Polypeptide for inhibiting and reversing pathological aggregation of human crystalline lens protein gamma D

Abstract

The invention discloses a polypeptide for inhibiting and reversing pathological aggregation of human crystalline lens protein gamma D. According to the invention, alpha B-crystalline lens protein is cut into small peptides, and the capability of inhibiting and reversing pathological aggregation of crystalline lens protein is detected, so that the optimal polypeptide targeting the gamma D-crystalline lens P23T mutant protein is screened out. The polypeptide screened by the invention can significantly inhibit the aggregation of the gammaD-lens P23T mutant protein, and can reverse the aggregation of the gammaD-lens P23T mutant protein, which indicates that the polypeptide provided by the invention has a potential effect of treating cataract, and has a wide application prospect clinically.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a polypeptide for inhibiting and reversing pathological aggregation of human crystalline protein γD. The polypeptide targets γD-crystalline P23T mutant protein. Background Art

[0002] Pathological aggregation of lens proteins is closely related to a variety of eye diseases, including but not limited to senile cataracts, congenital cataracts, secondary cataracts, traumatic cataracts, etc. The treatment of these cataracts usually relies on surgical removal of the cloudy lens and implantation of an artificial lens, and there is no effective targeted therapeutic drug. Since abnormal aggregation of lens proteins is one of the key mechanisms of cataract formation, it is of great significance to develop drugs targeting pathological aggregation of lens proteins. Currently, drugs for the treatment of cataracts include hormone drugs, antioxidants, lanosterol, etc., but some drugs can only be used to delay the progression of cataracts, and the therapeutic effect is limited. There is currently no targeted drug for the P23T point mutation of γD-crystallin.

[0003] In the research on crystallin as a therapeutic target, there are literature reports that lanosterol plays a key role in inhibiting crystallin aggregation and reducing cataract formation, but lanosterol has difficulties in drug delivery, and its stability and bioavailability in the body are low, making it difficult to achieve the ideal therapeutic concentration in the lens, thus failing to cure related cataract diseases. At present, there are studies trying to optimize the chemical structure of lanosterol, but due to the high cost of raw materials and expensive modification, subsequent drug development has encountered bottlenecks. Summary of the invention

[0004] In order to make up for the shortcomings of the prior art, the purpose of the present invention is to screen small molecule polypeptide drugs targeting pathological aggregation of crystallins and develop potential polypeptide drugs that can regulate crystallin self-assembly caused by γD-P23T mutation and inhibit or reverse protein aggregation.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a polypeptide targeting γD-crystallin P23T mutant protein or a pharmaceutically acceptable salt thereof.

[0007] Further, the polypeptide is selected from any one of the following:

[0008] 1) the polypeptide shown in SEQ ID NO: 11;

[0009] 2) the polypeptide shown in SEQ ID NO: 14;

[0010] 3) a polypeptide having 75%, 80%, 85%, 90%, 95% or 100% or more homology with the polypeptide shown in SEQ ID NO: 11;

[0011] 4) A polypeptide having 75%, 80%, 85%, 90%, 95% or 100% or more homology with the polypeptide shown in SEQ ID NO:14.

[0012] Furthermore, the N-terminus of the polypeptide is modified by acetylation and / or the C-terminus is modified by amidation.

[0013] Further, the polypeptide acts on the H16, H23, N25, R60, H66, Q67, G71, L72, S85, H88, E96, H122, Y154, I171 and D172 amino acid residues of the γD-crystallin P23T mutant protein.

[0014] In the present invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds having amino acid residues covalently linked by peptide bonds.

[0015] In some embodiments, the polypeptide described in the present invention also includes a polypeptide formed by replacing, deleting or adding one or more (2, 3, 4, 5, 6) amino acid residues of the polypeptide shown in SEQ ID NO: 11 or SEQ ID NO: 14, and having a polypeptide with specific binding function to the γD-crystallin P23T mutant protein.

[0016] In the present invention, the terms "homology" and "identity" are used interchangeably. To determine sequence identity, a sequence alignment can be performed, which can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required to achieve optimal alignment in the full-length sequences being compared.

[0017] In the present invention, the polypeptide sequence obtained by modification also belongs to the protection scope of the present invention. The term "modification" refers to any chemical modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to the change in the amino acid sequence resulting in the increase of one or more amino acids compared to the naturally occurring molecule.

[0018] A second aspect of the present invention provides a biomaterial.

[0019] Furthermore, the biomaterial comprises:

[0020] 1) A nucleic acid molecule encoding the polypeptide according to the first aspect of the present invention;

[0021] 2) A vector comprising the nucleic acid molecule described in 1);

[0022] 3) A host cell comprising the nucleic acid molecule described in 1) or the vector described in 2).

[0023] In the present invention, the terms "polynucleotide", "nucleic acid molecule" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules.

[0024] In the present invention, the term "vector" refers to a non-chromosomal nucleic acid containing a complete replicon so that when placed in a permissive cell, the vector can be replicated, for example, by a transformation process. The vector can replicate in one cell type (e.g., bacteria), but has limited ability to replicate in another cell (e.g., mammalian cells). The vector can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; and DNA complexed with cationic lipids alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles containing DNA condensed with cationic polymers (e.g., heterogeneous polylysine, fixed-length oligopeptides, and polyethyleneimine), in some cases also contained in liposomes.

[0025] In some embodiments, the vector of the present invention comprises a plasmid (expression plasmid, cloning vector, minicircle, microvector, double minichromosome), a lentiviral vector, an adenoviral vector or a retroviral vector.

[0026] Preferably, the lentiviral vector comprises a recombinant lentiviral vector of primates, namely a recombinant human immunodeficiency virus vector (human immunodeficiency virus, HIV) or a recombinant simian immunodeficiency virus vector (simian immunodeficiency virus, SIV).

[0027] Preferably, the lentiviral vector comprises a non-primate recombinant lentiviral vector, namely, a recombinant equine infectious anemia virus (EIAV), a recombinant feline immunodeficiency virus (FIV) or a recombinant caprine arthritis-encephalitis virus (CAEV).

[0028] Preferably, the vector further comprises one or more regulatory elements.

[0029] Preferably, the regulatory elements include a promoter, an enhancer, a ribosome binding site for translation initiation, a terminator, a polyadenylation sequence, and a selection marker gene.

[0030] Preferably, the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter or any combination thereof.

[0031] In some embodiments, the host cell of the present invention comprises one or more of Escherichia coli, Streptomyces, Agrobacterium, yeast cells, plant cells, animal cells or viruses.

[0032] The third aspect of the present invention provides a polypeptide derivative.

[0033] Furthermore, the polypeptide derivative is a complex obtained by modifying the polypeptide described in the first aspect of the present invention; the modification is modification by conjugation with a detectable marker or modification by conjugation with an imaging agent.

[0034] Preferably, the detectable marker comprises one or more of a fluorescent dye, a fluorescent molecule, a chemiluminescent marker, a dye molecule, a phosphorescent molecule, biotin, a radioactive isotope, a molecule that absorbs in the UV spectrum, a molecule that absorbs in the near infrared radiation, or a molecule that absorbs in the far infrared radiation.

[0035] Preferably, the fluorescent dye includes but is not limited to rhodamine, rhodamine, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, and thiorhodamine, cyanine, indolecarbocyanine, oxacarbocyanine, thiocarbocyanine, merocyanine, cyanine dyes (e.g., cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7), oxadiazole derivatives, pyridyloxazole, nitro Benzodiazole, benzonitrobenzene, pyrene derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavine, acridine orange, acridine yellow, arylmethine derivatives, auramine, thioxanthene dyes, sulfonated thioxanthene dyes, Alexa Fluor (e.g., Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 700), crystal violet, malachite green, tetrapyrrole derivatives, porphyrin, phthalocyanine, bilirubin, Cy5.5, indocyanine green (ICG), DyLight750 or IRdye800.

[0036] Preferably, the fluorescent molecule includes but is not limited to FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, TexasRed or LCRED460.

[0037] Preferably, the chemiluminescent marker includes but is not limited to peroxidase, alkaline phosphatase, luciferase, aequorin, functionalized iron-porphyrin derivatives, luminal, luminol, isoluminol, acridinium ester, sulfonamide and the like.

[0038] Preferably, the luciferase includes, but is not limited to, Gaussia luciferase, Renilla luciferase, dinoflagellate luciferase, firefly luciferase, fungal luciferase, bacterial luciferase and vargula luciferase.

[0039] A fourth aspect of the present invention provides a pharmaceutical composition for treating cataracts.

[0040] Furthermore, the pharmaceutical composition comprises the polypeptide or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention.

[0041] Preferably, the pharmaceutical composition can be a tablet (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled release preparations (e.g., instant release preparations, sustained release preparations, sustained release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosa-adhesive films), injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), intravenous drips, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), eye drops, etc., oral or parenteral preparations (e.g., intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, intrarectal, etc., administration to the vicinity of the lesion and direct administration to the lesion).

[0042] Preferably, the pharmaceutical composition may further include a pharmaceutically acceptable carrier and / or excipient.

[0043] Furthermore, the pharmaceutically acceptable carriers and / or excipients are described in detail in Remington's Pharmaceutical Sciences.

[0044] A fifth aspect of the present invention provides any one of the following methods.

[0045] Further, the method comprises:

[0046] 1) A method for preparing the polypeptide according to the first aspect of the present invention, the method comprising the following steps: culturing the host cell according to the second aspect of the present invention to obtain a culture product, and isolating and purifying the polypeptide according to the first aspect of the present invention from the culture product;

[0047] 2) A method for screening the polypeptide described in the first aspect of the present invention, the method comprising the following steps: using γD-crystallin P23T mutant protein as a target, and screening out the polypeptide described in the first aspect of the present invention by truncating a polypeptide with a length of 20 amino acids on αB-crystallin.

[0048] The sixth aspect of the present invention provides an in vitro method for inhibiting the pathological aggregation of γD-crystallin P23T mutant protein for non-therapeutic purposes.

[0049] Furthermore, the method comprises mixing and incubating the polypeptide described in the first aspect of the present invention with the γD-crystallin P23T mutant protein.

[0050] Furthermore, the concentration ratio of the polypeptide to the γD-crystallin P23T mutant protein is 12.5:1-20:1.

[0051] The seventh aspect of the present invention provides an in vitro non-therapeutic method for reversing the pathological aggregation of γD-crystallin P23T mutant protein.

[0052] Furthermore, the method comprises: firstly incubating the γD-crystallin P23T mutant protein to its aggregation plateau phase, and then mixing and incubating the polypeptide described in the first aspect of the present invention with the γD-crystallin P23T mutant protein.

[0053] Furthermore, the time for incubating the γD-crystallin P23T mutant protein to reach its aggregation plateau phase is 3 hours.

[0054] Furthermore, the concentration ratio of the polypeptide to the γD-crystallin P23T mutant protein is 12.5:1-20:1.

[0055] An eighth aspect of the present invention provides any one of the following applications.

[0056] Furthermore, the application includes:

[0057] 1) Use of the polypeptide according to the first aspect of the present invention in the preparation of polypeptide derivatives, nucleic acid molecules and vectors;

[0058] 2) Use of the polypeptide described in the first aspect, the biomaterial described in the second aspect, or the polypeptide derivative described in the third aspect of the present invention in the preparation of a product for inhibiting γD-crystallin aggregation;

[0059] 3) Use of the polypeptide described in the first aspect, the biomaterial described in the second aspect, and the polypeptide derivative described in the third aspect of the present invention in the preparation of a product for reversing γD-crystallin aggregation;

[0060] 4) Use of the polypeptide described in the first aspect, the biomaterial described in the second aspect, and the polypeptide derivative described in the third aspect of the present invention in the preparation of a product for treating cataracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 OD of different concentrations of HγD-P23T and HγD-WT 350 Trend graphs over time;

[0062] Figure 2 The morphological diagram of HγD-P23T with different concentrations changing with time under differential interference microscopy;

[0063] Figure 3 This is the result of HγD-P23T fluorescence bleaching experiment;

[0064] Figure 4 It is the fusion trend diagram of HγD-P23T aggregates changing with time;

[0065] Figure 5Schematic diagram of the polypeptide structure derived from αB-crystallin;

[0066] Figure 6 The result diagram of the inhibition of pathological aggregation of HγD-P23T by peptides derived from αB-crystallin;

[0067] Figure 7 This is a graph showing the ability of AB9 peptides of different concentrations to inhibit pathological aggregation of HγD-P23T at 37°C;

[0068] Figure 8 The OD of HγD-P23T at 37℃ was measured by different concentrations of AB9 peptide. 350 Impact result diagram;

[0069] Fig. 9 This is a graph showing the ability of AB9 at different concentrations to inhibit the pathological aggregation of HγD-P23T at room temperature;

[0070] Fig.10 The graph shows the ability of different concentrations of AB9 to reverse the pathological aggregation of HγD-P23T at 25°C;

[0071] Fig.11 This is a graph showing the results of regulating the pathological aggregation of HγD-P23T under different concentrations of NaCl;

[0072] Fig.12 The results of regulating the pathological aggregation of HγD-P23T by using Tris-HCl buffer at different concentrations;

[0073] Fig.13 The results of regulating the pathological aggregation of HγD-P23T by different concentrations of PB buffer;

[0074] Fig.14 The results of regulating the pathological aggregation of HγD-P23T by different concentrations of 1,6-hexanediol;

[0075] Fig.15 This is the result of regulating the pathological aggregation of HγD-P23T with different concentrations of PEG8000;

[0076] Fig.16 The results of regulating the pathological aggregation of HγD-P23T in solution environments with different pH values;

[0077] Fig.17 This is a graph showing the scattered light intensity results of static light scattering detection of AB9 polypeptide;

[0078] Fig.18 This is the result diagram of the autocorrelation function of the dynamic light scattering detection of AB9 polypeptide;

[0079] Fig.19 This is the result of dynamic light scattering detection of AB9 polypeptide aggregate size;

[0080] Fig. 20 This is a diagram showing the size of AB9 polypeptide detected by infrared spectroscopy;

[0081] Fig.21 This is the result of CCK-8 testing the cytotoxicity of AB9 peptide;

[0082] Fig. 22 This is the result diagram of the affinity between AB9 peptide and target protein detected by BLI;

[0083] Fig.23 This is the HSQC spectrum. DETAILED DESCRIPTION

[0084] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following embodiments are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0085] The experimental materials used in the following examples come from the following sources:

[0086] 1. Chemical reagents: Sodium chloride (NaCl), glucose, calcium chloride (CaCl2), magnesium sulfate (MgSO4) and phosphoric acid (H3PO4) 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 Science 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) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; 1,6-Hexanediol, polyethylene glycol 8000 (PEG8000), cobalt chloride hexahydrate (CoCl2·6H2O), choline chloride, biotin, thiamine, riboflavin, (NH4)6Mo7O24·4H2O were purchased from Shanghai MacLean Biochemical Reagent Co., Ltd.; D2O was purchased from Beijing Jinouxiang Science and Trade Co., Ltd.; 1,1,1,3,3,3-Hexafluoroisopropanol was purchased from China Inokai Technology Co., Ltd.; Guanidine hydrochloride was purchased from ACROS, Belgium; 96-well cell culture plates were purchased from Corning, USA; DMEM, Ham's F-12 cell culture medium, fetal bovine serum (FBS), phosphate buffered saline (PBS), and streptomycin-penicillin (PS, 100×) were purchased from Thermo Fisher Scientific, USA; kanamycin (kana) and ampicillin (amp) were purchased from Tiangen Biotechnology Co., Ltd.; yeast extract and trypsin were purchased from Thermo Fisher Scientific; isopropyl-beta-D-thiogalactoside (IPTG) was purchased from Beijing Quanshijin Company; Escherichia coli BL21 (DE3) P Lyss and BL21 (DE3) were purchased from Beijing Quanshijin Company; sodium dodecyl sulfate (SDS) was purchased from Sigma; ethylenediaminetetraacetic acid disodium (EDTA) was purchased from Sigma; salt, EDTA-2Na) was purchased from Beijing Inokai Technology Co., Ltd.; dithiothreitol (DTT), 2-Morpholinoethanesulfonic Acid (MES), Trometamol (Tris), glutathione and pyridoxal were purchased from Beijing Langbolide Trading Co., Ltd.; Coomassie brilliant blue dye (homemade);Peptide molecules: AB1: Ac-MDIAIHHPWIRRPFFPFHSP-NH2, AB2: Ac-SRLFDQFFGEHLLESDLFPT-NH2, AB3: Ac-STSLSPFYLRPPSFLRAPS-NH2, AB4: Ac-WFDTGLSEMRLEKDRFSVN-NH2, AB5: Ac-LDVKHFSPEELKVKVLGDV-NH2, AB6: Ac-IEVHGK HEERQDEHGFISR-NH2, AB7: Ac-EFHRKYRIPADVDPLTITS-NH2, AB8: Ac-SLSSDGVLTVNGP RKQVSGP-NH2, AB9: Ac-ERTIPITREEKPAVTAAPKK-NH2 were synthesized in Guoping Pharmaceutical Co., Ltd.;

[0087] 2. Consumables and instruments and equipment

[0088] Ni-NTA affinity chromatography column, anion exchange chromatography column, and cation exchange chromatography column were purchased from Si Tuo Fan Co., Ltd.; GST filler was purchased from Beijing Rambo Lead Trading Co., Ltd.; CCK-8 kit (Beijing Rambo Lead Co., Ltd., China) 10kDa concentrator tube was purchased from Millipore; electric thermostatic incubator (XMTD HH.B11-600); eight-well chamber cover glass (Hangzhou Xinyou Biotechnology Co., Ltd.), vertical pressure steam sterilizer (Shanghai Boxun, China); constant temperature bacterial incubator (Shanghai Boxun, China); desktop centrifuge (eppendorf, Centrifuge 5415D); electric thermostatic water tank (SHHW21600); micro-volume UV spectrophotometer (NanoDrop 2000); electronic balance (Sartorius 2000S); optical inverted microscope (XDS-1B); micropipette (eppendorf Researchplus); -80℃ ultra-low temperature freezer (SANYO, MDF-382E); high-speed refrigerated centrifuge (Beckman, Germany); pH meter (Thermo Orion 868); magnetic stirrer (IKARH-KT / C); ultrasonic disruptor; pure 25 protein purification system (superdex 75, GE Healthcare); SDS-PAGE electrophoresis instrument (Bio-Rad); gel imager (Tanon); 2 mL disposable sterile syringe (purchased from Becton Dickinson Medical Devices Co., Ltd.); electrophoresis instrument (Beijing Longfang Technology Co., Ltd., China); polyacrylamide gel electrophoresis precast gel (Beijing Qingke Biotechnology Co., Ltd., China), differential interference contrast microscope (DMI8, Leica, Germany); Fortibio OctetRed96 molecular interaction detection system (ForteBio); laser scanning confocal microscope Leica Stellar Leica Stellaris 5Confocal Microscope (Leica, Germany); shaker (Harbin Donglian Electronic Technology Development Co., Ltd.); small micro-volume oscillator, small vortex oscillator (Axygen, USA); DNA-free and RNAse-free centrifuge tubes 15 mL, 50 mL (Corning, USA); RNAse-free EP tubes (Axygen, USA); pipettes of various ranges (eppendorf, Germany); streptavidin (SA) biosensor (Sartorius, Germany); carbon dioxide cell culture incubator (Thermo, USA); biological safety cabinet (Haier, China); fume hood (Harbin Donglian, China); circulating water multi-purpose vacuum pump (Zhengzhou Great Wall, China); 0.22 μm filter membrane (Merck Millipore, USA); high-speed refrigerated centrifuge (eppendorf, Germany); 4× protein loading buffer (Beijing Rambo Lead, China); Fourier transform infrared spectrometer Nicolet iS50 (Thermo Fisher Scientific); Bruker Avance III 600 nuclear magnetic resonance instrument (Bruker Corporation, Germany); multifunctional microplate reader (BioTek, USA); Malvern Zetasizer Nano dynamic light scattering instrument (Malvern, UK).

[0089] Example 1 Characterization of HγD-P23T and HγD-WT Protein Properties

[0090] 1. Experimental methods

[0091] (1) Construction of vector

[0092] After codon optimization, the DNA encoding the HγD-P23T protein sequence, HγD-WT protein sequence, and HγD-His-P23T protein sequence were cloned into the pET-28a vector plasmid through the NcoI / XhoI double restriction sites to construct three vectors, pET-28a-HγD-P23T, pET-28a-HγD-His-P23T, and pET-28a-HγD-WT.

[0093] After codon optimization, the DNA encoding the HγD-P23T-eGFP protein sequence was cloned into the pGEX-6p-1 vector plasmid through the BamHI / EcoRI double restriction site to construct the pGEX-6p-1-γD-P23T-eGFP vector.

[0094] After codon optimization, the DNA encoding the αB protein sequence was cloned into the pET-28a vector plasmid through the NcoI / XhoI double restriction site to construct the pET-28a-αB vector.

[0095] The sequence of HγD-P23T protein is MGKITLYEDRGFQGRHYECSSDHPNLQPYLSRCNSARVDSGCWMLYEQPNYSGLQYFLRRGDYADHQQWMGLSDSVRSCRLIPHSGSHRIRLYEREDYRGQMI EFTEDCSCLQDRFRFNEIHSLNVLEGSWVLYELSNYRGRQYLLMPGDYRRYQDWGATNARV GSLRRVIDFS (SEQ ID NO:1); the sequence of HγD-WT protein is MGKTLYEDRGFQGRHYECSSD HPNLQPYLSRCNSARVDSGCWMLYEQPNYSGLQYFLRRGDYADHQQWMGLSDSVRSCRLI PHSGSHRIRLYEREDYRGQMIEFTEDCSCLQDRFRFNEIHSLNVLEGSWVLYELSNYRGRQY LLMPGDYRRYQDWGATNARVGSLRRVIDFS (SEQ ID NO:2); the sequence of HγD-His-P23T protein is MKHHHHHHQMGKITLYEDRGFQGRHYECSSDHTNLQPYLSRCNSARVDSGCWMLYEQPNYSGLQYFLRRGDYADHQQWMGLSDSVRSCRLIPHSGSHRIRLYEREDYRGQMIEFTEDCSCLQDRFRFNEIHSLNVLEGSWVLYELSNYRGRQYLLMPGDYRRYQDWGATNARVGSLRRVIDFS (SEQ ID NO:3);The sequence of HγD-P23T-eGFP protein is MGKITLYEDRGFQGRHYECSSDHTNLQ PYLSRCNSARVDSGCWMLYEQPNYSGLQYFLRRGDYADHQQWMGLSDSVRSCRLIPHSGSHRIRLYEREDYRGQMIEFTEDCSCLQDRFRFNEIHSLNVLEGSWVLYELSNYRGRQYLLMPGDYRRYQDWGATNARVGSLRRVIDFSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO: 4); the sequence of αB protein is MDIAIHHPWIRRPFFPFHS PSRLFDQFFGEHLLESDLFPTSTSLSPFYLRPPSFLRAPSWFDTGLSEMRLEKDRFSVNLDVK HFSPEELKVKVLGDVIEVHGKHEERQDEHGFISREFHRKYRIPADVDPLTITSSLSSDGVLTVNGPRKQVSGPERTIPITREEKPAVTAAPKK (SEQ ID NO: 5). ;

[0096] (2) Expression and purification of HγD-P23T, HγD-WT, HγD-eGFP-P23T, HγD-His-P23T and αB recombinant proteins

[0097] Transformation: BL21(DE3)PLyss and BL21(DE3) competent cells and pET-28a-HγD-P23T, pET-28a-HγD-WT, pET-28a-HγD-his-P23T, pET-28a-αB and pGEX-6p-1-HγD-P23T-eGFP recombinant plasmids were thawed on ice; pET-28a-HγD-P23T, pET-28a-HγD-WT, pET-28a-αB and

[0098] 0.5 μL (0.2 μg / μL) of pET-28a-HγD-his-P23T recombinant plasmid was added to BL21(DE3)PLyss competent cells, and pGEX-6p-1-HγD-P23T-eGFP was added to BL21(DE3) competent cells. The cells were incubated on ice for 20-30 min. Heat-shocked in a 42°C water bath for 90 s. The cells were quickly placed on ice and ice-bathed for 2 min. 500 μL of resistance-free LB was added and the cells were cultured in a 37°C constant temperature incubator for 45-60 min. in; Take the bacterial liquid, pET-28a-HγD-P23T, pET-28a-HγD-WT, pET-28a-αB and pET-28a-HγD-His-P23T are inoculated on LB solid medium containing Kanamycin (25 μg / mL), and pGEX-6p-1-HγD-P23T-eGFP is inoculated on LB solid medium containing Ampicillin (50 μg / mL), and cultured in a constant temperature incubator at 37°C overnight.

[0099] Bacterial culture: Using Kanamycin (25 μg / mL) as a selection marker, pick the positive monoclonal BL21 (DE3) Plyss / pET-28a-HγD-P23T, pET-28a-HγD-WT, pET-28a-αB and pET-28a-HγD-His-P23T on the solid culture medium in the previous step and inoculate them into 20 mL of LB liquid culture medium containing Kanamycin resistance, and culture them at 37°C and 210 r / min shaking overnight. The bacterial solution was inoculated into 1 L of LB liquid culture medium containing Kanamycin resistance at a ratio of 1:50. culture medium, and cultured under shaking conditions of 37°C and 210 r / min; using Ampicillin (50 μg / mL) as a selection marker, picking the positive monoclonal pGEX-6p-1-HγD-P23T-eGFP on the solid culture medium in the previous step, inoculating it in 20 mL of LB liquid culture medium containing Ampicillin resistance, and shaking and culturing it at 37°C and 210 r / min overnight on a shaker, inoculating the bacterial liquid in a ratio of 1:50 in 1 L of LB liquid culture medium containing pGEX-6p-1-HγD-P23T-eGFP resistance, and cultured under shaking conditions of 37°C and 210 r / min.

[0100] Induced expression and acquisition of target protein: When the optical density of E. coli at 600nm (OD 600) was 0.6-0.8, IPTG was added at a final concentration of 0.5 mM, and the cells were cultured at 37°C, 210 r / min shaking for 4 h (pET-28a-HγD-P23T, pET-28a-HγD-WT, pET-28a-αB, pET-28a-HγD-His-P23T), and cultured at 16°C, 210 r / min shaking for 16 h (pGEX-6p-1-HγD-P23T-eGFP) to induce the expression of the target protein; when the optical density of Escherichia coli at 600 nm (OD600) was 0.6-0.8 When the expression was complete, the culture medium was divided into 500 ml centrifuge bottles and centrifuged at 3260 rpm for five minutes. The culture medium was resuspended in 250 ml of M9 medium, and vitamins, trace elements, KANA, CaCl2, biotin, MgSO4, NH4Cl and glucose were added thereto. The culture medium was incubated at 37°C and 210 r / min for 1 hour. IPTG with a final concentration of 0.5 mM was added and the culture medium was incubated at 37°C and 210 r / min for 4 hours. The expressed bacterial culture medium was enriched, centrifuged at 6000 g and 4°C for 20 minutes, the supernatant was discarded, and the culture medium was resuspended in Lysis Buffer 20 mM MES (pH 6.0, containing 1 mM EDTA) (pET-28a-HγD-P23T and pET-28a-HγD-WT), and the culture medium was resuspended in Lysis Buffer 50 mM Tris, 150 mM NaCl (pH 7.5, containing 1 mM EDTA)

[0101] (pGEX-6p-1-HγD-P23T-eGFP) was resuspended in Lysis Buffer 20mM Tris-HCl (pH8.5) (pET-28a-αB) was resuspended in Lysis Buffer 10mM Tris-HCl, 0.5mM NaCl, 10mM imidazole (pH 8.0) (pET-28a-HγD-His-P23T) were resuspended and concentrated in a beaker with a volume of about 30 mL, and PMSF was added at a ratio of 1:100 (final concentration of 1 mM) to prevent the target protein from being degraded, and DTT was added at a ratio of 1:1000 (final concentration of 1 mM) (Due to the need for Ni affinity chromatography of pET-28a-HγD-His-P23T, DTT was not added) to prevent the target protein from aggregating; Ultrasonic disruption of bacteria: The beaker was placed on ice for ultrasonication at a power of 25%, a working time of 25 min, an ultrasonic on time of 3 s, and an ultrasonic off time of 9 s; Protein collection: Centrifugation (4°C, 12000 g, 30 min) was performed to remove cell debris and at the same time to break up the nucleic acid released after cell disruption, and the supernatant was collected. The soluble target protein was present in the supernatant.

[0102] Purification of target protein: HγD-P23T and HγD-WT proteins were purified by cation exchange chromatography: balance the cation exchange column, first pass about 2-3 column volumes of the cation exchange column with ddH2O, then pass about 2-3 column volumes of the cation exchange column with the corresponding LysisBuffer, and then start eluting the protein; load the supernatant obtained by centrifugation, pass it through the cation exchange column 2-3 times; wash the column with Lysis Buffer to elute the impurities; wash the column with Lysis Buffer containing 100mM NaCl and 1mM DTT to debind the target protein from the cation exchange column; concentration: place the target protein collected by the above elution into a 10kDa concentrator tube, centrifuge at 4°C, 4000×g for 50min, and add a small amount of DTT during the concentration process to prevent the target protein from aggregating and precipitating.

[0103] HγD-P23T-eGFP protein was purified by GST affinity chromatography: balance the GST affinity chromatography column: first, use ddH2O to pass through the GST affinity chromatography column for about 2-3 column volumes, and then use the corresponding Lysis Buffer to pass through the GST affinity chromatography column for about 2-3 column volumes to start eluting the protein; load the supernatant obtained by centrifugation and pass through the GST affinity chromatography column 2-3 times; wash the column with Lysis Buffer to elute the impurities; wash the column with Lysis Buffer containing 20mM glutathione to debind the target protein from the GST affinity chromatography column; concentrate: place the target protein collected by the above elution into a 10kDa concentration tube, centrifuge at 4°C, 4000×g for 50min, and add a small amount of DTT during the concentration process to prevent the target protein from aggregating and precipitating.

[0104] HγD-His-P23T protein was purified by Ni column affinity chromatography: Balance the Ni affinity column: first, use ddH2O to pass through the Ni affinity column for about 2-3 column volumes, and then use the corresponding Lysis Buffer to pass through the Ni affinity column for about 2-3 column volumes to start eluting the protein; load the supernatant obtained by centrifugation and pass through the Ni affinity column 2-3 times; wash the column with Lysis Buffer to elute the impurities; wash the column with Lysis Buffer containing 80mM imidazole to elute the impurities (non-specific binding proteins can be removed); wash the column with Lysis Buffer containing 250mM imidazole to debind the target protein from the Ni affinity column; concentrate: place the target protein collected by the above elution into a 10kDa concentration tube and centrifuge at 4°C, 4000×g for 50min.

[0105] αB protein was purified by anion exchange chromatography: balance the anion exchange column: first, use ddH2O to pass through the cation exchange column for about 2-3 column volumes, and then use the corresponding Lysis Buffer to pass through the anion exchange column for about 2-3 column volumes to start eluting the protein; load the supernatant obtained by centrifugation and pass through the anion exchange column 2-3 times; wash the column with Lysis Buffer to elute the impurities; wash the column with Lysis Buffer containing 200mM NaCl and 1mM DTT to debind the target protein from the anion exchange column; concentrate: place the target protein collected by the above elution into a 10kDa concentration tube, centrifuge at 4°C, 4000×g for 50min, and add a small amount of DTT during the concentration process to prevent the target protein from aggregating and precipitating.

[0106] pure 25 protein purification: Cleaning the chromatography column: Use 20% ethanol solution to clean the chromatography column, about 8 mL, then place the pump head in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution, (HγD-His-P23T protein uses 20mM sodium phosphate buffer, containing 5mM DTT (pH 6.2)), repeat the above steps and execute, about 24 mL of cleaning; parameters: System flow: 0.5mL / min; Columnposition: 1; Alarm delta column pressure enabled: 3.0; Alarmpre column pressure enabled: 5.0; Loading: First use 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution, (HγD-His-P23T protein uses 20mM sodium phosphate buffer, containing 5mM DTT (pH 6.2) Rinse the sample loop 2-3 times, and then inject about 2mL of the concentrated sample into the sample loop. Arrange the collection tube and select injectvalve: inject; Collection: When the UV 280 mark representing the protein content rises, use the sample automatic collector to collect the target protein, and set the collection volume of each tube to 0.5mL; Clean the chromatography column: After collecting the sample, place the pump head in 1×PBS (pH7.4, containing 1mM EDTA and 5mM DTT) solution (HγD-His-P23T protein uses 20mM sodium phosphate buffer, containing 5mM DTT (pH 6.2)), continue to run until 20mL passes, replace with sterile water for cleaning, run 10ml, and then replace with 20% ethanol solution to run 20mL, and then shut down; All proteins should be extracted immediately after use, because liquid nitrogen freezing will affect their properties, and they can be stored briefly in a 4-degree refrigerator.

[0107] SDS-PAGE identification of the expression product: Sample preparation: whole bacteria sample, supernatant sample after centrifugation, sample after the protein supernatant passes through the chromatography column, Lysis Buffer washed impurities sample, pass-through sample, protein sample collected at the head, peak tip and peak tail of the molecular sieve; Glue preparation: 5% concentrated gel; According to the size of the protein, choose 15% separation gel; Sample preparation: 4×LoadingBuffer and sample mixed at a ratio of 1:3; Sample loading: marker 5μL, other samples 10μL; Staining: Place the protein gel in Coomassie Brilliant Blue dye solution and microwave at medium-high heat for 2min; Decolorization: Place the stained protein gel in tap water and microwave at high heat for 20-40min.

[0108] Determination of HγD-P23T protein concentration: The concentration of γD-P23T was measured using a One-Drop spectrophotometer; in some experiments, 6 M guanidine hydrochloride was used to disassemble the protein, and the protein concentration was measured using a One-Drop spectrophotometer.

[0109] (3) Characterization of HγD-P23T and HγD-WT protein properties: HγD-P23T and HγD-WT were dissolved in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and the concentrations of HγD-P23T were adjusted to 0.03 mM, 0.06 mM, 0.09 mM, 0.12 mM, and 0.15 mM; the concentration of HγD-WT was adjusted to 0.15 mM; 100 μL of HγD-P23T and HγD-WT of different concentrations were added to a 96-well plate, and three replicates were made for each concentration. The plates were incubated at 37°C for 3 h in an ELISA reader, and the OD was measured every five minutes. 350 ; HγD-P23T was incubated in a 37°C incubator, and 5 μL of sample was added to an eight-well chamber coverslip every half an hour and observed using a differential interference contrast microscope. The total incubation time was three hours; origin 2021 was used to process the data, and the resulting images were processed using Photoshop, the same area was captured, and finally Adobe Illustrator 2023 was used to draw the graph.

[0110] (4) HγD-P23T fluorescence photobleaching recovery experiment: HγD-P23T was dissolved in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT), and the concentration of HγD-P23T was adjusted to 0.15mM; eGFP-P23T was added thereto and mixed with HγD-P23T in a certain proportion (1%-3%, 3% was used in this experiment); the protein solution was divided and placed in a 37°C incubator for incubation for 3 hours to allow it to aggregate and reach the plateau phase; the protein solution was shaken and mixed, and an appropriate amount of the solution was added to the eight-well chamber coverslip; a confocal microscope was used to perform a FRAP experiment, and full and partial photobleaching were performed; origin2021 was used to process the data, Photoshop was used to process the resulting images, the same area was captured, and Adobe Illustrator2023 was used to draw the graph.

[0111] (5) HγD-P23T protein fusion experiment: HγD-P23T was dissolved in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) and the concentration of HγD-P23T was adjusted to 0.15 mM; an appropriate amount of the protein solution was placed in a 37°C incubator and incubated for 3 hours to allow it to aggregate and reach the plateau phase; the protein solution was vortexed to mix, and an appropriate amount of the solution was added to the eight-well chamber cover slip; the protein fusion state was observed using a DMI8 inverted microscope; the obtained image was processed using Photoshop, the same area was captured, and finally Adobe Illustrator 2023 was used to draw the image.

[0112] 2. Experimental results

[0113] (1) OD of different concentrations of HγD-P23T and HγD-WT 350 Results: OD values ​​of different concentrations of HγD-P23T and HγD-WT 350 The trend graph over time is as follows Figure 1 As shown. 350 It can be used to reflect the ability of crystallin to form aggregates. A high value means more aggregates are formed. The OD of 0.06mM-0.15mM HγD-P23T 350 The aggregation of 0.03 mM HγD-P23T and 0.15 mM HγD-WT did not occur. The middle graph was taken at the OD value of 3 hours. 350 The numerical value can reflect that the pathological aggregation of HγD-P23T is concentration-dependent, and the t1 / 2 obtained by overfitting can also reflect that the pathological aggregation of HγD-P23T is concentration-dependent.

[0114] (2) Differential interference microscopy results: The results of the morphological changes of HγD-P23T with different concentrations over time under differential interference microscopy are shown in Figure 2. Figure 2 As shown, and the above detection OD 350 The experiments are self-consistent, and the pathological aggregation of HγD-P23T is time- and concentration-dependent.

[0115] (3) Fluorescence bleaching experiment results: FRAP experiments can be used to measure the fluidity of protein droplets. The results of fluorescence bleaching experiments are as follows: Figure 3 As shown, the fluorescence intensity of HγD-P23T still did not recover to the platform after 200 seconds, and the fluorescence intensity did not recover to 100%, indicating that the HγD-P23T protein aggregates have some solid properties, but are not pure droplets (for example, droplets with strong fluidity will recover the fluorescence intensity to 100% within a few seconds to more than ten seconds, and will also reach the platform).

[0116] (4) HγD-P23T protein fusion experiment results: The fusion experiment results are as follows Figure 4 As shown. Fusion experiments and fluorescence photobleaching recovery experiments corroborate each other and can prove whether protein aggregates are liquid. Figure 4 As shown, within a sufficiently long period of 10 minutes, the two aggregates did not fuse, which also shows that the HγD-P23T protein aggregate has partial solid properties.

[0117] Example 2 Screening of αB-crystallin-derived polypeptides that inhibit HγD-P23T aggregation

[0118] 1. Experimental methods

[0119] (1) Design of αB-crystallin derived peptides: The three-dimensional structure of αB-crystallin with the code number PDB No. 3J07 was used for analysis and mapping using PYMOL software. The peptides were cut with a length of about 20 amino acids to ensure that the peptides had the secondary structure of αB-crystallin as much as possible.

[0120] (2) Screening of αB-crystallin-derived peptides that can inhibit HγD-P23T aggregation: HγD-P23T was dissolved in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) to adjust the concentration of HγD-P23T to 0.30mM; the above nine peptides were taken out from the -20°C refrigerator, 100ul of hexafluoroisopropanol was added to completely dissolve them, and they were completely blown dry with nitrogen gas, and the peptides were dissolved in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution with a molar concentration gradient of 10mM; an appropriate amount of peptide was mixed with HγD-P23T in a 1:1 ratio, incubated at 37°C for 3 hours, and observed using a DMI8 inverted microscope; the obtained image was processed with Photoshop, the same area was cut, and finally Adobe Illustrator 2023 was used to draw the image.

[0121] 2. Experimental results

[0122] (1) Design results of αB-crystallin-derived peptides

[0123] like Figure 5 As shown, a total of 9 small molecule peptides were designed, and their peptide names and sequences are shown in Table 1.

[0124] Table 1 Names and sequences of peptides derived from αB-crystallin

[0125]

[0126]

[0127] The sequences of the AB1, AB2, AB3, AB4, AB5, AB6, AB7, AB8, and AB9 polypeptides whose N-termini are not acetylated and whose C-termini are not amidated are MDIAIHHPWIRRPFFPFHSP (SEQ ID NO: 6), SRLFDQFFGEHLLESDLFPT (SEQ ID NO: 7), STSLSPFYLRPPSFLRAPS (SEQ ID NO: 8), WFDTGLSEMRLEKDRFSVN (SEQ ID NO: 9), LDVKHFSPEELKVKVLGDV (SEQ ID NO: 10), IEVHGKHEERQDEHGFISR (SEQ ID NO: 11), EFHRKYRIPADVDPLTITS (SEQ ID NO: 12), SLSSDGVLTVNGPRKQVSGP (SEQ ID NO: 13), and ERTIPITREEKPAVTAAPKK (SEQ ID NO: 14), respectively.

[0128] (2) Filter results

[0129] Filter results such as Figure 6 As shown, both AB6 and AB9 have the ability to inhibit the pathological aggregation of HγD-P23T, among which AB9 has the strongest ability to inhibit the pathological aggregation of HγD-P23T.

[0130] Example 3 In vitro characterization experiment of AB9 polypeptide inhibiting pathological aggregation of HγD-P23T

[0131] 1. Experimental methods

[0132] (1) In vitro characterization experiment of AB9 peptide inhibiting pathological aggregation of HγD-P23T at 37°C: HγD-P23T was dissolved in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT), and the concentration of γD-P23T was adjusted to 0.24mM; AB9 peptide was taken out from the -20°C refrigerator, 100ul of hexafluoroisopropanol was added to completely dissolve it, and it was completely blown dry with nitrogen gas. The peptide molecule (Ac-ERTIPITREEKPAVTAAPKK-NH2) was dissolved in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution, the molar concentration gradient is 10.0mM, 6.0mM, 2.0mM, 1mM; the polypeptide molecule is mixed with HγD-P23T in a certain proportion, the final concentration of HγD-P23T is 0.12mM, and it is placed in an electric constant temperature incubator for incubation at 37°C. Samples are dropped on the crystal plate every half an hour for observation under an optical microscope. The total incubation time is three hours; the obtained image is processed with Photoshop, the same area is intercepted, and finally Adobe Illustrator 2023 is used to draw the image; the polypeptide molecule is mixed with HγD-P23T in a certain proportion, added to a 96-well plate, incubated at 37°C for 3 hours in an enzyme reader, and the OD is measured every five minutes 350； Use origin 2021 to process the data, and finally use Adobe Illustrator 2023 to create the graphics.

[0133] (2) In vitro characterization experiment of 25℃AB9 polypeptide inhibiting pathological aggregation of HγD-P23T: The experiment was the same as method (1) in Example 3, except that the polypeptide molecule was mixed with HγD-P23T in a volume ratio of 1:1, incubated at room temperature (25℃), and then samples were taken every half an hour and dropped onto a crystal plate for observation under an optical microscope. The total incubation time was three hours.

[0134] (3) In vitro characterization experiment of AB9 peptide reversing pathological aggregation of HγD-P23T: HγD-P23T was dissolved in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT), and the concentration of HγD-P23T was adjusted to 0.24mM; the AB9 peptide was taken out of the -20℃ refrigerator, 100ul of hexafluoroisopropanol was added to completely dissolve it, and it was completely blown dry with nitrogen gas. The dissolved peptide molecule (Ac-ERTIPITREEKPAVTAAPKK-NH2) was dissolved in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution with a molar concentration gradient of 5.0 mM, 3.0 mM, and 1.0 mM; 0.24 mM HγD-P23T was incubated in a 25°C incubator until it aggregated to the plateau phase; the polypeptide molecule was mixed with the HγD-P23T that reached the plateau phase in a volume ratio of 1:1, incubated at room temperature, and samples were dropped on a crystal plate every half an hour for observation under an optical microscope. The total incubation time was three hours; the obtained images were processed with Photoshop, and finally Adobe Illustrator 2023 was used to draw the graph.

[0135] 2. Experimental results

[0136] (1) In vitro characterization results of AB9 peptide inhibiting pathological aggregation of HγD-P23T at 37℃

[0137] The ability of different concentrations of AB9 to inhibit pathological aggregation of HγD-P23T at 37°C Figure 7 As shown, OD 350 The results are as follows Figure 8 As shown, OD 350 Results and Figure 7 The light microscopy images are self-consistent, indicating that AB9 has the ability to inhibit the pathological aggregation of HγD-P23T.

[0138] (2) In vitro characterization results of 25℃ AB9 peptide inhibiting pathological aggregation of HγD-P23T

[0139] At room temperature, the ability of AB9 peptide to inhibit pathological aggregation of HγD-P23T was Fig. 9 As shown, 3mM to 5mM AB9 can inhibit the pathological aggregation of HγD-P23T.

[0140] (3) In vitro characterization results of AB9 peptide reversing pathological aggregation of HγD-P23T

[0141] The ability of different concentrations of AB9 to reverse the pathological aggregation of HγD-P23T at 25°C is shown in Figure 2 Fig.10As shown, at 25°C, 3 mM AB9 can reverse the pathological aggregation of HγD-P23T, and 5 mM AB9 can better reverse the pathological aggregation of HγD-P23T.

[0142] Example 4 In vitro characterization experiment of regulating pathological aggregation of HγD-P23T by different solutions

[0143] (1) In vitro characterization of the regulation of pathological aggregation of HγD-P23T by different concentrations of NaCl

[0144] HγD-P23T was dissolved in 20mM PB (pH 7.4, containing 1mM EDTA and 5mM DTT), and the concentration of HγD-P23T was adjusted to 0.06mM, 0.12mM, 0.18mM, 0.24mM, and 0.30mM; 20mM PB (pH 7.4, containing 1mM EDTA and 5mM DTT) containing 400mM, 800mM, 1200mM, 1600mM, 2000mM, 2400mM, 2800mM, and 3200mM NaCl was mixed with HγD-P23T in a volume ratio of 1:1, incubated at 37°C for three hours, and the sample was dropped on a crystal plate for observation under an optical microscope; the obtained image was processed with Photoshop, and finally Adobe Illustrator 2023 was used to draw the figure.

[0145] (2) In vitro characterization experiment of regulating pathological aggregation of HγD-P23T using Tris-HCl buffer at different concentrations

[0146] HγD-P23T was dissolved in 1mM, 5mM, 10mM, 15mM, 20mM Tris-HCl (pH 7.4, containing 1mM EDTA and 5mM DTT) buffer, and the concentration was adjusted to make HγD-P23T concentration 0.03mM, 0.06mM, 0.09mM, 0.12mM, 0.15mM; incubated at 37°C for three hours, sampled and dropped on a crystal plate for observation under an optical microscope; the obtained image was processed with PhotoShop, and finally drawn using Adobe Illustrator 2023.

[0147] (3) In vitro characterization experiment of regulating pathological aggregation of HγD-P23T using PB buffer at different concentrations

[0148] HγD-P23T was dissolved in 1mM, 5mM, 10mM, 15mM, 20mM PB (pH 7.4, containing 1mM EDTA and 5mM DTT) buffer, and the concentration was adjusted to make HγD-P23T concentration 0.03mM, 0.06mM, 0.09mM, 0.12mM, 0.15mM; incubated at 37°C for three hours, sampled and dropped on a crystal plate, observed under an optical microscope, and the obtained image was processed with Photoshop, and finally drawn using Adobe Illustrator 2023.

[0149] (4) In vitro characterization of different concentrations of 1,6-hexanediol regulating HγD-P23T pathological aggregation

[0150] HγD-P23T was dissolved in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) buffer containing 0%, 5%, and 10% 1,6-hexanediol, and the concentration of HγD-P23T was adjusted to 0.03 mM, 0.06 mM, 0.09 mM, 0.12 mM, and 0.15 mM; an appropriate amount of protein solution was taken and incubated at 37°C for three hours, and a sample was dropped on a crystal plate for observation under an optical microscope; the obtained image was processed with Photoshop, and finally Adobe Illustrator 2023 was used to draw the image.

[0151] (5) In vitro characterization of different concentrations of PEG8000 regulating HγD-P23T pathological aggregation

[0152] HγD-P23T was dissolved in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) containing 0%, 1%, 3%, 10%, and 15% PEG8000, and the concentration was adjusted to make HγD-P23T concentrations of 0.03 mM, 0.04 mM, 0.05 mM, and 0.6 mM; an appropriate amount of protein solution was taken, incubated at 37°C for three hours, and a sample was dropped on a crystal plate for observation under an optical microscope; the obtained image was processed with Photoshop, and finally Adobe Illustrator 2023 was used to draw the image.

[0153] (6) In vitro characterization of pathological aggregation of HγD-P23T regulated by different pH values

[0154] HγD-P23T was dissolved in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) buffer, and the concentration of HγD-P23T was adjusted to 0.10mM, 0.20mM, 0.30mM, 0.40mM, and 0.30mM; Britton–Robinson universal buffer solution was prepared, and its pH value was adjusted to prepare solutions with pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, and 11; Britton–Robinson buffer and HγD-P23T were mixed in a ratio of 4:1 and incubated at room temperature for three hours, and a sample was dropped on a crystal plate for observation under an optical microscope; the obtained image was processed with Photoshop, and finally Adobe Illustrator 2023 was used for drawing.

[0155] 2. Experimental results

[0156] Effects of different concentrations of NaCl on pathological aggregation of HγD-P23T Fig.11 As shown, at 37°C, the pathological aggregation of HγD-P23T was not dependent on the concentration of NaCl.

[0157] Effects of different concentrations of Tris-HCl buffer on pathological aggregation of HγD-P23T Fig.12 As shown, HγD-P23T can still aggregate, and the aggregation behavior is not dependent on the buffer concentration.

[0158] Effects of different concentrations of PB buffer on pathological aggregation of HγD-P23T Fig.13 As shown, by changing the concentration of PB buffer, the results show that the aggregation behavior of the protein is not regulated by the salt ion concentration of PB buffer.

[0159] Effects of different concentrations of 1,6-hexanediol on pathological aggregation of HγD-P23T Fig.14 As shown in the figure, 1,6-hexanediol can affect the pathological aggregation of proteins by interfering with or destroying the hydrophobic interaction in proteins. Therefore, it was found that 5% 1,6-hexanediol can interfere with the pathological aggregation of HγD-P23T at different concentrations. Hydrophobic interaction refers to the phenomenon that non-polar molecules or groups spontaneously aggregate together in aqueous solution due to the repulsion of water molecules. Hydrophobic interaction is one of the main forces driving biological processes such as protein folding and cell membrane formation.

[0160] PEG8000 refers to PEG with a molecular weight of about 8000. PEG is often used to study protein folding, aggregation and stability because it can regulate protein interactions by changing the volume exclusion effect and viscosity of the solution. PEG8000 is an impermeable polymer compound that occupies volume in the solution, resulting in a reduction in the available volume of the aqueous solution. The effects of different concentrations of PEG8000 on the pathological aggregation of HγD-P23T are shown in Figure 2. Fig.15 As shown in the figure, with the increase of PEG8000 concentration, the effective concentration between HγD-P23T proteins increased, which promoted the pathological aggregation of HγD-P23T. As can be seen from the figure, after adding 1% PEG8000 to the low concentration of 0.03mM HγD-P23T, partial aggregation occurred. With the increase of PEG8000 concentration, the aggregation trend became more obvious.

[0161] The surface charge of a protein depends on the pH value of the solution. When the pH value of the solution is far away from the isoelectric point of the protein, the protein surface will carry more net charge (positive or negative). Electrostatic repulsion usually occurs between charged proteins, thus inhibiting aggregation. On the contrary, when the pH value of the solution is close to the isoelectric point of the protein, the protein surface charge is less, the electrostatic repulsion is weakened, and the hydrophobic interaction may dominate, which often promotes aggregation; and the change in pH value may affect the secondary and tertiary structure of the protein, causing the protein to partially or completely unfold. Under extreme pH conditions (overly acidic or overly alkaline), the protein may unfold, which may expose more hydrophobic areas and increase the possibility of aggregation. Fig.16 As shown, the protein aggregated near the isoelectric point of HγD-P23T.

[0162] Example 5 Characterization of AB9 properties

[0163] 1. Experimental methods

[0164] (1) Static light scattering detection of AB9 aggregates: Take out the AB9 peptide from the -20℃ refrigerator, add 100uL hexafluoroisopropanol to completely dissolve it, blow it completely dry with nitrogen, dissolve the peptide molecules in 1×PBS (pH7.4, containing 1mM EDTA and 5mM DTT) solution, and the molar concentration gradient is 10mM, 5.0mM, 3.0mM, 1.0mM, 0.5mM; use static light scattering to detect AB9; use origin 2021 to process the data, and finally use Adobe Illustrator 2023 to draw the graph.

[0165] (2) Static light scattering detection of AB9 aggregate size: Take out the AB9 polypeptide from the -20℃ refrigerator, add 100uL hexafluoroisopropanol to completely dissolve it, blow it completely dry with nitrogen, dissolve the polypeptide molecules in 1×PBS (pH7.4, containing 1mM EDTA and 5mM DTT) solution, and the molar concentration gradient is 10Mm, 5.0mM, 3.0mM, 1.0mM, 0.5mM; use dynamic light scattering to detect AB9; use origin 2021 to process the data, and finally use Adobe Illustrator 2023 to draw the graph.

[0166] (3) Detection of AB9 secondary structure by infrared spectroscopy: Take out the AB9 peptide from the -20℃ refrigerator, add 100uL hexafluoroisopropanol to completely dissolve it, blow it completely dry with nitrogen, dissolve the peptide molecules in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution, and the molar concentration gradient is 10Mm, 5.0mM, 3.0mM, 1.0mM; take 10μL of AB9 solution of different concentrations and drop it on the calcium fluoride window and dry overnight; place the sample at the detection position of the instrument and detect AB9 by infrared spectroscopy; use PeakFit to fit and process the data, use origin 2021 to draw the graph, and finally use Adobe Illustrator2023 to organize it.

[0167] (4) CCK8 detection of AB9 toxicity to cells: prepare 100 μL of a suspension of 5000 Hela and HLEB3 cells in a 96-well plate and culture for 24 hours according to experimental requirements; add different concentrations of AB9 stimulation to the culture plate to make the concentrations 1000 μM, 300 μM, 100 μM, 30 μM, 10 μM, 30 μM, and 1 μM, respectively; incubate the culture plate in an incubator for 24 hours; add 10 μL of CKK-8 solution to each well; continue incubating in the cell culture incubator for 1-4 hours; use an ELISA reader to measure the absorbance at 450 nm, and use a wavelength of 630 nm as a reference wavelength for dual-wavelength measurement; use origin 2021 to draw the graph, and finally use Adobe Illustrator 2023 to organize it.

[0168] (5) BLI detection of affinity between AB9 and target protein: Take out the AB9 peptide with biotin from the -20℃ refrigerator, add 100ul hexafluoroisopropanol to completely dissolve it, blow it completely dry with nitrogen, dissolve the peptide molecules in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution, and the molar concentration is 2μM; first activate the streptavidin (SA) biosensor in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) solution; dissolve HγD-P23T, HγD-WT and αB crystallin proteins in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) buffer, and concentrate and dilute the three proteins to appropriate concentrations; the streptavidin (SA) biosensor is first immersed in 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) buffer as the baseline; the streptavidin (SA) biosensor was immersed in a 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) buffer containing the AB9 polypeptide of biotin to bind to the polypeptide; the streptavidin (SA) biosensor was immersed in a 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) buffer to wash away the unbound polypeptide; the streptavidin (SA) biosensor was immersed in a 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) buffer containing the target protein to bind the protein to the polypeptide; the streptavidin (SA) biosensor was immersed in a 1×PBS (pH 7.4, containing 1mM EDTA and 5mM DTT) buffer to dissociate the target protein; the experimental data were analyzed; the graphs were drawn using origin 2021, and finally organized using Adobe Illustrator 2023.

[0169] (6) Detection of the interaction site between AB9 and HγD-His-P23T by liquid NMR HSQC spectrum: HγD-His-P23T was dissolved in 20 mM sodium phosphate buffer, 5 mM DTT (pH 6.2), and a buffer containing 5% (v / v) D2O, and the concentration of HγD-P23 was adjusted to 0.12 mM; the AB9 polypeptide was taken out of the -20°C refrigerator, 100 ul of hexafluoroisopropanol was added to completely dissolve it, and it was completely blown dry with nitrogen gas, and the polypeptide molecules were dissolved in 20 mM sodium phosphate buffer, 5 mM DTT (pH 6.2), and 5% (v / v) D2O. The AB9 peptide concentration was adjusted to an appropriate concentration in a buffer solution; the sample was transferred to an NMR tube to ensure that there were no bubbles; the Bruker Avance III 950MHz NMR spectrometer was turned on at 298K; the prepared sample tube was taken out and placed on the sample rack, and the sample was introduced into the spectrometer; the heteronuclear single quantum correlation spectroscopy (HSQC) and NOESY experimental parameters were set to collect data; after the experiment, the data was saved and HSQC and NOESY spectra were analyzed using NMRPipe and NMRView; the signals in the spectra were identified to determine the residues with chemical shift changes.

[0170] 2. Experimental results

[0171] In a static light scattering experiment, when a laser beam passes through a sample solution, particles in the solution (such as protein molecules or aggregates) scatter light. By detecting the intensity of the scattered light, information related to the size of the aggregates can be obtained. The results of static light scattering detection of AB9 scattered light intensity are as follows Fig.17 As shown, it can be found that as the concentration of AB9 increases, its density increases, and kcps changes. After fitting, it can be found in the right figure that there is a linear correlation.

[0172] In a DLS experiment, particles or molecules in the sample move randomly in a Brownian motion in a solution. When the laser beam hits these moving particles, the intensity of the scattered light fluctuates slightly over time. By performing autocorrelation analysis on these fluctuations, the diffusion coefficient of the particles can be obtained, and the diffusion coefficient is converted into a hydrated diameter (Dh) using the Stokes-Einstein equation to obtain the particle size distribution of AB9 particles. It can be found that AB9 forms aggregates with a size of about 100 to 1000nm ( Fig.18 , Fig.19 ).

[0173] The AB9 sample was scanned using an infrared spectrometer to obtain its absorption spectrum in the infrared region, focusing on the amide I band between 1700-1600 cm-1, because this region is closely related to the secondary structure of the protein. The infrared spectrum data was imported into the PeakFit software. PeakFit is a software specifically used for peak fitting and deconvolution. First, the data was baseline corrected to eliminate the influence of background noise and instrument errors on the data. In the amide I band region, Gaussian was used to fit the absorption peak of the infrared spectrum. Through deconvolution, the absorption peaks corresponding to different secondary structures (such as α-helix, β-fold, β-turn and random coil) can be separated. After fitting, the area of ​​each fitted peak is calculated, and these areas are proportional to the corresponding secondary structure content, such as Fig. 20 As shown, the AB9 polypeptide has four secondary structures, and the proportions are similar.

[0174] CCK-8 (Cell Counting Kit-8) is a commonly used cytotoxicity detection method used to evaluate the cytotoxicity of AB9 peptide. CCK-8 reagent is based on the reduction reaction of tetrazolium salt (WST-8) and can quantitatively detect cell survival rate by colorimetry. Fig.21 As shown, 1000 μM caused about 40% cell death in Hela cells and about 25% cell death in HLEB3 cells. The survival rate was dependent on the AB9 concentration.

[0175] BLI detection of AB9 and target protein affinity results are as follows Fig. 22 As shown, AB9 has a strong affinity for HγD-P23T.

[0176] HSQC (Heteronuclear Single Quantum Coherence) spectra are a nuclear magnetic resonance (NMR) technique commonly used to study protein-protein interactions. By observing the chemical shift changes of the cross peaks between the labeled atom nitrogen-15 and the hydrogen atom, it is possible to determine which residues on the protein are involved in the interaction with AB9. Using HSQC spectra to detect the interaction sites between AB9 and HγD-His-P23T, the action sites and structural changes of AB9 on the HγD-His-P23T protein can be accurately identified. The residues on the protein that have been identified to have significant changes in the chemical environment or conformation before and after binding with AB9 are: H16, H23, N25, R60, H66, Q67, G71, L72, S85, H88, E96, H122, Y154, I171, D172 ( Fig.23 ).

[0177] In summary, by changing the solution environment of HγD-P23T, we found that electrostatic interaction and hydrophobic interaction play a key role in the pathological aggregation of HγD-P23T. We successfully found the peptide AB9 that can inhibit and reverse the pathological aggregation of HγD-P23T crystallin in vitro through screening, and detected the affinity of the interaction between the peptide and the protein and the residues on HγD-P23T that may be involved in the interaction. In summary, the peptide AB9 screened in this study has potential clinical medicinal value.

[0178] It should be pointed out that the specific implementation methods are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-mentioned embodiments, and there may be many variations. Those skilled in the art who can obtain the invention without objection based on what is clearly disclosed in the present invention or based on the written description of the document should be considered to be within the scope of protection of this patent.

Claims

1. A polypeptide targeting γD-crystallin P23T mutant protein or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide is selected from any one of the following: 1) the polypeptide shown in SEQ ID NO: 11; 2) the polypeptide shown in SEQ ID NO: 14; 3) a sequence having 75%, 80%, 85%, 90%, 95% or 100% or more homology with the polypeptide shown in SEQ ID NO: 11; 4) A sequence having 75%, 80%, 85%, 90%, 95% or 100% or more homology with the polypeptide shown in SEQ ID NO:

14.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The N-terminus of the polypeptide is modified by acetylation and / or the C-terminus is modified by amidation; The polypeptide acts on the H16, H23, N25, R60, H66, Q67, G71, L72, S85, H88, E96, H122, Y154, I171 and D172 amino acid residues of the γD-crystallin P23T mutant protein.

3. A biomaterial, characterized in that: The biological material comprises: 1) A nucleic acid molecule encoding the polypeptide according to claim 1; 2) A vector comprising the nucleic acid molecule described in 1); 3) A host cell comprising the nucleic acid molecule described in 1) or the vector described in 2).

4. A polypeptide derivative, characterized in that: The polypeptide derivative is a complex obtained by modifying the polypeptide according to any one of claims 1 to 2; the modification is modification by conjugation with a detectable marker or modification by conjugation with an imaging agent.

5. A pharmaceutical composition for treating cataracts, characterized in that: The pharmaceutical composition comprises the polypeptide according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof.

6. Any of the following methods, comprising: 1) A method for preparing the polypeptide of claim 1, characterized in that the method comprises the following steps: culturing the host cell of claim 3 to obtain a culture product, and isolating and purifying the polypeptide of claim 1 from the culture product; 2) A method for screening the polypeptide according to claim 1, characterized in that the method comprises the following steps: using γD-crystallin P23T mutant protein as a target, and screening out the polypeptide according to claim 1 by cutting a polypeptide with a length of 20 amino acids on αB-crystallin.

7. A method for inhibiting pathological aggregation of γD-crystallin P23T mutant protein for non-therapeutic purposes in vitro, characterized in that: The method comprises incubating the polypeptide according to claim 1 with the γD-crystallin P23T mutant protein.

8. A method for reversing pathological aggregation of γD-crystallin P23T mutant protein for non-therapeutic purposes in vitro, characterized in that: The method comprises: firstly incubating the γD-crystallin P23T mutant protein to its aggregation plateau phase, and then mixing and incubating the polypeptide according to claim 1 with the γD-crystallin P23T mutant protein; The time for incubating the γD-crystallin P23T mutant protein until its aggregation plateau is 3 hours.

9. The method according to claim 7 or claim 8, characterized in that: The concentration ratio of the polypeptide to the γD-crystallin P23T mutant protein is 12.5:1-20:

1.

10. Any of the following applications, characterized in that: The applications include: 1) Use of the polypeptide according to claim 1 in the preparation of polypeptide derivatives, nucleic acid molecules, and vectors; 2) Use of the polypeptide according to any one of claims 1 to 2, the biomaterial according to claim 3, or the polypeptide derivative according to claim 4 in the preparation of a product for inhibiting γD-crystallin aggregation; 3) Use of the polypeptide according to any one of claims 1 to 2, the biomaterial according to claim 3, or the polypeptide derivative according to claim 4 in the preparation of a product for reversing γD-crystallin aggregation; 4) Use of the polypeptide according to any one of claims 1 to 2, the biomaterial according to claim 3, or the polypeptide derivative according to claim 4 in the preparation of a product for treating cataracts.

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