buffer with the sample at a ratio of 1:3;

[0122] 4) Sample loading: 5 μL for marker, 10 μL for the remaining samples;

[0123] 5) Electrophoresis: Electrophoresis buffer MOPS, constant voltage 100V electrophoresis until the protein sample migrates to the bottom of the tank;

[0124] 6) Staining: Place the protein gel in Coomassie brilliant blue staining solution and stain over medium-high heat for 2 minutes;

[0125] 7) Decolorization: Place the stained protein gel in tap water and decolorize over high heat for 25 minutes;

[0126] 8) Imaging and analysis: The electrophoresis results were analyzed using a gel imaging system. The target protein showed a clear band at the molecular sieve peak with few other proteins, which allowed for subsequent experiments.

[0127] 7. Protein concentration determination:

[0128] 1) In the experiment, 6M guanidine hydrochloride was used to deassemble the protein. The concentrations of γC-WT and γC-G129C proteins were measured using a One Drop spectrophotometer and corrected using the extinction coefficient.

[0129] 2) Adjust the protein concentration to the target level according to experimental needs.

[0130] Example 2: Characterization of the oxidative stress response properties of γC-WT and γC-G129C proteins

[0131] I. Experimental Methods

[0132] 1) Dilute γC-WT and γC-G129C or hydrogen peroxide (3%) solutions with 1×PBS (pH 7.4, containing 1mM EDTA, without DTT); after mixing the protein with the hydrogen peroxide solution, the final protein concentration is 0.1mM (100μM), or 50μM, 30μM, 20μM, 10μM, 5μM, 1μM; the final hydrogen peroxide concentration is 1%, 0.1%, 0.01%, 0.001%.

[0133] 2) Add 150 μL of the mixture of γC-WT or γC-G129C and hydrogen peroxide to a 96-well plate, with three replicates per group. Monitor the absorbance at 350 nm in a microplate reader at 37 °C for 3 hours, with measurements taken every 3 minutes.

[0134] 3) Incubate the mixture of γC-WT or γC-G129C and hydrogen peroxide in a 37°C incubator. Take a sample every 30 min and add it to a coverslip coated with 0.2 mg / mL BSA. Observe the sample using a differential interference contrast microscope (DIC) for a total of 3 h.

[0135] 4) The data was processed using GraphPad Prism 9, and the resulting images were processed using Adobe Illustrator 2023.

[0136] II. Experimental Results

[0137] The trends of absorbance at 350 nm of hydrogen peroxide solutions of different concentrations and γC-WT / G129C over time are as follows: Figure 1 As shown, the absorbance at 350 nm can reflect the degree of turbidity in the system and characterize the ability of lens proteins to form aggregates. A higher value indicates a higher degree of aggregation. Responsive c(H2O2) is the minimum hydrogen peroxide concentration required for protein aggregation, and Onset time is the time it takes for proteins to begin to aggregate under a 1% hydrogen peroxide concentration. It can be seen that compared with γC-WT, the γC-G129C mutant protein requires a very low hydrogen peroxide concentration to aggregate, and aggregation can occur when the hydrogen peroxide concentration is higher than 0.001%, and the aggregation time is also shorter, indicating that the γC-G129C mutant protein is more likely to aggregate. Figure 2 The results show the morphological changes of hydrogen peroxide solutions of different concentrations and γC-WT / G129C over time under differential interference microscopy. It can be seen that γC-WT exhibits obvious aggregation behavior when the hydrogen peroxide concentration is higher than 0.1%. The results of differential interference microscopy are consistent with the absorbance monitoring results of the microplate reader. Figure 3The graph shows the absorbance at 350 nm over time for different concentrations of γC-WT / G129C and 0.1% hydrogen peroxide solution. Under the same hydrogen peroxide pressure, the minimum protein concentration for γC-WT aggregation is 5 μM, while the minimum protein concentration for γC-G129C aggregation is 1 μM. At the same hydrogen peroxide and protein concentrations, γC-G129C aggregates faster than γC-WT, and its absorbance is higher at the plateau phase. These results demonstrate that G129C has different responses to hydrogen peroxide, and that G129C mutations affect the oxidative tolerance of γC. Without hydrogen peroxide stimulation, neither γC-WT nor G129C aggregates, suggesting that the extremely low tolerance of γC-G129C to oxidants may be the reason for early-onset disease.

[0138] Example 3: Characterization of the properties of γC-WT and γC-G129C aggregates

[0139] I. Fluorescence bleaching recovery experiment to determine protein aggregate fluidity

[0140] 1. Experimental Methods

[0141] 1) Adjust the protein concentration of γC-WT and γC-G129C to 0.2mM with 1×PBS (pH 7.4, containing 1mM EDTA);

[0142] 2) After labeling γC-WT and γC-G129C with a Protein Labeling Kit (Alexa Fluor 488, A10235 Invitrogen), they were mixed with an equal volume of 0.2% hydrogen peroxide solution.

[0143] 3) Incubate the system solution in a 37°C incubator for 1 hour. During the rapid aggregation phase, shake the solution to mix well and add 10 μL of the solution to an eight-well coverslip.

[0144] 4) Photobleaching recovery experiment using laser confocal microscopy: Select a region of interest (ROI), record baseline fluorescence with low laser intensity through imaging, and then perform photobleaching on the selected ROI using a high-intensity laser pulse; monitor the recovery fluorescence after bleaching by acquiring time-lapse images over a long period of time until the fluorescence in the ROI reaches a plateau; generate fluorescence recovery curves by normalizing the fluorescence intensity of the bleached area to the intensity before bleaching and correcting the photobleaching using an unbleached reference ROI during imaging; all experiments are repeated at least 3 times.

[0145] 5) The data was processed using GraphPad Prism 9, and the resulting images were processed using Adobe Illustrator 2023.

[0146] 2. Experimental Results

[0147] The results of the fluorescence bleaching recovery experiment of γC-WT (left) and γC-G129C (right) aggregates are as follows: Figure 4 As shown in the full bleaching experiment, γC-G129C has a lower recovery rate compared to γC-WT, indicating that γC-G129C aggregates have a more solid tendency.

[0148] II. Fusion Experiments of γC-WT and γC-G129C Condensates

[0149] 1. Experimental Methods

[0150] 1) Incubate the mixture of γC-WT or γC-G129C and hydrogen peroxide (final protein concentration 0.1mM, final hydrogen peroxide solution concentration 1% or 0.1%) in a 37°C incubator for 1 hour.

[0151] 2) Every 30 minutes, a sample was added to an eight-well coverslip coated with 0.2 mg / mL BSA, and the fusion phenomenon was observed using a differential interference contrast microscope (DIC).

[0152] 2. Experimental Results

[0153] The fusion trends of γC-WT and γC-G129C aggregates over time are as follows: Figure 5 As shown, droplet fusion is a fast process on a time scale. The results show that no fusion of condensates occurred within 10 minutes. The fusion experiment and the fluorescence bleaching recovery experiment corroborate each other, and the condensates formed by γC-WT or γC-G129C are more like solid amorphous aggregates.

[0154] III. Fourier Transform Infrared (FTIR) γC-G129C Secondary Structure

[0155] 1. Experimental Methods

[0156] 1) Incubate the mixture of γC-G129C and hydrogen peroxide solution (final protein concentration 0.1 mM, final hydrogen peroxide solution concentration 1% or 0.1%) in a 37°C incubator for 3 hours.

[0157] 2) Shake the turbid liquid to mix well, take 100 μL of γC-G129C solution and drop it onto the calcium fluoride window and dry it completely;

[0158] 3) Use an FTIR spectrometer to record spectra in the range of 400 to 4500 cm⁻¹, with a resolution of 2 cm⁻¹. Perform three scans and average the results to improve the signal-to-noise ratio. Analyze the amide I band (1600-1700 cm⁻¹). -1 To determine the secondary structure of protein aggregates;

[0159] 4) PeakFit software was used for deconvolution and second derivative analysis to analyze overlapping peaks and quantify the contributions of different secondary structure elements. GraphPad Prism 9 was used to process the data, and Adobe Illustrator 2023 was used to process the resulting images.

[0160] 2. Experimental Results

[0161] The secondary structure ratio of γC-G129C aggregates is as follows: Figure 6 As shown, the left figure represents the condition with a hydrogen peroxide concentration of 0.1%, and the right figure represents the condition with a hydrogen peroxide concentration of 1%. As the concentration of hydrogen peroxide solution increases, the proportion of random coils in the γC-G129C aggregate decreases, reflecting the effect of hydrogen peroxide solution on the structure of the γC-G129C protein aggregate.

[0162] Example 4: Screening for peptide inhibitors targeting γC-G129C aggregates

[0163] I. γC-G129C aggregates cannot be destroyed by reducing agents or hydrophobic interaction inhibitors.

[0164] 1. Experimental Methods

[0165] 1) Dilute γC-G129C with 1×PBS (pH 7.4) to adjust the protein concentration to 0.1 mM, while also containing 0.1% hydrogen peroxide solution; incubate the system in a 37°C incubator for 1 hour;

[0166] 2) Divide the incubated solution into equal portions, centrifuge at 12000g for 10 min at 4℃, and remove the supernatant; then centrifuge the precipitate again with an equal volume of 1×PBS (pH 7.4) or 1×PBS (pH 7.4) containing 5mM DTT, 1% / 2% / 5% / 10% 1-6 Hexanediol, and incubate at 37℃ for 210 rpm for 3 h.

[0167] 3) After incubation, take a sample and drop it onto a coverslip coated with 0.2 mg / mL BSA, and image it using a differential interference contrast microscope.

[0168] 2. Experimental Results

[0169] The results are as follows Figure 7 As shown, DTT and 1,6-hexanediol have no reversible effect on γC-G129C aggregates.

[0170] II. Screening for peptides that specifically bind to γC-G129C using microarray chips

[0171] 1. Experimental Methods

[0172] 1) After labeling γC-WT and γC-G129C with a Protein Labeling Kit (Alexa Fluor 488, A10235 Invitrogen), the concentration was adjusted to 0.2 mg / mL;

[0173] 2) The fluorescently labeled protein was incubated with the microarray chip at room temperature for 3 hours, washed three times with 1×PBS (pH 7.4), and fluorescence imaging was performed.

[0174] 3) Six peptides with a binding strength to γC-G129C that is much stronger than that to γC-WT were selected as potential candidates (see Table 1).

[0175] Table 1 Potential peptides obtained after microarray chip screening.

[0176] Name Sequence No. 1 Biotin-PESFDGDPASNTAPLQPEQLQ (SEQ ID NO: 1) No. 2 Biotin-YGRKKRRQRRR (SEQ ID NO: 2) No. 3 Biotin-FIHHIIGGLFSIGKIIHRLIRRRRR (SEQ ID NO: 3) No. 4 EEGFFSA[cit]GHRPLDKK-Biotin (SEQ ID NO: 4) No. 5 Biotin-CPRGNYVNEKPY (SEQ ID NO: 5) No. 6 Biotin-GGRSFILLRIIQGCRRRNTVDD (SEQ ID NO: 6)

[0177] 4) Dilute γC-G129C with 1×PBS (pH 7.4) to adjust the protein concentration to 0.1 mM, while also containing 0.1% hydrogen peroxide solution; incubate the system in a 37°C incubator for 1 hour, then divide the solution into equal portions, centrifuge at 12000g for 10 min at 4°C, and remove the supernatant; then re-centrifuge the precipitate with an equal volume of 1×PBS (pH 7.4) containing 1 mM of the peptide candidate, and monitor the absorbance change at 350 nm at 210 cpm using a microplate reader at 37°C;

[0178] 5) After the enzyme-linked immunosorbent assay (ELISA) monitoring is completed, take samples and observe them under an optical microscope.

[0179] 2. Experimental Results

[0180] The absorbance at 350 nm after mixing the peptide candidate with γC-G129C aggregates changes over time as follows: Figure 9 As shown, optical microscopy images of the peptide candidate at the end of incubation with γC-G129C aggregates are as follows. Figure 10 As shown, based on the results of kinetics and optical microscopy, the turbidity of candidate peptide No.2 in the co-incubation system with γC-G129C aggregates showed a monotonically decreasing trend. After incubation, the turbidity observed under an optical microscope was significantly better than that of the control group and other candidates. Peptide No.2 was selected as the target for subsequent research.

[0181] Example 5: In vitro characterization experiments of peptide No. 2

[0182] I. Concentration-dependent experiment on the reversal of γC-G129C aggregates by peptide No. 2

[0183] 1. Experimental Methods

[0184] 1) Dilute γC-G129C with 1×PBS (pH 7.4) to adjust the protein concentration to 0.1mM, while also containing 0.1% hydrogen peroxide solution; incubate the system in a 37°C incubator for 1 hour, then divide the solution into equal parts, centrifuge at 12000g for 10min at 4°C, and remove the supernatant.

[0185] 2) The precipitate after centrifugation with equal volumes of 1×PBS (pH 7.4) containing 1 mM, 300 μM, 100 μM, 30 μM, 10 μM, and 3 μM peptide No.2 was monitored at 350 nm at 37 °C and 210 cpm.

[0186] 3) Compare the absorbance at 350 nm before adding peptide No.2 and after 3 hours of incubation, and perform statistical analysis and plotting using GraphPad Prism 9.

[0187] 2. Experimental Results

[0188] The concentration-dependent experimental results of peptide No. 2 reversing γC-G129C aggregates are as follows: Figure 11 As shown, for aggregates formed by 0.1 mM γC-G129C protein at 37℃ for 1 h, peptide No.2 in the system showed a significant reversal trend at concentrations above 10 μM.

[0189] II. Biomembrane Interference (BLI) Detection of Affinity Between Peptide No.2 and γC-G129C Protein

[0190] 1. Experimental Methods

[0191] 1) Peptide No.2 (Biotin-YGRKKRRQRRR) was dissolved in 1×PBS (pH 7.4) to a stock solution concentration of 250 nM, and then serially diluted; γC-WT and γC-G129C were diluted to appropriate concentrations with 1×PBS (pH 7.4);

[0192] 2) Activation of streptavidin (SA) probe: Incubate in 1×PBS (pH 7.4) solution for 10 min;

[0193] 3) The SA probe was first soaked in 1×PBS (pH 7.4) buffer to obtain baseline data;

[0194] 4) Then soak the SA probe in a buffer solution containing peptide No.2 in 1×PBS (pH 7.4) to allow it to bind to the peptide;

[0195] 5) Transfer the SA probe to 1×PBS (pH 7.4) buffer to remove unbound peptides;

[0196] 6) Immerse the SA probe in a buffer solution containing the target protein in 1×PBS (pH 7.4) to allow the protein to bind to the polypeptide, and measure the binding rate constant.

[0197] 7) Transfer the SA probe to 1×PBS (pH 7.4) buffer to dissociate the protein from the peptide and measure the dissociation rate constant;

[0198] 8) Analyze the experimental data and calculate the dissociation constant KD.

[0199] 2. Experimental Results

[0200] The affinity assay results for γC-G129C, γC-WT, and peptide No.2 are as follows: Figure 12 As shown, the dissociation constant of peptide No.2 with γC-G129C is one order of magnitude lower than that of γC-WT, indicating that peptide No.2 binds more tightly to G129C compared to the wild type.

[0201] Example 6 Intracellular characterization experiment of peptide No. 2

[0202] I. Cell transfection experiment to express exogenous γC-G129C / WT

[0203] 1. Experimental Results

[0204] 1) Cell culture and preparation: HeLa cells were seeded in 24-well plates at a density of approximately 2 × 10⁵ cells / well and cultured in DMEM medium containing 10% FBS and 1% PS until 80% confluence.

[0205] 2) Before transfection, change the culture medium to Opti-MEM and starve the cells with serum for 2 hours;

[0206] 3) Dissolve plasmids peGFP-N1-γC-WT and peGFP-N1-γC-G129C in TEA Buffer; transfect the plasmids using Lipofectamine 3000 transfection reagent at a ratio of 1:1.5 (100 ng plasmid vector / 1.5 μL reagent);

[0207] 4) Four hours after transfection, replace the medium with DMEM containing 10% FBS and 1% PS and continue culturing for 24-48 hours;

[0208] 5) After washing the cells 2-3 times with 1×PBS (pH 7.4), fix them with 4% paraformaldehyde at room temperature for 30-40 min, then wash them 2-3 times with 1×PBS (pH 7.4) and observe them under a fluorescence microscope.

[0209] 2. Experimental Results

[0210] like Figure 13As shown, without exogenous oxidative stimulation, γC-WT was uniformly distributed in the cytoplasm after intracellular expression, while γC-G129C was observed to accumulate significantly in the cytoplasm after intracellular expression.

[0211] II. Exogenous oxidative stimulation increases the intracellular aggregation rate of γC-G129C.

[0212] 1) Cell culture and preparation: HeLa cells were seeded in 24-well plates at a density of approximately 1×10⁵ to 2×10⁵ cells / well and cultured in DMEM medium containing 10% FBS and 1% PS until 80% confluence.

[0213] 2) Before transfection, change the culture medium to Opti-MEM and starve the cells with serum for 2 hours;

[0214] 3) Dissolve plasmids peGFP-N1-γC-WT and peGFP-N1-γC-G129C in TEA Buffer; transfect the plasmids using Lipofectamine 3000 transfection reagent at a ratio of 1:1.5 (100 ng plasmid vector / 1.5 μL reagent);

[0215] 4) Four hours after transfection, replace the medium with DMEM containing 10% FBS and 1% PS and continue culturing for 24-48 hours;

[0216] 5) Replace the culture medium with a complete culture medium containing 100 μM or 500 μM hydrogen peroxide or 100 μM or 200 μM ascorbic acid, and culture for 4–6 hours;

[0217] 6) After washing the cells 2-3 times with 1×PBS (pH 7.4), fix them with 4% paraformaldehyde at room temperature for 30-40 min, then wash them 2-3 times with 1×PBS (pH 7.4) and observe them under a fluorescence microscope.

[0218] 2. Experimental Results

[0219] γC-G129C exists in two states within the cell: in some cells, fluorescence is uniformly distributed in the cytoplasm, indicating that γC-G129C is normally expressed but does not aggregate. Figure 14 (Left image) These cells are defined as aggregation-negative cells; simultaneously, some cells exhibit non-uniform fluorescence distribution in the cytoplasm, forming micron-sized aggregates. Figure 14 (See right figure) These cells are defined as aggregation-positive cells (Aggregation+). To quantitatively describe the degree of aggregation of γC-G129C, the intracellular aggregation rate is defined as the ratio of the number of aggregation-positive cells to the total number of observed cells.

[0220] Following stimulation with exogenous hydrogen peroxide, cells expressing exogenous γC-WT did not exhibit intracellular aggregation even at concentrations up to 500 μM hydrogen peroxide; however, for cells expressing exogenous γC-G129C, the intracellular aggregation rate increased with increasing exogenous hydrogen peroxide concentration, and this aggregation was not reversed by exogenous small molecule antioxidants. Figure 15 , Figure 16 ).

[0221] III. Detection of Cytotoxicity of Peptide No. 2 by CCK-8 Assay

[0222] 1. Experimental Methods

[0223] 1) Cell culture and preparation: HeLa or HLEB3 cells were seeded in 96-well plates at a density of approximately 5×10⁴ to 1×10⁵ cells / well; culture medium: HeLa (DMEM containing 10% FBS and 1% PS) or HLEB3 (MEM containing 10% FBS and 1% PS) were cultured to 80% confluence.

[0224] 2) Add gradient concentrations of peptide No.2 to the culture wells, with final concentrations of 1000 μM, 300 μM, 100 μM, 30 μM, 10 μM, 30 μM, and 1 μM;

[0225] 3) After culturing the cells for 48 hours, add 10 μL of CCK-8 solution to each well and continue culturing for 3 hours;

[0226] 4) Measure the absorbance at 450 nm using an ELISA reader, and perform dual-wavelength measurement using a wavelength of 630 nm as a reference wavelength.

[0227] 5) Use GraphPad Prism 9 for statistical analysis and graphing.

[0228] 2. Experimental Results

[0229] CCK-8 (Cell Counting Kit-8) is a commonly used method for cytotoxicity detection. The CCK-8 reagent is based on the reduction reaction of tetrazolium salt (WST-8) and can relatively quantify cell viability colorimetrically. The safe concentration of peptide No. 2 on HeLa cells is below 300 μM, and the safe concentration on HLEB3 cells is below 100 μM. Figure 17 ).

[0230] IV. Peptide No. 2 reverses intracellular aggregation of γC-G129C

[0231] 1. Experimental Methods

[0232] 1) Cell Culture and Preparation: HeLa cells were seeded in 48-well plates at a density of approximately 1 × 10⁻⁶ cells / well. 4~2×10⁵ / well, cultured in DMEM medium containing 10% FBS and 1% PS until 80% confluence;

[0233] 2) Before transfection, change the culture medium to Opti-MEM and starve the cells with serum for 2 hours;

[0234] 3) Dissolve plasmid peGFP-N1-γC-G129C in TEA Buffer; transfect the plasmid using Lipofectamine 3000 transfection reagent at a ratio of 1:1.5 (100 ng plasmid vector / 1.5 μL reagent);

[0235] 4) Four hours after transfection, replace the medium with DMEM containing 10% FBS and 1% PS and continue culturing for another 24 hours;

[0236] 5) Replace with complete culture medium containing 50μM, 100μM, 200μM, and 500μM peptide No.2, and continue culturing in a long-term cell observation and analysis instrument for 24 hours, with continuous live cell imaging at 1-hour intervals.

[0237] 6) After the experiment, wash the cells 2-3 times with 1×PBS (pH 7.4), fix them with 4% paraformaldehyde at room temperature for 30-40 min, wash them 2-3 times with 1×PBS (pH 7.4), and then immerse them in it for imaging with a fluorescence microscope.

[0238] 2. Experimental Results

[0239] Peptide No. 2 reduced intracellular aggregation in HeLa cells in a dose-dependent manner. Figure 18 Live-cell real-time imaging results showed that in the peptide No.2 treatment group, the aggregates that had formed gradually decreased in size over time and even redistributed uniformly within the cells, while in the control group (without peptide No.2 treatment), aggregates gradually formed. Figure 19 , Figure 20 This demonstrates the potential of peptide No.2 in reversing γC-G129C aggregates.

[0240] In summary, we demonstrated that the high sensitivity of γC-G129C to oxidants is the main reason for the formation of insoluble aggregates. Using a self-made microarray chip, we screened for a polypeptide with high affinity for γC-G129C and capable of reversing γC-G129C aggregates. We demonstrated its potential as a polypeptide inhibitor for the treatment of cataract-related diseases in vitro and intracellularly.

[0241] It should be noted that the specific embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there can be many variations. All those who are skilled in the art and who obtain the invention based on the explicit disclosure of the present invention or the written description of the document without objection should be considered to be within the scope of protection of this patent.

Claims

1. The use of the polypeptide shown in SEQ ID NO.2 in the preparation of products for treating cataracts.

Images