Use of crybb2 recombinant protein in treatment and / or prevention of diabetic retinopathy

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to application of Crybb2 recombinant protein in treatment and / or prevention of diabetic retinopathy. The present application finds that the use of Crybb2 recombinant protein can improve the activity of müller cells, reduce the level of GFAP, restore the level of GS, reduce inflammation, reduce apoptosis of retinal cells and restore the structure and function of the retina. It can be used for treatment and / or prevention of diabetic retinopathy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of recombinant Crybb2 protein in the treatment and / or prevention of diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is an irreversible blinding eye disease. Currently, the clinical diagnosis and staging of DR are primarily based on vascular lesions. Neurodegeneration or damage plays a crucial role in DR. Studies have found that in animal models of DR and in the retinas of diabetic patient donors, neurodegeneration or damage exists before obvious microvascular complications, including the hallmark glial cell proliferation (Müller cells and astrocytes) and retinal cell apoptosis. The structural and functional changes in the retina caused by neurodegeneration lead to corresponding clinical manifestations, including delayed dark adaptation, decreased color vision, decreased contrast sensitivity, and visual field abnormalities, ultimately resulting in a decline in the patient's vision-related quality of life.

[0003] Current research on retinal-related diseases largely focuses on mechanical injury models. However, there are significant differences in the neurological damage mechanisms and treatment mechanisms between diabetic retinopathy and mechanical injury. Diabetic retinopathy is caused by chronic metabolic disorders such as long-term hyperglycemia, with damage to the retinal neurovascular unit (NVU) as the core, simultaneously affecting retinal microvessels and various types of nerve cells in a diffuse, bilateral, progressive disease. The mechanism involves the synergistic effects of abnormal glucose metabolism, apoptosis, oxidative stress, and ischemia-hypoxia. Mechanical injury, on the other hand, is an acute mechanical injury caused by localized damage to the unilateral optic nerve trunk, directly resulting in axonal or myelin sheath destruction. In terms of treatment, for mechanical injury, the core approach is to repair axons by secreting neurotrophic factors, while for diabetic retinopathy, which is a chronic metabolic neurological injury disease, the focus needs to be on regulating the metabolic microenvironment and protecting nerve cell function by reducing Müller cell activation, inhibiting inflammation, and improving retinal apoptosis. For example, patent document (CN118685500A) discloses the application of LECT2 as a target in the preparation of drugs for the prevention and treatment of diabetic retinopathy-related diseases. It discloses the STMP1 regulation mechanism, aiming to alleviate key pathological processes of diabetic retinopathy by reducing STMP1 expression levels, such as: reducing microglial cell activation and inflammatory responses, improving mitochondrial function, inhibiting oxidative stress, and protecting retinal neurons from damage. However, there is currently no existing technology that uses recombinant Crybb2 protein as a therapeutic target for the treatment of diabetic retinopathy. Summary of the Invention

[0004] In a first aspect, the present invention provides the use of recombinant Crybb2 protein in the preparation of medicaments for the treatment and / or prevention of diabetic retinopathy.

[0005] Crybb2 as described in this application refers to βB2-crystal protein.

[0006] Preferably, the treatment and / or prevention of diabetic retinopathy includes intravitreal injection of recombinant Crybb2 protein.

[0007] Preferably, the injection dose of recombinant Crybb2 protein includes 20-2000 ng / eye, such as 20, 50, 100, 200, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1200, 1500 or 2000 ng / eye.

[0008] Preferably, the concentration of the Crybb2 recombinant protein includes 1-1000 ng / μL, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 ng / μL.

[0009] Preferably, the amino acid sequence of the Crybb2 recombinant protein includes SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence having more than 90% homology with SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence with the same function obtained by substitution, deletion and / or insertion of one or more amino acid residues of SEQ ID NO: 1 or SEQ ID NO: 3.

[0010] Preferably, amino acid sequences with more than 90% homology include amino acid sequences with more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.9% homology.

[0011] Preferably, substitution, deletion and / or insertion of one or more amino acid residues includes substitution, deletion and / or insertion of 1 to 20 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues.

[0012] MASDHQTQAGKPQPLNPKIIIFEQENFQGHSHELSGPCPNLKETGMEKAGSVLVQAGPWVGYEQANCKGEQFVFEKGEYPRWDSWTSSRRTDSLSSLRPIKVDS QEHKIILYENPNFTGKKMEIVDDDVPSFHAHGYQEKVSSVRVQSGTWVGYQYPGYRGLQYLLEKGDYKDNSDFGAPHPQVQSVRRIRDMQWHQRGAFHPSS (SEQ ID NO: 3).

[0013] Preferably, the nucleotide sequence encoding the Crybb2 recombinant protein includes SEQ ID NO: 2, or a complementary or degenerate sequence of the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence that hybridizes with the above sequence and is capable of encoding an amino acid sequence with the same function.

[0014] Preferably, the complementary sequence is a complementary sequence formed according to the base complementary pairing principle, and more preferably, it can be a nucleotide sequence that is completely or partially complementary to SEQ ID NO: 2 and can encode the same functional amino acid sequence.

[0015] Preferably, the degenerate sequence refers to a sequence in which the amino acid type encoded by the nucleotide sequence at the changed position remains unchanged after changing one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) in SEQ ID NO: 2, and does not affect the function and expression level of the nucleotide sequence.

[0016] Preferably, a nucleotide sequence with more than 90% homology refers to a nucleotide sequence with more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.9% homology.

[0017] Preferably, the Crybb2 recombinant protein is recombinantly expressed using an insect baculovirus expression system.

[0018] Preferably, after recombinant expression using an insect baculovirus expression system, the method further includes cleaving cells and purifying the protein. Preferably, the protein purification includes column chromatography.

[0019] Preferably, the Crybb2 recombinant protein treats and / or prevents diabetic retinopathy through neuroprotective effects.

[0020] Preferably, the Crybb2 recombinant protein treats and / or prevents diabetic retinopathy by increasing Müller cell viability, reducing GFAP levels, restoring GS levels, alleviating inflammation (e.g., reducing TNF-α levels), and reducing retinal cell apoptosis (reducing the Bax / Bcl-2 ratio).

[0021] The diabetes mentioned is type 2 diabetes.

[0022] The diabetic retinopathy mentioned is diabetic retinal neurodegeneration.

[0023] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0024] Preferably, the pharmaceutically acceptable excipients include one or more of the following: diluents, buffers, wetting agents, absorption enhancers, surfactants, lubricants, antioxidants, antibacterial agents, isotonic regulators, or pH regulators.

[0025] Preferably, the drug can be any dosage form, such as one or more of the following: injection, tablet, powder, gel, or emulsion.

[0026] Preferably, the treatment and / or prevention of diabetic retinopathy includes administration of the drug in the early stages or in mild nonproliferative diabetic retinopathy.

[0027] The severity stages of diabetic retinopathy include the non-proliferative stage (mild, moderate, or severe) and the proliferative stage, as referenced from Table 14-1 on page 334 of the 3rd edition of the Ophthalmology textbook.

[0028] In one specific embodiment of the invention, the absence of microvascular complications constitutes the early stage. Preferably, the administration includes 1-10 doses, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0029] In a second aspect, the present invention provides a Crybb2 recombinant protein, wherein the amino acid sequence of the Crybb2 recombinant protein includes SEQ ID NO: 1, or an amino acid sequence having more than 90% homology with SEQ ID NO: 1, or an amino acid sequence having the same function obtained by substitution, deletion and / or insertion of one or more amino acid residues of SEQ ID NO: 1.

[0030] A third aspect of the present invention provides a method for preparing recombinant Crybb2 protein.

[0031] The preparation method includes using the baculovirus expression vector system (BEVS).

[0032] Preferably, the preparation method includes:

[0033] 1) Construct an expression vector containing the Crybb2 gene sequence, transfect it into competent cells for amplification, and obtain competent cells containing recombinant rod granules;

[0034] 2) Extract rod-particle DNA from competent cells containing recombinant rod particles, infect insect cells (e.g., sf9 cell line) to obtain P1 recombinant baculovirus, and further transfer the P1 generation virus into insect cells for continued amplification to obtain high-titer P2 generation recombinant baculovirus. Preferably, depending on the specific implementation plan, the P2 generation recombinant baculovirus can be further transferred into insect cells for continued amplification to obtain P3 generation, as well as amplify more generations of recombinant baculovirus.

[0035] 3) Infect insect cells (e.g., Hi-5 cell line) in the logarithmic growth phase with the recombinant baculovirus obtained in step 2) and culture them to express the Crybb2 protein.

[0036] Preferably, the preparation method further includes purifying the protein after cell lysis. The purification is preferably performed using column chromatography.

[0037] In a fourth aspect, the present invention provides a medicament for treating and / or preventing diabetic retinopathy, said medicament comprising the above-described recombinant Crybb2 protein or the recombinant Crybb2 protein obtained by the above-described preparation method.

[0038] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0039] Preferably, the pharmaceutically acceptable excipients include one or more of the following: diluents, buffers, wetting agents, absorption enhancers, surfactants, lubricants, antioxidants, antibacterial agents, isotonic regulators, or pH regulators.

[0040] Preferably, the drug can be any dosage form, such as one or more of the following: injection, tablet, powder, gel, or emulsion.

[0041] Preferably, the drug can be administered via any suitable route of administration, such as intraocular administration, preferably intravitreal injection.

[0042] Preferably, the drug may contain an active substance (e.g., the Crybb2 recombinant protein of this application) in a weight or volume ratio of 0.01-99.5% (0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.5%).

[0043] Preferably, the concentration of the Crybb2 recombinant protein in the drug ranges from 1 to 1000 ng / μL.

[0044] Preferably, the drug can be a human drug or a veterinary drug.

[0045] A fifth aspect of the invention provides a method for treating and / or preventing diabetic retinopathy, the method comprising administering an effective amount of recombinant Crybb2 protein or the aforementioned drugs to a subject in need.

[0046] Preferably, the method comprises injecting an effective amount of recombinant Crybb2 protein or the aforementioned drug into the vitreous cavity of a subject in need.

[0047] Preferably, the effective amount of the Crybb2 recombinant protein includes 500-1000 ng / eye, such as 20, 50, 100, 200, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1200, 1500 or 2000 ng / eye.

[0048] In a sixth aspect, the present invention provides a method for improving Müller cell viability, reducing GFAP levels, restoring GS levels, alleviating inflammation (e.g., reducing TNF-α levels), and reducing retinal cell apoptosis (reducing the Bax / Bcl-2 ratio), the method comprising using recombinant Crybb2 protein or the aforementioned drugs.

[0049] The term "treatment" as used in this invention refers to slowing down, interrupting, preventing, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the onset of the disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0050] The term "prevention" as used in this invention refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in the body.

[0051] The term "effective amount" as used in this invention refers to the amount or dose of the product of this invention that provides the desired treatment after being administered to a subject in one or more doses.

[0052] The "subject" described in this invention can be a human or a non-human mammal. The non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, or a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, donkeys, minks, jackals, foxes, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, or squirrels, etc.) or monkeys, etc.

[0053] The "method" or "application" described in this invention may be for therapeutic purposes or for non-therapeutic purposes.

[0054] The term "comprising" or "including" as used in this invention is an open-ended expression. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still having the same or similar activity as the original sequence.

[0055] The term "and / or" as used in this invention includes all combinations of items connected by the term, and should be regarded as each combination having been individually listed in this application. For example, "A and / or B" includes "A", "B" and "A and B", and "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0056] The term "pharmaceutically acceptable" as used in this invention refers to the biological activity and characteristics of the active substance in the applied product that neither significantly irritates the organism nor inhibits it.

[0057] The beneficial effects of this invention are:

[0058] This invention constructs a db / db mouse model (a classic type 2 diabetes animal model) and, through continuous monitoring using ERG and OCT, reveals that db / db mice exhibit neurodegeneration as early as 12 weeks of age, displaying a diabetic retinopathy phenotype. Intravitreal injection of recombinant Crybb2 protein into db / db mice showed that the recombinant Crybb2 protein has a certain neuroprotective effect on db / db mice exhibiting the diabetic retinopathy phenotype, including maintaining retinal structure (including increased retinal thickness detected by OCT) and function (including shortened b-wave latency detected by ERG), reducing retinal cell apoptosis (reduced retinal cell apoptosis detected by Tnuel staining), alleviating inflammation (e.g., reducing TNF-α expression levels), and reducing glial cell activation (restoring GS expression levels and reducing GFAP levels). Attached Figure Description

[0059] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0060] Figure 1 Example 1 shows the small-scale expression and purification results of the Crybb2 recombinant protein prepared in Example 1. In the example, M represents the protein molecular weight marker, Lysate represents the cell lysis supernatant, Load represents the sample loaded onto the chromatography column, FT represents the sample flowed through the chromatography column, E1 and E2 represent different concentrations of imidazole elution buffer, and the imidazole concentration of the elution buffer is E2 (500 mM) > E1 (200 mM). Method 1 corresponds to the result of lysis condition 1, Method 2 corresponds to the result of lysis condition 2, and Method 3 corresponds to the result of lysis condition 3.

[0061] Figure 2 Example 1 shows the amplified expression and purification results of the Crybb2 recombinant protein prepared in Example 1. In the example, M represents the protein molecular weight marker, Lysate represents the cell lysis supernatant, Load represents the sample loaded onto the chromatography column, FT represents the sample flowed through the chromatography column, and E1 and E2 represent different concentrations of imidazole elution buffer. The imidazole concentration of the elution buffer is E2 > E1.

[0062] Figure 3 The results of the Crybb2 recombinant protein prepared in Example 1 on Müller cell CCK-8 assay;

[0063] Figure 4 Example 1: Immunofluorescence staining results of recombinant Crybb2 protein prepared in Example 1 on Müller cells (A) and db / db mouse retina (B);

[0064] Figure 5 The results of TUNEL staining of the retina of db / db mice with the recombinant Crybb2 protein prepared in Example 1;

[0065] Figure 6 Western Blot results of the Crybb2 recombinant protein prepared in Example 1 on Müller cells (A) and db / db mouse retina (B);

[0066] Figure 7 OCT examination results of the retina of db / db mice using the recombinant Crybb2 protein prepared in Example 1;

[0067] Figure 8 Example 1: ERG examination results of the retina of db / db mice using recombinant Crybb2 protein.

[0068] In each figure, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p<0.0001, ns indicates no significant difference. Detailed Implementation

[0069] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] Example 1: Preparation of Crybb2 recombinant protein

[0071] Recombinant Crybb2 protein (SEQ ID NO: 1) was expressed and purified using the Baculovirus Expression Vector System (BEVS).

[0072] MHHHHHHHHHHLEVLFQGPMASDHQTQAGKPQPLNPKIIIFEQENFQGHSHELSGPCPNLKETGMEKAGSVLVQAGPWVGYEQANCKGEQFVFEKGEYPRWDSWTSSRRTDSLS SLRPIKVDSQEHKIILYENPNFTGKKMEIVDDDVPSFHAHGYQEKVSSVRVQSGTWVGYQYPGYRGLQYLLEKGDYKDNSDFGAPHPQVQSVRRIRDMQWHQRGAFHPSS (SEQ ID NO: 1)

[0073] 1. Construction of expression vector

[0074] (1) The Crybb2 gene sequence (SEQ ID NO: 2) was obtained by PCR.

[0075] (SEQ ID NO: 2).

[0076] (2) The Crybb2 gene sequence and the vector were ligated to obtain 10H-3C-mCrybb2(1-205aa) (pFastBac);

[0077] (3) Design primers (Bac-1) to sequence the constructed expression vector and obtain plasmids containing the correct sequence;

[0078] (4) Transfect plasmids containing the correct sequence into competent E. coli cells for amplification to obtain a sufficient amount of recombinant plasmids.

[0079] 2. Virus Packaging

[0080] (1) Host cells: The virus packaging and amplification were performed using the sf9 insect cell line;

[0081] (2) Extracting rod-granule DNA from competent Escherichia coli cells containing recombinant rod-granules and infecting insect cells to obtain P1 recombinant baculovirus;

[0082] (3) The P1 generation virus was transferred into sf9 insect cells and amplified to obtain a high-titer P2 generation recombinant baculovirus.

[0083] 3. Cell culture and protein expression

[0084] (1) Host cells: Protein expression was performed using the Hi-5 insect cell line;

[0085] (2) Viral generation: P2 generation recombinant baculovirus;

[0086] (3) Hi-5 insect cells in logarithmic growth phase were infected with P2 generation recombinant baculovirus at a multiplicity of infection (MOI) of 1 / 200. The infected cells were cultured at 27°C for 2-4 days to express the Crybb2 recombinant protein.

[0087] 4. Protein purification

[0088] The chromatography column was equilibrated with equilibration buffer. The cell lysate supernatant after centrifugation and filtration was loaded and washed with equilibration buffer to remove some contaminating proteins. The target protein was then eluted with buffers containing different concentrations of elution solvent. The eluted fraction was collected and desalted into storage buffer. The eluted fraction containing the target protein was dialyzed using a stepwise dialysis method to remove denaturing agents and renature the protein. Finally, the renatured protein was desalted into storage buffer. Protein concentration was determined using UVOD 280 for the process samples and the final protein product. Protein purity was determined using Coomassie Brilliant Blue staining and SDS-PAGE (reduced SDS-PAGE).

[0089] (1) Protein small-scale expression and purification conditions and reagents

[0090] Conditions: The host cell line was Hi-5, the MOI was 1 / 200, the culture period was 2 days, the culture volume was 80 mL for each condition, the protein expression was intracellular, and the cells were collected for purification.

[0091] Cell lysis method: ultrasonic disruption.

[0092] Lysis buffer: 20mM Tris, pH 8.0, NaCl, 10% glycerol, Triton X-100, protease inhibitor cocktail, nuclease.

[0093] Pyrolysis conditions: Condition 1 is low-salt pyrolysis + low-salt extraction; Condition 2 is high-salt pyrolysis + high-salt extraction; Condition 3 is low-salt pyrolysis + high-salt extraction.

[0094] Protein purification: Ni ion affinity chromatography.

[0095] Buffer: Equilibration buffer: 20mM Tris, pH 8.0, NaCl, 10% glycerol, imidazole;

[0096] Elution buffer: 20mM Tris, pH 8.0, NaCl, 10% glycerol, imidazole.

[0097] Elution gradient: Step elution.

[0098] The results of the small-scale expression purification and reduced SDS-PAGE are shown below. Figure 1 .

[0099] (2) Protein amplification, expression, and purification conditions and reagents

[0100] Conditions: The host cell line was Hi-5, the MOI was 1 / 200, the culture period was 2 days, the culture volume was 80 mL for each condition, the protein expression was intracellular, and the cells were collected for purification.

[0101] Cell lysis method: ultrasonic disruption.

[0102] Lysis buffer: 20mM Tris, pH 8.0, NaCl, 10% glycerol, Triton X-100, protease inhibitor cocktail, nuclease.

[0103] Pyrolysis conditions: high-salt pyrolysis + high-salt extraction.

[0104] Protein purification: Ni ion affinity chromatography.

[0105] Buffer: Equilibration buffer: 20mM Tris, pH 8.0, NaCl, 10% glycerol, imidazole;

[0106] Elution buffer: 20mM Tris, pH 8.0, NaCl, 10% glycerol, imidazole.

[0107] Elution gradient: Step elution.

[0108] For the results of scaled-up expression purification, see the reduced-state SDS-PAGE results. Figure 2 .

[0109] 5. The basic physicochemical properties of the Crybb2 recombinant protein (SEQ ID NO: 1) are shown in Table 1.

[0110] Table 1

[0111]

[0112] Example 2: Effects of recombinant Crybb2 protein on Müller cells in a high-glucose environment

[0113] Biocompatibility: The recombinant Crybb2 protein prepared in Example 1 (the recombinant Crybb2 protein after amplification and expression purification) showed no significant toxicity to normal cells at appropriate doses and had good biocompatibility.

[0114] Human retinal Müller cells (MIO-M1) were cultured and divided into a normal control group (Glu: 5mM; Crybb2: 0ng / μL) and experimental groups, including a high glucose group (Glu: 35mM; Crybb2: 0ng / μL) and a high glucose + Crybb2 group (Glu: 35mM; Crybb2: 10ng / μL; the volume of recombinant Crybb2 protein added was 2-200μL depending on the actual culture conditions). The stimulation time was 24h. Cell morphology was observed under an inverted microscope.

[0115] Müller cell viability was determined using the CCK-8 assay: 100 μL of basal culture medium and 10 μL of CCK-8 solution were added to each well, and the cells were incubated at 37°C for 1-2 h. The absorbance (A value) at 450 nm was read using a microplate reader, and cell viability was calculated using the following formula: Cell viability = (A value of each experimental group / A value of the control group) × 100%. See results below. Figure 3 The results indicate that the addition of 10 ng / μL of recombinant Crybb2 protein can significantly improve Müller cell viability and alleviate the high glucose-induced decline in Müller cell viability.

[0116] Immunofluorescence staining: Fix with 4% paraformaldehyde at room temperature for 20-30 min, incubate with freshly prepared 0.1% Triton X-100 solution at room temperature for 15 min, block with 2% BSA for 1 h, incubate with primary antibodies (glutamine synthase (GS) and glial fibrillary acidic protein (GFAP)): Add primary antibody diluted with 2% BSA at a certain ratio, incubate overnight at 4℃, add secondary antibody working solution diluted with 2% BSA at a certain ratio, incubate at 37℃ in the dark for 1 h, mount with mounting medium, observe and photograph under a confocal microscope, analyze fluorescence intensity using ImageJ software, repeat the experiment 3 times. Results are as follows. Figure 4As shown in Figure A, 5 mM represents the normal control group, 35 mM represents the high glucose group, and 35 mM + Crybb2 represents high glucose + 10 ng / μL of recombinant Crybb2 protein. The results showed that in the 35 mM + Crybb2 group, green fluorescence (GFAP) was reduced and red fluorescence (GS) was increased, indicating that recombinant Crybb2 protein can reduce GFAP levels and restore GS levels, thus alleviating Müller cell activation.

[0117] Western blot: Total cellular protein was extracted, and protein concentration was determined using the BCA method. The protein loading volume was 20-40 μg, and 5× sample buffer was added. After 12.5% ​​SDS-PAGE gel electrophoresis, the sample was transferred to a PVDF membrane and blocked with TBST containing 5% skim milk powder at room temperature for 1 h. Primary antibody was added and incubated overnight at 4°C. The membrane was washed three times with TBST for 10 min each time. HRP-labeled secondary antibody was added and incubated at room temperature for 1 h, followed by three TBST washes for 5 min each time. The membrane was developed using an ECL kit, and grayscale values ​​were analyzed using ImageJ software. The experiment was repeated three times. Results are as follows: Figure 6 As shown in Figure A, 5mM represents the normal control group (Glu: 5mM; Crybb2: 0ng / μL), 35mM represents the high glucose group (Glu: 35mM; Crybb2: 0ng / μL), and 35mM+Crybb2 represents the high glucose+Crybb2 recombinant protein group (Glu: 35mM; Crybb2: 10ng / μL). The results indicate that in the 35mM+Crybb2 group, the gray value of the GFAP band decreased, the gray value of the GS band increased, and the gray value of the TNF-α band decreased. The bar graph also clearly shows that the Crybb2 recombinant protein can significantly reduce GFAP levels, restore GS levels, and reduce TNF-α levels, thus alleviating Müller cell activation and reducing inflammation.

[0118] Example 3: Effect of recombinant Crybb2 protein on early retinal neurodegeneration in db / db mice

[0119] Twelve-week-old db / db mice were selected as animal models, and four groups (n=6) were set up: a normal control group (db / m, labeled N), a db / db mouse group (labeled db / db), a db / db mouse + PBS group (labeled db / db + PBS), and a db / db mouse + Crybb2 group (labeled db / db + Crybb2). Changes in relevant indicators were observed at 13 weeks of age. Mice in the db / db mouse + Crybb2 group received a single intravitreal injection of 1.5 μL of recombinant Crybb2 protein (prepared in Example 1, concentration 500 ng / μL), while mice in the db / db mouse + PBS group received a single intravitreal injection of 1.5 μL of PBS.

[0120] ERG detection of visual function changes: Phoenix ERG module for small animal imaging. Mice were placed in a dark-adapted room overnight. After thorough anesthesia, the pupils were dilated, topical anesthesia was administered, and antibiotic eye drops and artificial tears were applied to keep the cornea moist. The reference electrode and ground electrode were placed subcutaneously behind the ear and tail, respectively, and the test electrode contacted both corneas. Light intensity stimulation parameters were set (-1.7, 1.0, and 3.1 log cd sec / m). 2 ERG tests were performed on mice in each group, and the data were recorded and quantitatively analyzed. ERG results showed that the b-wave latency in the retina of mice in the db / db+Crybb2 group was shortened, indicating that recombinant Crybb2 protein can improve retinal function (see results). Figure 8 ).

[0121] OCT detection of retinal structural changes: Mice were mydriatic, topically anesthetized, and fixed to an OCT scanner (Heidelberg HRA2, Germany). SD-OCT (Spectral domain OCT) was used to automatically segment retinal images to distinguish different retinal layers and quantify their thickness. OCT results showed that the total retinal thickness was increased in the db / db+Crybb2 group, indicating that recombinant Crybb2 protein can improve retinal structure (see results). Figure 7 ).

[0122] Immunofluorescence staining: Frozen sections were prepared from mouse eyeballs. The sections were first fixed and blocked, then incubated with primary antibody (GFAP) overnight at 4°C. Secondary antibody working solution diluted with 2% BSA was added and incubated for 1 hour. The sections were then mounted with mounting media, observed under a fluorescence microscope, and photographed for analysis. Results are as follows: Figure 4 As shown in Figure B, the green fluorescence (GFAP) was significantly reduced in the db / db+Crybb2 group, indicating that the recombinant Crybb2 protein can also reduce GFAP levels in mice, thus alleviating Müller cell activation.

[0123] TUNEL staining: Frozen sections were prepared from mouse eyeballs. After fixation and sealing, the sections were stained according to the TUNEL detection kit instructions. The staining was observed and imaged under a fluorescence microscope, and the number of TUNEL-positive cells was counted. Results are as follows: Figure 5 As shown, the db / db mice injected with intravitreal Crybb2 recombinant protein showed reduced retinal green fluorescence, indicating that Crybb2 recombinant protein can reduce retinal cell apoptosis.

[0124] Western blot: retinal tissue lysate proteins were collected, and the rest was the same as described in Example 2. Results are as follows: Figure 6As shown in Figure B, in the db / db+Crybb2 group, the gray value of the GFAP band decreased, and the gray value ratio of the bax / bcl-2 band decreased. The bar graph clearly shows that the recombinant Crybb2 protein can significantly reduce GFAP levels and decrease the bax / bcl-2 ratio, that is, reduce Müller cell activation and reduce retinal cell apoptosis.

[0125] The above results indicate that the Crybb2 recombinant protein can maintain the viability of Müller cells under high glucose conditions, reduce Müller cell activation, decrease retinal cell apoptosis, improve retinal structure and function, and alleviate inflammation, thus exhibiting neuroprotective effects on the retina and can be used for the treatment and prevention of diabetic retinopathy. The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the inventive concept, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0126] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Application of recombinant Crybb2 protein in the preparation of drugs for the treatment of diabetic retinopathy; The amino acid sequence of the Crybb2 recombinant protein is SEQ ID NO: 1 or SEQ ID NO:

3.

2. The application according to claim 1, characterized in that, The treatment for diabetic retinopathy includes intravitreal injection of recombinant Crybb2 protein.

3. The application according to claim 2, characterized in that, The injection dose of recombinant Crybb2 protein is 20-2000 ng / eye.

4. The application according to claim 2, characterized in that, The concentration of recombinant Crybb2 protein is 1-1000 ng / μL.

5. The application according to claim 1, characterized in that, The nucleotide sequence encoding the recombinant Crybb2 protein includes SEQ ID NO:

2.

6. The application according to claim 1, characterized in that, The Crybb2 recombinant protein was recombinantly expressed using an insect baculovirus expression system.

7. The application according to claim 6, characterized in that, Recombinant expression via an insect baculovirus expression system also includes protein purification after cell lysis.

8. The application according to claim 1, characterized in that, The drug further includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are selected from one or more of the following: diluents, buffers, wetting agents, absorption enhancers, surfactants, lubricants, antioxidants, antibacterial agents, isotonic regulators, or pH regulators.

9. The application according to claim 1, characterized in that, The dosage form of the drug is selected from one or more of the following: injection, powder, gel, or emulsion.

Images