A polypeptide that inhibits and reverses the pathological aggregation of human lens protein γD

Abstract

The present invention discloses a polypeptide that inhibits and reverses the pathological aggregation of human lens protein γD. By truncating αB-crystallin into small peptides, the present invention detects their ability to inhibit and reverse the pathological aggregation of crystallin, thereby screening out the optimal polypeptide targeting the γD-crystallin P23T mutant protein. The polypeptide screened out by the present invention can significantly inhibit the aggregation of the γD-crystallin P23T mutant protein and can reverse the aggregation of the γD-crystallin P23T mutant protein, indicating that the polypeptide provided by the present invention has the potential to treat cataracts and has broad application prospects in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a polypeptide that inhibits and reverses the pathological aggregation of human lens protein γD, and the polypeptide targets the γD-lens P23T mutant protein. Background Art

[0002] The pathological aggregation of lens proteins is closely related to various eye diseases, including but not limited to senile cataract, congenital cataract, secondary cataract, traumatic cataract, 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 yet. Since the abnormal aggregation of lens proteins is one of the key mechanisms for cataract formation, it is of great significance to develop drugs targeting the pathological aggregation of lens proteins. Currently, drugs for the treatment of cataracts include hormonal drugs, antioxidants, lanosterol, etc., but some drugs can only be used to delay the progression of cataracts, and the treatment effect is limited. Currently, there is no targeted drug for the P23T point mutation of γD-lens protein.

[0003] Regarding the research with lens proteins as the therapeutic target, there have been literature reports that lanosterol plays a key role in inhibiting the aggregation of lens proteins and reducing cataract formation. However, lanosterol has problems in drug delivery, and its stability and bioavailability in vivo are low, making it difficult to reach the ideal therapeutic concentration in the lens, thus unable to cure related cataract diseases. Currently, there are studies attempting to optimize the chemical structure of lanosterol, but due to the high raw material cost and expensive modification cost, subsequent drug development has encountered bottlenecks. Summary of the Invention

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

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

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

[0007] Furthermore, 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 more than 100% homology with the polypeptide shown in SEQ ID NO: 11;

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

[0012] Furthermore, the N-terminus of the polypeptide is acetylated and / or the C-terminus is amidated.

[0013] Furthermore, 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 a compound having amino acid residues covalently linked by: a peptide bond.

[0015] 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, 6) amino acid residues to the polypeptide shown in SEQ ID NO: 11 or SEQ ID NO: 14 and having the 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, sequence alignment can be performed, which can be carried out 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 the appropriate parameters for alignment, including any algorithms required to achieve optimal alignment in the full-length sequences being compared.

[0017] In the present invention, the polypeptide sequences obtained by modification also fall within 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 with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase in one or more amino acids compared to the naturally occurring molecule.

[0018] The second aspect of the present invention provides a biological material.

[0019] Furthermore, the biomaterial includes:

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

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

[0022] 3) A host cell containing 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 and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. The polynucleotide can 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 such that when placed within 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; 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 (such as heteropolylysine, defined-length oligopeptides, and polyethyleneimine), and in some cases also contained within liposomes.

[0025] In some embodiments, the vector described in the present invention includes plasmids (expression plasmids, cloning vectors, minicircles, microcarriers, dicentric minichromosomes), lentiviral vectors, adenoviral vectors, or retroviral vectors.

[0026] Preferably, the lentiviral vector includes a recombinant lentiviral vector of primates, i.e., 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 includes a recombinant lentiviral vector of non - primates, namely recombinant equine infectious anemia virus (EIAV), recombinant feline immunodeficiency virus (FIV), or recombinant caprine arthritis - encephalitis virus (CAEV).

[0028] Preferably, the vector further includes 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 cells of the present invention include 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 conjugation modification with a detectable label or a imaging agent.

[0034] Preferably, the detectable label includes one or more of a fluorescent dye, a fluorescent molecule, a chemiluminescent marker, a dye molecule, a phosphorescent molecule, biotin, a radioisotope, 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 dyes include, but are not limited to, rhodamine, p-methylaminophenol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfo-fluorescein, aminop-methylaminophenol, carboxyp-methylaminophenol, chlorop-methylaminophenol, methylp-methylaminophenol, sulfop-methylaminophenol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, and thiorhodamine, cyanine, indocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine dyes (such as cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7), oxadiazole derivatives, pyridyl oxazole, nitrobenzoxadiazole, 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 (such as 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), DyLight 750 or IRdye 800.

[0036] Preferably, the fluorescent molecules include, but are not limited to, FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LCRED460.

[0037] Preferably, the chemiluminescent markers include, but are not limited to, peroxidase, alkaline phosphatase, luciferase, aequorin, functionalized iron-porphyrin derivatives, luminol, luminol, isoluminol, acridinium ester, sulfonamide, etc.

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

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

[0040] Furthermore, the pharmaceutical composition includes the polypeptide described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0041] Preferably, the pharmaceutical composition may be tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, oral tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled-release preparations (e.g., immediate-release preparations, sustained-release preparations, sustained-release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosa-adhesive films), injections (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous drip agents, 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, intra-organ, intranasal, intradermal, drip, intracerebral, intrarectal, etc. administration forms, administration near the lesion and direct administration to the lesion).

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

[0043] Furthermore, the pharmaceutically acceptable carrier and / or excipient is described in detail in Remington's Pharmaceutical Sciences.

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

[0045] Furthermore, the method comprises:

[0046] 1) A method for preparing the polypeptide described in the first aspect of the present invention, the method comprising the following steps: culturing the host cell 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;

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

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

[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 a method for reversing the pathological aggregation of γD-crystallin P23T mutant protein for non-therapeutic purposes in vitro.

[0052] Further, the method includes: first incubating the γD-crystallin P23T mutant protein until 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] Further, the time for incubating the γD-crystallin P23T mutant protein until its aggregation plateau phase is 3 h.

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

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

[0056] Further, the application includes:

[0057] 1) The application of the polypeptide described in the first aspect of the present invention in the preparation of polypeptide derivatives, nucleic acid molecules, and vectors;

[0058] 2) The application of the polypeptide described in the first aspect of the present invention, the biological material described in the second aspect, and the polypeptide derivative described in the third aspect in the preparation of products for inhibiting the aggregation of γD-crystallin;

[0059] 3) The application of the polypeptide described in the first aspect of the present invention, the biological material described in the second aspect, and the polypeptide derivative described in the third aspect in the preparation of products for reversing the aggregation of γD-crystallin;

[0060] 4) The application of the polypeptide described in the first aspect of the present invention, the biological material described in the second aspect, and the polypeptide derivative described in the third aspect in the preparation of products for treating cataracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 is a trend graph of the OD of different concentrations of HγD-P23T and HγD-WT changing with time; 350 changing with time;

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

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

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

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

[0066] Figure 6 Result diagram of the polypeptide derived from αB-crystallin inhibiting the pathological aggregation of HγD-P23T;

[0067] Figure 7 Diagram of the ability of different concentrations of AB9 polypeptide to inhibit the pathological aggregation of HγD-P23T at 37°C;

[0068] Figure 8 For the effect of different concentrations of AB9 polypeptide on HγD-P23T OD 350 Result diagram of the effect;

[0069] Figure 9 Diagram of the ability of different concentrations of AB9 to inhibit the pathological aggregation of HγD-P23T at room temperature;

[0070] Figure 10 Diagram of the ability of different concentrations of AB9 to reverse the pathological aggregation of HγD-P23T at 25°C;

[0071] Figure 11 Result diagram of different concentrations of NaCl regulating the pathological aggregation of HγD-P23T;

[0072] Figure 12 Result diagram of different concentrations of Tris-HCl buffer regulating the pathological aggregation of HγD-P23T;

[0073] Figure 13 Result diagram of different concentrations of PB buffer regulating the pathological aggregation of HγD-P23T;

[0074] Figure 14 Result diagram of different concentrations of 1,6-hexanediol regulating the pathological aggregation of HγD-P23T;

[0075] Figure 15 Result diagram of different concentrations of PEG8000 regulating the pathological aggregation of HγD-P23T;

[0076] Figure 16 Result diagram of solution environments with different pH values regulating the pathological aggregation of HγD-P23T;

[0077] Figure 17 Result diagram of the scattered light intensity of AB9 polypeptide detected by static light scattering;

[0078] Figure 18 Result diagram of the autocorrelation function of AB9 polypeptide detected by dynamic light scattering;

[0079] Figure 19 Result diagram of the aggregate size of AB9 polypeptide detected by dynamic light scattering;

[0080] Figure 20 It is a size result diagram of the AB9 polypeptide detected by infrared spectroscopy;

[0081] Figure 21 It is a result diagram of the cytotoxicity of the AB9 polypeptide detected by CCK-8;

[0082] Figure 22 It is a result diagram of the affinity of the AB9 polypeptide and the target protein detected by BLI;

[0083] Figure 23 It is an HSQC spectrum. Detailed implementation manners

[0084] The following further elaborates the present invention in conjunction with specific embodiments, which are only used to explain the present invention and cannot be construed as a limitation to the present invention. Those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods without specific conditions noted in the following embodiments are generally implemented according to conventional conditions or conditions recommended by the manufacturers.

[0085] The sources of the experimental materials used in the following embodiments are as follows:

[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; absolute 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), copper sulfate pentahydrate (CuSO4·5H2O) were purchased from Shanghai Aladdin Biochemical Technology 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 Macklin Biochemical Co., Ltd.; D2O was purchased from Beijing Jinouxiang Science & Trade Co., Ltd.; 1,1,1,3,3,3-hexafluoroisopropanol was purchased from China InnoChem Technology Co., Ltd.; guanidine hydrochloride was purchased from ACROS (Belgium); 96-well cell culture plates were all purchased from Corning (USA); DMEM, Ham’s F-12 cell culture medium, fetal bovine serum (FBS), phosphate buffer solution (PBS), streptomycin-penicillin double antibody (PS, 100×) were purchased from Thermo Fisher Scientific (USA); kanamycin (Kanamycin, kana) and ampicillin (Ampicillin, amp) were 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 and BL21(DE3) were purchased from Beijing TransGen Biotech Co., Ltd.; sodium dodecyl sulfate (SDS) was purchased from Sigma; disodium ethylenediaminetetraacetate (Ethylenediaminetetraacetic acid disodium salt, EDTA-2Na) was purchased from Beijing InnoChem Technology Co., Ltd.; dithiothreitol (DTT), 2-morpholinoethanesulfonic acid (MES), tris (hydroxymethyl) aminomethane (Trometamol, Tris), glutathione, and pyridoxal were purchased from Beijing Lamboid Trading Co., Ltd.; Coomassie Brilliant Blue staining solution (self-made);Polypeptide 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 by Guoping Pharmaceutical Co., Ltd.

[0087] 2. Consumables, instruments and equipment

[0088] Ni-NTA affinity chromatography column, anion exchange chromatography column, cation exchange chromatography column were purchased from Cytiva; GST packing material was purchased from Beijing Lamboid Trading Co., Ltd.; CCK-8 kit (Beijing Lamboid Co., Ltd., China), 10 kDa concentrator tube was purchased from Millipore; Electrothermal constant temperature incubator (XMTD HH.B11-600); Eight-well chamber cover glass (Hangzhou Xinyou Biotechnology Co., Ltd.), vertical autoclave (Shanghai Boxun, China); Constant temperature bacterial incubator (Shanghai Boxun, China); Desktop centrifuge (eppendorf, Centrifuge 5415D); Electrothermal constant temperature water bath (SHHW21600); Micro ultraviolet spectrophotometer (NanoDrop 2000); Electronic balance (Sartorius 2000S); Inverted optical microscope (XDS-1B); Micropipette (eppendorf Researchplus); -80 °C ultra-low temperature freezer (SANYO, MDF-382E); High-speed refrigerated centrifuge (Beckman, Germany); pH meter (Thermo Orion 868); Magnetic stirrer (IKARH-KT / C); Ultrasonic crusher Pure 25 protein purification system (Superdex 75, GE Healthcare); SDS-PAGE electrophoresis apparatus (Bio-Rad); Gel imaging apparatus (Tanon); 2 mL disposable sterile syringe (purchased from BD Medical Devices Co., Ltd.); Electrophoresis apparatus (Beijing Longfang Technology Co., Ltd., China); Polyacrylamide gel electrophoresis precast gel (Beijing Tsingke Biotechnology Co., Ltd., China), Differential interference contrast microscope (DMI8, Leica Microsystems GmbH, Germany); Fortibio Octet Red96 molecular interaction detection system (ForteBio Corporation); Laser scanning confocal microscope Leica Stellar Leica Stellaris 5 Confocal Microscope (Leica Microsystems GmbH, Germany); Shaker (Harbin Donglian Electronic Technology Development Co., Ltd.); Small micro oscillator, small vortex oscillator (Axygen, USA); DNA-free and RNA-free centrifuge tubes of 15 mL and 50 mL (Corning, USA); RNA-free EP tubes (Axygen, USA); Pipettes of various ranges (eppendorf, Germany); Streptavidin (SA) biosensor (Sartorius, Germany); Carbon dioxide cell incubator (Thermo, USA); Biological safety cabinet (Haier, China); Fume hood (Harbin Donglian Harbin, 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 Lamboid, China); Fourier transform infrared spectrometer Nicolet iS50 (Thermo Fisher Scientific); Bruker Avance III 600 nuclear magnetic resonance spectrometer (Bruker Corporation, Germany); Multifunctional microplate enzyme labeler (BioTek, USA); Malvern Zetasizer Nano dynamic light scattering instrument (Malvern, UK).

[0089] Example 1 Characterization of the properties of HγD-P23T and HγD-WT proteins

[0090] 1. Experimental method

[0091] (1) Construction of vectors

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

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

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

[0095] The sequence of HγD-P23T protein is MGKITLYEDRGFQGRHYECSSDHPNLQPYLSRCNSARVDSGCWMLYEQPNYSGLQYFLRRGDYADHQQWMGLSDSVRSCRLIPHSGSHRIRLYEREDYRGQMIEFTEDCSCLQDRFRFNEIHSLNVLEGSWVLYELSNYRGRQYLLMPGDYRRYQDWGATNARVGSLRRVIDFS (SEQ ID NO:1); the sequence of HγD-WT protein is MGKTLYEDRGFQGRHYECSSD HPNLQPYLSRCNSARVDSGCWMLYEQPNYSGLQYFLRRGDYADHQQWMGLSDSVRSCRLIPHSGSHRIRLYEREDYRGQMIEFTEDCSCLQDRFRFNEIHSLNVLEGSWVLYELSNYRGRQYLLMPGDYRRYQDWGATNARVGSLRRVIDFS (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 PYLSRCNSARVDSGCWMLYEQPNYSGLQYFLRRGDYADHQQWMGLSDSVRSCRLIPHSGSHRIRLYEREDYRGQMIEFTEDCSCLQDRFRFNEIHSLNVLEGSWVLYELSNYRGRQYLLMPGDYRRYQDWGATNARVGSLRRVIDFSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (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: Thaw the competent cells of BL21(DE3)PLyss and BL21(DE3) and the recombinant plasmids of pET-28a-HγD-P23T, pET-28a-HγD-WT, pET-28a-HγD-his-P23T, pET-28a-αB and pGEX-6p-1-HγD-P23T-eGFP on ice; Take pET-28a-HγD-P23T, pET-28a-HγD-WT, pET-28a-αB and

[0098] 0.5 μL (0.2 μg / μL) of the 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. Incubate on ice for 20 - 30 min; heat shock in a 42°C water bath for 90 s; quickly place on ice and incubate on ice for 2 min; add 500 μL of antibiotic-free LB and culture in a 37°C constant temperature incubator for 45 - 60 min; take the bacterial solution, and inoculate pET-28a-HγD-P23T, pET-28a-HγD-WT, pET-28a-αB, and pET-28a-HγD-His-P23T on LB solid medium containing Kanamycin (25 μg / mL), and inoculate pGEX-6p-1-HγD-P23T-eGFP on LB solid medium containing Ampicillin (50 μg / mL), and culture overnight in a 37°C constant temperature incubator.

[0099] Bacterial culture: Using Kanamycin (25 μg / mL) as the 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 medium in the previous step and inoculate them into 20 mL of LB liquid medium containing Kanamycin resistance respectively, and culture overnight in a shaker at 37°C and 210 r / min. Then inoculate the bacterial solution into 1 L of LB liquid medium containing Kanamycin resistance at a ratio of 1:50 and culture under the conditions of 37°C and 210 r / min oscillation; using Ampicillin (50 μg / mL) as the selection marker, pick the positive monoclonal pGEX-6p-1-HγD-P23T-eGFP on the solid medium in the previous step, inoculate it into 20 mL of LB liquid medium containing Ampicillin resistance, and culture overnight in a shaker at 37°C and 210 r / min. Then inoculate the bacterial solution into 1 L of LB liquid medium containing pGEX-6p-1-HγD-P23T-eGFP resistance at a ratio of 1:50 and culture under the conditions of 37°C and 210 r / min oscillation.

[0100] Induction expression and obtaining of the target protein: When the optical density (OD 600) When it is 0.6 - 0.8, add IPTG with a final concentration of 0.5 mM, and culture for 4 h under the conditions of shaking at 37°C and 210 r / min (pET-28a-HγD-P23T, pET-28a-HγD-WT, pET-28a-αB, pET-28a-HγD-His-P23T), and culture for 16 h under the conditions of shaking at 16°C and 210 r / min (pGEX-6p-1-HγD-P23T-eGFP) to induce the expression of the target protein; when the optical density (OD600) of Escherichia coli at 600 nm is 0.6 - 0.8, aliquot it into 500 ml centrifuge bottles and centrifuge at 3260 rpm for five minutes, resuspend with 250 mL of M9 medium, add vitamins, trace elements, KANA, CaCl2, biotin, MgSO4, NH4Cl and glucose, culture for 1 h under the conditions of shaking at 37°C and 210 r / min, add IPTG with a final concentration of 0.5 mM, and culture for 4 h under the conditions of shaking at 37°C and 210 r / min respectively; enrich the expressed bacterial culture solution, centrifuge at 6000 g and 4°C for 20 min, discard the supernatant, resuspend with buffer LysisBuffer 20 mM MES (pH 6.0, containing 1 mM EDTA) (pET-28a-HγD-P23T and pET-28a-HγD-WT), and resuspend with buffer Lysis Buffer 50 mM Tris, 150 mM NaCl (pH 7.5, containing 1 mM EDTA)

[0101] (pGEX-6p-1-HγD-P23T-eGFP), resuspend with buffer Lysis Buffer 20 mM Tris-HCl (pH8.5) (pET-28a-αB), resuspend with buffer Lysis Buffer 10 mM Tris-HCl, 0.5 mM NaCl, 10 mM imidazole (pH 8.0) (pET-28a-HγD-His-P23T), respectively concentrate them in a beaker with a volume of about 30 mL, add PMSF (final concentration of 1 mM) at a ratio of 1:100 to prevent the degradation of the target protein, add DTT (final concentration of 1 mM) at a ratio of 1:1000 (since pET-28a-HγD-His-P23T requires Ni affinity chromatography, DTT is not added), to prevent the aggregation of the target protein; ultrasonically disrupt the bacteria: place the beaker on ice for ultrasonic treatment, with 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; collect the protein: centrifuge (at 4°C, 12000 g, 30 min) to remove cell debris, and at the same time break up the nucleic acids released after cell disruption, collect the supernatant, and the soluble target protein exists in the supernatant.

[0102] Purification of the target protein: HγD-P23T and HγD-WT proteins were purified by cation exchange chromatography: Equilibrate the cation exchange column. First, pass ddH2O through the cation exchange column for about 2 - 3 column volumes, and then pass the corresponding Lysis Buffer through the cation exchange column for about 2 - 3 column volumes to start eluting the protein; Load the supernatant obtained by centrifugation, and pass it through the cation exchange column 2 - 3 times; Wash the column with Lysis Buffer to elute the impurity proteins; Wash the column with Lysis Buffer containing 100 mM NaCl and 1 mM DTT to dissociate the target protein from the cation exchange column; Concentration: Place the target protein eluted and collected above into a 10 kDa concentrator tube, centrifuge at 4°C and 4000×g for 50 min. Add a small amount of DTT during the concentration process to prevent the aggregation and precipitation of the target protein.

[0103] The HγD-P23T-eGFP protein was purified by GST affinity chromatography: Equilibrate the GST affinity column: First, pass ddH2O through the GST affinity column for about 2 - 3 column volumes, and then pass the corresponding Lysis Buffer through the GST affinity column for about 2 - 3 column volumes to start eluting the protein; Load the supernatant obtained by centrifugation, and pass it through the GST affinity column 2 - 3 times; Wash the column with Lysis Buffer to elute the impurity proteins; Wash the column with Lysis Buffer containing 20 mM glutathione to dissociate the target protein from the GST affinity column; Concentration: Place the target protein eluted and collected above into a 10 kDa concentrator tube, centrifuge at 4°C and 4000×g for 50 min. Add a small amount of DTT during the concentration process to prevent the aggregation and precipitation of the target protein.

[0104] The HγD-His-P23T protein was purified by Ni column affinity chromatography: Equilibrate the Ni affinity column: First, pass ddH2O through the Ni affinity column for about 2 - 3 column volumes, and then pass the corresponding Lysis Buffer through the Ni affinity column for about 2 - 3 column volumes to start eluting the protein; Load the supernatant obtained by centrifugation, and pass it through the Ni affinity column 2 - 3 times; Wash the column with Lysis Buffer to elute the impurity proteins; Wash the column with Lysis Buffer containing 80 mM imidazole to elute the impurity proteins (which can remove non-specifically bound proteins); Wash the column with Lysis Buffer containing 250 mM imidazole to dissociate the target protein from the Ni affinity column; Concentration: Place the target protein eluted and collected above into a 10 kDa concentrator tube, centrifuge at 4°C and 4000×g for 50 min.

[0105] Purification of αB protein by anion exchange chromatography: Balancing the anion exchange column: First, pass ddH2O through the cation exchange column at a volume of about 2 - 3 column volumes, and then pass the corresponding Lysis Buffer through the anion exchange column at a volume of about 2 - 3 column volumes to start eluting the protein; load the supernatant obtained by centrifugation, and pass it through the anion exchange column 2 - 3 times; wash the column with Lysis Buffer to elute the miscellaneous proteins; wash the column with Lysis Buffer containing 200 mM NaCl and 1 mM DTT to dissociate the target protein from the anion exchange column; Concentration: Place the target protein eluted and collected above into a 10 kDa concentrator tube, centrifuge at 4°C and 4000×g for 50 min, and add a small amount of DTT during the concentration process to prevent the aggregation and precipitation of the target protein.

[0106] Purification of pure 25 protein: Cleaning the chromatography column: Clean the chromatography column with 20% ethanol solution, about 8 mL, and then place the pump head in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution, (for HγD-His-P23T protein, use 20 mM sodium phosphate buffer containing 5 mM DTT (pH 6.2)), repeat the above steps and perform, about 24 mL; Parameters: System flow: 0.5 mL / min; Column position: 1; Alarm delta column pressure enabled: 3.0; Alarm pre column pressure enabled: 5.0; Loading: First, rinse the sample loop 2 - 3 times with 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution, (for HγD-His-P23T protein, use 20 mM sodium phosphate buffer containing 5 mM DTT (pH 6.2)), and then inject about 2 mL of the concentrated sample into the sample loop. Arrange the collection tubes, and select inject valve: inject; Collection: When the UV 280 identification line representing the protein content rises, collect the target protein using an automatic sample collector, and set the collection volume per tube to 0.5 mL; Cleaning the chromatography column: After collecting the sample, place the pump head in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution, (for HγD-His-P23T protein, use 20 mM sodium phosphate buffer containing 5 mM DTT (pH 6.2)), continue to run until 20 mL passes through, replace it with sterile water for cleaning, run 10 mL, and then replace it with 20% ethanol solution and run 20 mL to shut down; All proteins are extracted immediately before use because liquid nitrogen freezing will affect their properties, and they can be stored briefly in a 4°C refrigerator.

[0107] SDS-PAGE Identification of the Expression Product: Sample Preparation: Whole cell samples, supernatant samples after centrifugation, samples after the protein supernatant passed through the chromatography column, samples washed with Lysis Buffer, samples passed through, protein samples collected at the peak head, peak tip, and peak tail in the molecular sieve; Gel Preparation: 5% stacking gel; Select 15% separating gel according to the size of the protein; Sample Preparation: Mix 4×Loading Buffer with the sample at a ratio of 1:3; Loading: 5 μL of marker, 10 μL of the remaining samples; Staining: Place the protein gel in Coomassie Brilliant Blue staining solution and microwave at high power for 2 min; Decoloring: Place the stained protein gel in tap water and microwave at high power for 20 - 40 min.

[0108] Determination of HγD-P23T Protein Concentration: Use a One-Drop spectrophotometer to measure the concentration of γD-P23T; In some experiments, 6 M guanidine hydrochloride was used to disassemble the protein, and a One-Drop spectrophotometer was used to measure the protein concentration.

[0109] (3) Characterization of the Protein Properties of HγD-P23T and HγD-WT: Dissolve HγD-P23T and HγD-WT in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT), adjust the concentration of HγD-P23T to 0.03 mM, 0.06 mM, 0.09 mM, 0.12 mM, 0.15 mM; adjust the concentration of HγD-WT to 0.15 mM; Take 100 μL of different concentrations of HγD-P23T and HγD-WT and add them to a 96-well plate. Each concentration is repeated three times. Incubate at 37°C in an enzyme-linked immunosorbent assay (ELISA) reader for 3 hours, and measure the OD every five minutes. 350 ; Incubate HγD-P23T in a 37°C incubator. Take 5 μL of the sample every half hour and add it to an eight-well chamber cover glass and observe it using differential interference contrast microscopy. The total incubation time is 3 hours; Use Origin 2021 to process the data, use Photoshop to process the obtained images, intercept the same area, and finally use Adobe Illustrator 2023 to create the graph.

[0110] (4) HγD-P23T Fluorescence Recovery After Photobleaching (FRAP) Experiment: Dissolve HγD-P23T in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration of HγD-P23T to 0.15 mM; add eGFP-P23T to it and mix with HγD-P23T at a certain ratio (any ratio from 1% - 3% can be used, and a ratio of 3% is used in this experiment); aliquot the protein solution and incubate it in an incubator at 37°C for 3 hours until aggregation reaches a plateau; shake and mix the protein solution evenly, take an appropriate amount of the solution and add it onto an eight-well chambered cover glass; perform frap experiments using a confocal microscope for full bleaching and partial bleaching; process the data using origin2021, process the obtained images using Photoshop, intercept the same area, and finally create graphs using Adobe Illustrator 2023.

[0111] (5) HγD-P23T Protein Fusion Experiment: Dissolve HγD-P23T in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration of HγD-P23T to 0.15 mM; take an appropriate amount of the protein solution and incubate it in an incubator at 37°C for 3 hours until aggregation reaches a plateau; shake and mix the protein solution evenly, take an appropriate amount of the solution and add it onto an eight-well chambered cover glass; observe the protein fusion state using a DMI8 inverted microscope; process the obtained images using Photoshop, intercept the same area, and finally create graphs using Adobe Illustrator 2023.

[0112] 2. Experimental Results

[0113] (1) OD of HγD-P23T and HγD-WT at Different Concentrations 350 Results: The trend graphs of the OD of HγD-P23T and HγD-WT at different concentrations 350 changing with time are as shown in Figure 1 . OD 350 can be used to reflect the ability of lens proteins to form aggregates. A higher value represents more aggregates formed. Among them, the OD of HγD-P23T at 0.06 mM - 0.15 mM 350 will increase with time, while 0.03 mM of HγD-P23T and 0.15 mM of HγD-WT do not aggregate. The OD 350 values at the three-hour time point in the middle graph can reflect that the pathological aggregation of HγD-P23T is concentration-dependent, and the t1 / 2 obtained through 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 for αB-crystallin-derived polypeptides that can inhibit the aggregation of HγD-P23T: Dissolve HγD-P23T in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration of HγD-P23T to 0.30 mM; Take out the above nine polypeptides from the -20°C refrigerator, add 100 μl of hexafluoroisopropanol to completely dissolve them, and completely dry them with nitrogen. Dissolve the polypeptides in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution with a molar concentration gradient of 10 mM; Take an appropriate amount of polypeptide and mix it with HγD-P23T in a 1:1 ratio, incubate at 37°C for 3 hours, and observe it using a DMI8 inverted microscope; Process the obtained images with Photoshop, intercept the same area, and finally use Adobe Illustrator 2023 to create a graph.

[0121] 2. Experimental results

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

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

[0124] Table 1 Polypeptide names and sequences derived from αB-crystallin

[0125]

[0126]

[0127] The sequences of AB1, AB2, AB3, AB4, AB5, AB6, AB7, AB8, and AB9 polypeptides without N-terminal acetylation modification and C-terminal amidation modification 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) Screening results

[0129] The screening results are as Figure 6 shown. Both AB6 and AB9 have the ability to inhibit the pathological aggregation of HγD-P23T, and among them, AB9 has the strongest ability to inhibit the pathological aggregation of HγD-P23T.

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

[0131] 1. Experimental method

[0132] (1) In vitro characterization experiment of AB9 polypeptide inhibiting the pathological aggregation of HγD-P23T at 37°C: Dissolve HγD-P23T in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration so that the concentration of γD-P23T is 0.24 mM; Take out the AB9 polypeptide from the -20°C refrigerator, add 100 μl of hexafluoroisopropanol to completely dissolve it, and completely dry it with nitrogen. Dissolve the polypeptide molecule (Ac-ERTIPITREEKPAVTAAPKK-NH2) in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution, and the molar concentration gradient is 10.0 mM, 6.0 mM, 2.0 mM, 1 mM; Mix the polypeptide molecule with HγD-P23T in a certain ratio, and the final concentration of HγD-P23T is 0.12 mM. Put it into an electrothermal constant temperature incubator and incubate at 37°C. Take samples every half hour and drop them on a crystal plate for observation with an optical microscope. The total incubation time is three hours; Process the obtained images with Photoshop, intercept the same area, and finally use Adobe Illustrator 2023 to draw graphs; Mix the polypeptide molecule with HγD-P23T in a certain ratio, add it to a 96-well plate, and incubate at 37°C in an enzyme-labeled instrument for 3 hours. Measure the OD every five minutes 350； Use origin 2021 to process the data, and finally use Adobe Illustrator 2023 to draw graphs.

[0133] (2) In vitro characterization experiment of AB9 polypeptide inhibiting the pathological aggregation of HγD-P23T at 25°C: The method is the same as that in method (1) of Example 3 above. The difference is that after mixing the polypeptide molecule with HγD-P23T at a volume ratio of 1:1, incubate it at room temperature (25°C), and then take samples every half hour and drop them on a crystal plate for observation with an optical microscope. The total incubation time is three hours.

[0134] (3) In vitro characterization experiment of AB9 polypeptide reversing the pathological aggregation of HγD-P23T: Dissolve HγD-P23T in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration to make the concentration of HγD-P23T 0.24 mM; Take out the AB9 polypeptide from the -20°C refrigerator, add 100 μl of hexafluoroisopropanol to completely dissolve it, and completely dry it with nitrogen. Dissolve the polypeptide molecule (Ac-ERTIPITREEKPAVTAAPKK-NH2) in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution, with a molar concentration gradient of 5.0 mM, 3.0 mM, and 1.0 mM; Incubate 0.24 mM of HγD-P23T in a 25°C incubator until it aggregates to a plateau; Mix the polypeptide molecule with HγD-P23T that has reached the plateau at a volume ratio of 1:1, incubate at room temperature, take samples every half hour and drop them on a crystal plate for observation with an optical microscope, and the total incubation time is three hours; Process the obtained images with Photoshop, and finally use Adobe Illustrator 2023 to draw graphs.

[0135] 2. Experimental results

[0136] (1) Results of in vitro characterization experiment of AB9 polypeptide inhibiting the pathological aggregation of HγD-P23T at 37°C

[0137] The ability of AB9 at different concentrations to inhibit the pathological aggregation of HγD-P23T at 37°C is as Figure 7 shown, OD 350 The results are as Figure 8 shown, OD 350 The results are consistent with Figure 7 the light microscope images, all indicating that AB9 has the ability to inhibit the pathological aggregation of HγD-P23T.

[0138] (2) Results of in vitro characterization experiment of AB9 polypeptide inhibiting the pathological aggregation of HγD-P23T at 25°C

[0139] At room temperature, the ability of AB9 polypeptide to inhibit the pathological aggregation of HγD-P23T is as Figure 9 shown, AB9 at 3 mM to 5 mM can inhibit the pathological aggregation of HγD-P23T.

[0140] (3) Results of in vitro characterization experiment of AB9 polypeptide reversing the pathological aggregation of HγD-P23T

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

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

[0143] (1) In vitro characterization experiment of regulating the pathological aggregation of HγD-P23T with different concentrations of NaCl

[0144] Dissolve HγD-P23T in 20 mM PB (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration to make the concentration of HγD-P23T 0.06 mM, 0.12 mM, 0.18 mM, 0.24 mM, 0.30 mM; Mix 20 mM PB (pH 7.4, containing 1 mM EDTA and 5 mM DTT) containing 400 mM, 800 mM, 1200 mM, 1600 mM, 2000 mM, 2400 mM, 2800 mM, 3200 mM NaCl with HγD-P23T at a volume ratio of 1:1, incubate at 37°C for three hours, take samples and drop them on a crystal plate for observation with an optical microscope; Process the obtained images with Photoshop, and finally use Adobe Illustrator 2023 to make graphs.

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

[0146] Dissolve HγD-P23T in buffers of 1 mM, 5 mM, 10 mM, 15 mM, 20 mM Tris-HCl (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration to make the concentration of HγD-P23T 0.03 mM, 0.06 mM, 0.09 mM, 0.12 mM, 0.15 mM; Incubate at 37°C for three hours, take samples and drop them on a crystal plate for observation with an optical microscope; Process the obtained images with PhotoShop, and finally use Adobe Illustrator 2023 to make graphs.

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

[0148] Dissolve HγD-P23T in buffers of 1 mM, 5 mM, 10 mM, 15 mM, 20 mM PB (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration so that the HγD-P23T concentration is 0.03 mM, 0.06 mM, 0.09 mM, 0.12 mM, 0.15 mM; incubate at 37 °C for three hours, take samples and drop them on a crystal plate, observe with an optical microscope, process the obtained images with Photoshop, and finally use Adobe Illustrator 2023 to create graphs.

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

[0150] Dissolve HγD-P23T in buffers of 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) with 0%, 5%, 10% 1,6-hexanediol concentrations, and adjust the concentration so that the HγD-P23T concentration is 0.03 mM, 0.06 mM, 0.09 mM, 0.12 mM, 0.15 mM; take an appropriate amount of the protein solution, incubate at 37 °C for three hours, take samples and drop them on a crystal plate, observe with an optical microscope; process the obtained images with Photoshop, and finally use Adobe Illustrator 2023 to create graphs.

[0151] (5) In vitro characterization experiment of the regulation of HγD-P23T pathological aggregation by different concentrations of PEG8000

[0152] Dissolve HγD-P23T in 1×PBS containing 0%, 1%, 3%, 10%, 15% PEG8000 (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration so that the HγD-P23T concentration is 0.03 mM, 0.04 mM, 0.05 mM, 0.6 mM; take an appropriate amount of the protein solution, incubate at 37 °C for three hours, take samples and drop them on a crystal plate, observe with an optical microscope; process the obtained images with Photoshop, and finally use Adobe Illustrator 2023 to create graphs.

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

[0154] Dissolve HγD-P23T in a buffer of 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT), and adjust the concentration to make the concentration of HγD-P23T 0.10 mM, 0.20 mM, 0.30 mM, 0.40 mM, 0.30 mM; prepare Britton–Robinson universal buffer solution, adjust its pH value, and prepare solutions with pH 3, 4, 5, 6, 7, 8, 9, 10, 11; mix the Britton–Robinson buffer and HγD-P23T in a ratio of 4:1, and incubate at room temperature for three hours, take samples and drop them on a crystal plate for observation with an optical microscope; process the obtained images with Photoshop, and finally use Adobe Illustrator 2023 to draw graphs.

[0155] 2. Experimental Results

[0156] The effects of different concentrations of Nacl on the pathological aggregation of HγD-P23T are as Figure 11 shown. At 37 °C, the pathological aggregation of HγD-P23T does not show a concentration dependence on NaCl.

[0157] The effects of different concentrations of Tris-HCl buffer on the pathological aggregation of HγD-P23T are as Figure 12 shown. HγD-P23T can still aggregate, and the aggregation behavior does not depend on the buffer concentration.

[0158] The effects of different concentrations of PB buffer on the pathological aggregation of HγD-P23T are as Figure 13 shown. By changing the concentration of the PB buffer, the results show that the aggregation behavior of the protein is not regulated by the salt ion concentration of the PB buffer.

[0159] The effects of different concentrations of 1,6-hexanediol on the pathological aggregation of HγD-P23T are as Figure 14 shown. 1,6-Hexanediol can affect the pathological aggregation of proteins by interfering with or disrupting the hydrophobic interactions in proteins. Therefore, it is found that 5% 1,6-hexanediol can interfere with the pathological aggregation of different concentrations of HγD-P23T. Hydrophobic interaction refers to the phenomenon that non-polar molecules or groups spontaneously aggregate together in an aqueous solution due to repelling 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 approximately 8000. PEG is commonly used in the study of protein folding, aggregation, and stability because it can modulate protein-protein interactions by altering the volume exclusion effect and viscosity of the solution. PEG8000 is an impermeable macromolecule 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 as Figure 15 shown. As the concentration of PEG8000 increases, the effective concentration between HγD-P23T proteins increases, promoting the pathological aggregation of HγD-P23T. As shown in the figure, it can be found that at a low concentration of 0.03 mM of HγD-P23T, partial aggregation occurred after adding 1% PEG8000, and as the concentration of PEG8000 increased, 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 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 approaches the isoelectric point of the protein, the protein surface charge is less, the electrostatic repulsion weakens, and hydrophobic interactions may dominate, which often promotes aggregation; and changes in the pH value may affect the secondary and tertiary structures of the protein, resulting in partial or complete unfolding of the protein. Under extreme pH conditions (too acidic or too basic), the protein may unfold, which may expose more hydrophobic regions and increase the possibility of aggregation. As Figure 16 shown, aggregation occurs near the isoelectric point of HγD-P23T.

[0162] Characterization of the properties of Example 5 AB9

[0163] 1. Experimental method

[0164] (1) Static light scattering detection of AB9 aggregates: Take out the AB9 polypeptide from the -20 °C refrigerator, add 100 uL of hexafluoroisopropanol to completely dissolve it, and completely dry it with nitrogen. Dissolve the polypeptide molecules in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution with a molar concentration gradient of 10 mM, 5.0 mM, 3.0 mM, 1.0 mM, 0.5 mM; detect AB9 using static light scattering; process the data using origin 2021, and finally plot the graph using Adobe Illustrator 2023.

[0165] (2) Static light scattering detection of the size of AB9 aggregates: Take out the AB9 polypeptide from the -20°C refrigerator, add 100 μL of hexafluoroisopropanol to completely dissolve it, and completely dry it with nitrogen. Dissolve the polypeptide molecules in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution with molar concentration gradients of 10 mM, 5.0 mM, 3.0 mM, 1.0 mM, and 0.5 mM; use dynamic light scattering to detect AB9; use Origin 2021 to process the data, and finally use Adobe Illustrator 2023 to plot the graph.

[0166] (3) Infrared spectroscopy detection of the secondary structure of AB9: Take out the AB9 polypeptide from the -20°C refrigerator, add 100 μL of hexafluoroisopropanol to completely dissolve it, and completely dry it with nitrogen. Dissolve the polypeptide molecules in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution with molar concentration gradients of 10 mM, 5.0 mM, 3.0 mM, and 1.0 mM; Take 10 μL of AB9 solutions with different concentrations and drop them on the calcium fluoride window, and let them dry overnight; Place the sample at the detection location of the instrument, and use infrared spectroscopy to detect AB9; Use PeakFit to fit and process the data, use Origin 2021 to plot the graph, and finally use Adobe Illustrator 2023 to organize it.

[0167] (4) CCK8 detection of the cytotoxicity of AB9 to cells: Prepare 100 μL of suspensions of 5000 Hela and HLEB3 cells in a 96-well plate, and culture them for 24 hours according to experimental requirements; Add different concentrations of AB9 stimulation to the culture plate, with concentrations of 1000 μM, 300 μM, 100 μM, 30 μM, 10 μM, 30 μM, and 1 μM respectively; Incubate the culture plate in the incubator for 24 hours; Add 10 μL of CKK-8 solution to each well; Continue to incubate in the cell incubator for 1 - 4 hours; Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader, and use a wavelength of 630 nm as the reference wavelength for dual-wavelength measurement; Use Origin 2021 to plot the graph, and finally use Adobe Illustrator 2023 to organize it.

[0168] (5) BLI detection of the affinity between AB9 and the target protein: Take out the biotinylated AB9 polypeptide from the -20°C refrigerator, add 100 μl of hexafluoroisopropanol to completely dissolve it, and completely dry it with nitrogen. Dissolve the polypeptide molecule in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution at a molar concentration of 2 μM; First, activate the streptavidin (SA) biosensor in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) solution; Dissolve HγD-P23T, HγD-WT, and αB-crystallin proteins in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) buffer, and concentrate and dilute the concentrations of the three proteins to appropriate concentrations; The streptavidin (SA) biosensor is first soaked in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) buffer as the baseline; The streptavidin (SA) biosensor is soaked in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) buffer containing biotinylated AB9 polypeptide to allow it to bind to the polypeptide; The streptavidin (SA) biosensor is soaked in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) buffer to wash away the unbound polypeptide; The streptavidin (SA) biosensor is soaked in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) buffer containing the target protein for the binding of the protein and the polypeptide; The streptavidin (SA) biosensor is soaked in 1×PBS (pH 7.4, containing 1 mM EDTA and 5 mM DTT) buffer to dissociate the target protein; Analyze the experimental data; Use origin 2021 to plot the graph, and finally use Adobe Illustrator 2023 to organize it.

[0169] (6) Detection of the interaction site between AB9 and HγD-His-P23T by liquid nuclear magnetic resonance HSQC spectrum: Dissolve HγD-His-P23T in a buffer of 20 mM sodium phosphate, 5 mM DTT (pH 6.2), containing 5% (v / v) D2O, and adjust the concentration to make the concentration of HγD-P23 0.12 mM; Take out the AB9 polypeptide from the -20 °C refrigerator, add 100 μl of hexafluoroisopropanol to completely dissolve it, blow it dry completely with nitrogen, dissolve the polypeptide molecule in a buffer of 20 mM sodium phosphate, 5 mM DTT (pH 6.2), containing 5% (v / v) D2O, and adjust the concentration of the AB9 polypeptide to an appropriate concentration; Transfer the sample to an NMR tube to ensure there are no bubbles; Turn on the Bruker Avance III 950 MHz NMR spectrometer at a temperature of 298 K; Take out the prepared sample tube and place it on the sample rack, and introduce the sample into the spectrometer; Set the experimental parameters of heteronuclear single quantum correlation spectroscopy (HSQC) and NOESY, and perform data acquisition; After the experiment, save the data and use NMRPipe and NMRView for HSQC and NOESY spectrum analysis; Identify the signals in the spectrum to determine the residues with chemical shift changes.

[0170] 2. Experimental results

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

[0172] In the DLS experiment, the particles or molecules in the sample move randomly in the solution in a Brownian motion manner. When a laser beam irradiates these moving particles, the intensity of the scattered light will fluctuate slightly with time. By performing autocorrelation analysis on these fluctuations, the diffusion coefficient of the particles can be obtained, and the Stokes-Einstein equation is used to convert the diffusion coefficient into the hydrodynamic diameter (Dh), so as to obtain the particle size distribution of AB9. It can be found that AB9 will form aggregates, and their size is about 100 to 1000 nm ( Figure 18 、 Figure 19 ).

[0173] The AB9 sample was scanned using an infrared spectrometer to obtain its absorption spectrum in the infrared region, with a focus on the amide I band between 1700 - 1600 cm-1, as this region is closely related to the secondary structure of proteins. The infrared spectral data was imported into 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 error on the data. In the amide I band region, the absorption peaks of the infrared spectrum were fitted using Gaussian. Through deconvolution, the absorption peaks corresponding to different secondary structures (such as α-helix, β-sheet, β-turn, and random coil) could be separated. After fitting, the areas of each fitted peak were calculated, and these areas are proportional to the content of the corresponding secondary structure. As Figure 20 shown, the AB9 polypeptide has four secondary structures and their proportions are similar.

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

[0175] The results of BLI detection of the affinity between AB9 and the target protein are as Figure 22 shown, AB9 has a strong affinity for HγD-P23T.

[0176] HSQC (Heteronuclear Single Quantum Coherence) spectra are a type of 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 hydrogen atoms, it is possible to obtain 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 can precisely identify the interaction sites and structural changes of AB9 with the HγD-His-P23T protein. The residues on the protein that have been identified to have significant changes in chemical environment or conformation before and after binding to AB9 are: H16, H23, N25, R60, H66, Q67, G71, L72, S85, H88, E96, H122, Y154, I171, D172( Figure 23 ).

[0177] In summary, by changing the solution environment of HγD-P23T, we found that electrostatic interaction and hydrophobic interaction play key roles in the pathological aggregation of HγD-P23T. Through screening, we successfully found the polypeptide AB9 that can inhibit and reverse the pathological aggregation of HγD-P23T lens protein in vitro, and detected the affinity of the polypeptide-protein interaction and the residues on HγD-P23T that may participate in the interaction. In summary, the polypeptide AB9 screened in this study has potential clinical medicinal value.

[0178] It should be noted that the specific implementation manners are only relatively representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments, and there can be many variations. Those of ordinary skill in the art who obtain, without any doubt, based on what is clearly disclosed in the present invention or according to the written description of the document, should be considered as the scope protected by this patent.

Claims

1. A method for inhibiting the pathological aggregation of γD-crystallin P23T mutant protein for non-therapeutic purposes in vitro, characterized in that, The method includes mixing and incubating the polypeptide shown in SEQ ID NO: 14 with the γD-crystallin P23T mutant protein.

2. A method for reversing the pathological aggregation of γD-crystallin P23T mutant protein for non-therapeutic purposes in vitro, characterized in that, The method includes: first incubating the γD-crystallin P23T mutant protein until its aggregation plateau phase, and then mixing and incubating the polypeptide shown in SEQ ID NO: 14 with the γD-crystallin P23T mutant protein; The time for incubating the γD-crystallin P23T mutant protein until its aggregation plateau phase is 3 h.

3. The method according to claim 1 or claim 2, characterized in that, When the polypeptide shown in SEQ ID NO: 14 is mixed and incubated with the γD-crystallin P23T mutant protein, the concentration ratio of the polypeptide to the γD-crystallin P23T mutant protein is 12.5:1 - 20:1.

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