Glucagon-like peptide-1-human interleukin 2 fusion protein as well as preparation method, product and application thereof

Abstract

The invention belongs to the field of genetic engineering and immunology, and particularly relates to a glucagon-like peptide-1-human interleukin 2 fusion protein as well as a preparation method, a product and application of the glucagon-like peptide-1-human interleukin 2 fusion protein. Specifically, a gene molecule cloning technology is utilized, a gene sequence of a fusion protein molecule of GLP-1-IL2-Fc and GLP-1-IL2 (glucagon-like peptide-1-human interleukin 2) introduced into an HEK293F cell is optimized, and two fusion proteins of GLP-1-IL2 and GLP-1-IL2 are successfully constructed and expressed. The GLP-1-IL2-Fc and GLP-1-IL2 bifunctional fusion protein prepared by the invention can bias stimulate the retention and proliferation of Foxp3 + T cells in pancreas and reduce the infiltration of CD8 + T cells, and meanwhile, the GLP-1 can also promote the proliferation of pancreatic beta cells and increase the quality of the pancreatic beta cells, and can be used for preventing the development of type 1 diabetes mellitus.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and immunology, specifically relating to glucagon-like peptide-1-human interleukin-2 fusion protein and its preparation method, products and applications. Background Technology

[0002] Diabetes is a chronic metabolic disease characterized by polydipsia, polyuria, and hyperglycemia due to insufficient insulin production or insulin insensitivity of the pancreas. There are two main types of diabetes: type 1 diabetes, caused by autoimmune cells attacking beta cells and damaging their function, and type 2 diabetes, caused by decreased insulin sensitivity. Type 1 diabetes typically develops in childhood or adolescence; therefore, it is also known as juvenile diabetes. From the onset of the disease, patients suffer from diabetes and its complications for a long period, and their life expectancy is significantly reduced.

[0003] Currently, there is no cure for type 1 diabetes, and most patients require lifelong insulin injection therapy. However, insulin has a narrow therapeutic index, and blood sugar levels fluctuate with daily diet, exercise, and physical condition, making it difficult to determine the correct insulin dosage. Excessive insulin dosage can cause severe hypoglycemia, even life-threatening situations. Insufficient insulin dosage can lead to various complications such as diabetic ketoacidosis and diabetic nephropathy.

[0004] The main bioactive fragment of glucagon-like peptide-1 (GLP-1) is a 30- or 31-amino acid polypeptide derived from the post-translational processing of proglucagon peptide (amino acids 7-36 or 7-37 of GLP-1). In humans, GLP-1 is a hormone primarily produced by intestinal cells and belongs to the incretin class. GLP-1 receptor agonists are a novel type of hypoglycemic drug that lowers blood sugar by activating the GLP-1 receptor (GLP-1R) in a glucose concentration-dependent manner, enhancing insulin secretion, inhibiting glucagon secretion, and delaying gastric emptying. This results in reduced food intake through central appetite suppression, thereby lowering blood sugar. Endogenous GLP-1 has a half-life of only about 2 minutes, so diabetic patients need to supplement with exogenous GLP-1 to achieve the goal of lowering blood sugar.

[0005] An imbalance between effector T cells and regulatory Treg cells can cause type 1 diabetes. Effector T cells attack pancreatic β cells, leading to their death, while regulatory Treg cells can suppress the function of regulatory T cells and protect pancreatic β cells. Numerous studies have shown that patients with type 1 diabetes have deficiencies in the number or function of regulatory Treg cells. Therefore, to prevent type 1 diabetes, it is essential to promote Treg cell proliferation as much as possible and maintain the balance between effector T cells and regulatory Treg cells. Interleukin-2 (IL-2) is a key cytokine for inducing Treg cell proliferation and maintaining Treg cell function, and it can reduce the attack of autoimmune cells on pancreatic β cells. Currently, IL-2 therapy is undergoing clinical trials for type 1 diabetes. However, IL-2 still has drawbacks such as rapid clearance, difficulty in dose control, and potential toxicity. Therefore, IL-2 therapy still needs further exploration and optimization to obtain drugs with longer half-lives and better efficacy. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides two fusion proteins comprising glucagon-like peptide-1 (GLP-1) and an IL-2 mutant, wherein the GLP-1 and the IL-2 mutant are connected by a linker, and the IL-2 mutant can selectively stimulate the proliferation of pancreatic Tregs. This fusion protein can be effectively used for the treatment of type 1 diabetes and has high safety.

[0007] The technical solution of this invention is as follows: On the one hand, the present invention provides a bifunctional fusion protein comprising, in the order from the N-terminus to the C-terminus of amino acids, glucagon-like peptide-1 (GLP-1), human interleukin-2 (IL2), and an IgG1 Fc fragment (Fc), forming a fusion protein A; Alternatively, it could be a fusion protein B consisting of GLP-1 and IL2, arranged in the order of amino acids from the N-terminus to the C-terminus.

[0008] Specifically, the fusion protein A and fusion protein B may also include a signal peptide.

[0009] Preferably, the amino acid sequence of the GLP-1 is as shown in SEQ ID NO: 4; the amino acid sequence of the IL2 is as shown in SEQ ID NO: 8; the amino acid sequence of the Fc is as shown in SEQ ID NO: 10; and the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 2.

[0010] Specifically, GLP-1 and IL2 are connected via a Linker.

[0011] More specifically, the Linker is (GGGGS)n, where n = 1-4.

[0012] Preferably, n=3.

[0013] Preferably, the amino acid sequence of the Linker is shown in SEQ ID NO: 6.

[0014] Preferably, the amino acid sequence of the fusion protein A is shown in SEQ ID NO: 12; and the amino acid sequence of the fusion protein B is shown in SEQ ID NO: 16.

[0015] Specifically, the bifunctional fusion protein also includes bioactive proteins or functional fragments thereof that assist in its expression and / or secretion, or prolong its half-life in vivo. Preferably, the bioactive protein or its functional fragment is selected from at least one of the following: immunoglobulin Fc domain, serum albumin, albumin-binding polypeptide, prealbumin, carboxyl-terminal peptide, elastin-like polypeptide, His tag, GST tag, MBP tag, FLAG tag, or SUMO tag.

[0016] On the other hand, the present invention provides a nucleic acid encoding the aforementioned bifunctional fusion protein.

[0017] Specifically, the nucleotide sequence of GLP-1 is shown in SEQ ID NO: 3; the nucleotide sequence of IL2 is shown in SEQ ID NO: 7; the nucleotide sequence of Fc is shown in SEQ ID NO: 9; the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1; and the nucleotide sequence of the linker is shown in SEQ ID NO: 5.

[0018] Preferably, the nucleotide sequence of the fusion protein A is shown in SEQ ID NO: 11; and the nucleotide sequence of the fusion protein B is shown in SEQ ID NO: 15.

[0019] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0020] Specifically, the expression vectors include, but are not limited to: plasmids, bacteriophages, viruses, artificial chromosomes, transposons, Cos plasmid vectors, integrative vectors, or free vectors.

[0021] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or expression vector.

[0022] Specifically, the host cells include, but are not limited to, prokaryotic cells or eukaryotic cells.

[0023] Preferably, the prokaryotic cells include, but are not limited to, Escherichia coli.

[0024] Preferably, the eukaryotic cells include, but are not limited to, yeast cells, insect cells, or animal cells.

[0025] Preferably, the yeast cells include, but are not limited to, Saccharomyces cerevisiae or Pichia pastoris; the insect cells include, but are limited to, Sf9 cells or St21 cells; and the animal cells include, but are limited to, CHO cells, BHK cells, HEK293 cells, COS cells, MDCK cells, Vero cells, Namalwa cells, or C127 cells.

[0026] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned bifunctional fusion protein or nucleic acid or expression vector or host cell.

[0027] Specifically, the bifunctional fusion protein, nucleic acid, expression vector, or host cell is the only or main active ingredient.

[0028] Specifically, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0029] Preferably, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0030] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0031] Specifically, the drug dosage forms include, but are not limited to: liquid solutions only, lyophilized powders, pre-filled syringes only, pre-filled syringes, lyophilized powders, tablets, capsules, granules, and sprays.

[0032] Specifically, the routes of administration include, but are not limited to: intraocular injection, intravenous injection, intramuscular injection, subcutaneous injection, nasal inhalation, and oral administration.

[0033] In another aspect, the present invention provides a kit comprising the aforementioned bifunctional fusion protein or nucleic acid or expression vector or host cell.

[0034] Specifically, the kit also includes, but is not limited to, a solid-phase support, a detection label, a detection substrate, and / or a buffer solution.

[0035] More specifically, the solid-phase carrier can be an affinity material that can immobilize specific antibodies on its surface.

[0036] The detection marker may be an enzyme marker, which is an enzyme that can bind to an antibody and is used to detect the binding of the antibody to the antigen.

[0037] The detection substrate can be the reaction product of an enzyme-labeled substance, which can generate a measurable signal under enzyme catalysis.

[0038] Furthermore, the present invention provides a method for preparing the aforementioned bifunctional fusion protein, comprising the following steps: S1. Culture the aforementioned host cells, centrifuge the cells, and collect the culture medium; S2. Pass the collected culture medium through polycarbonate membranes with different pore sizes; S3. Pass the filtered culture medium through an adsorption column to adsorb the bifunctional fusion protein; S4. Elution buffer is used to elute the bifunctional fusion protein adsorbed on the adsorption column to obtain a bifunctional fusion protein solution. S5. Ultrafiltration of the bifunctional fusion protein solution obtained by ultrafiltration with an ultrafiltration tube yields a high-concentration bifunctional fusion protein solution.

[0039] In another aspect, the present invention provides the use of the aforementioned bifunctional fusion protein or nucleic acid or expression vector or host cell or pharmaceutical composition or kit or fusion protein GLP-1-Fc in the preparation of products for the prevention and / or treatment of autoimmune diseases.

[0040] Specifically, the amino acid sequence of the fusion protein GLP-1-Fc is shown in SEQ ID NO: 19.

[0041] Specifically, the autoimmune disease can be type 1 diabetes.

[0042] The beneficial effects of this invention are as follows: (1) The bifunctional fusion protein provided by the present invention can selectively stimulate the residence and proliferation of Foxp3+ T cells in the pancreatic islet region.

[0043] (2) The bifunctional fusion protein provided by this invention can effectively reduce CD8 + T-cell infiltration.

[0044] (3) The bifunctional fusion protein provided by the present invention can promote the proliferation of pancreatic β cells and increase the mass of pancreatic β cells.

[0045] (4) The bifunctional fusion protein provided by the present invention can significantly reduce the incidence of type 1 diabetic mice, among which GLP-1-IL2-Fc has the best effect. Attached Figure Description

[0046] Figure 1This is a schematic diagram of the GLP-1-IL2-Fc fusion protein gene sequence.

[0047] Figure 2 This is a schematic diagram of the GLP-1-IL2 fusion protein gene sequence.

[0048] Figure 3 This is a schematic diagram of the IL2-Fc fusion protein gene sequence.

[0049] Figure 4 This is a schematic diagram of the GLP-1-Fc fusion protein gene sequence.

[0050] Figure 5 This is a schematic diagram of the GLP-1-Fc-IL2 fusion protein gene sequence.

[0051] Figure 6 The graph shows the percentage of pSTAT5 in CD8+ T cells of NOD mice after different treatments (a), the percentage of pSTAT5 in CD4+ T cells of NOD mice after different treatments (b), the percentage of pSTAT5 in CD4+ Treg cells of NOD mice after different treatments (c), and the average fluorescence intensity of pSTAT5 in spleen cells of three different NOD mice after different treatments (d).

[0052] Figure 7 The graphs show the blood glucose levels of NOD mice under different treatments, where a represents group I, b represents group II, c represents group III, d represents group IV, and e represents group V.

[0053] Figure 8 The graph shows the changes in body weight of NOD mice under different treatments, where a represents group I, b represents group II, c represents group III, d represents group IV, and e represents group V.

[0054] Figure 9 In vivo imaging of various organs in NOD mice after 2 h and 6 h of different treatments.

[0055] Figure 10 The radiation efficiency of various organs in NOD mice after 2 h of different treatments is shown in (a) and (b) respectively.

[0056] Figure 11 Blood biochemical levels in NOD mice after 50 days of different treatments.

[0057] Figure 12 Immunofluorescence staining images of the pancreas (glucagon and insulin) of mice after different treatments.

[0058] Figure 13 H&E staining images of mouse pancreas after different treatments.

[0059] Figure 14 H&E staining images of major organs (heart, liver, spleen, lung, and kidney) of mice after different treatments.

[0060] Figure 15 Tunel fluorescence staining images of mouse pancreas after different treatments. Detailed Implementation

[0061] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0062] "Host cell" refers to a cell that has been transformed with a nucleic acid sequence or is capable of being transformed and thereby expressing the target gene. This term includes the offspring of the parent cell, regardless of whether the offspring are identical to the original parent cell in morphology or genetic composition, as long as the target gene is present.

[0063] "Operationally linked" refers to an arrangement of elements in which such components are configured to perform their normal function. Thus, a given signal peptide, effectively linked to a polypeptide, directs the polypeptide from the cell for secretion. In the case of a promoter, a promoter effectively linked to a coding sequence will direct the expression of the coding sequence. The promoter or other control element need not be contiguous with the coding sequence, as long as it serves to direct its expression. For example, an inserted untranslated but transcribed sequence may exist between the promoter and coding sequences, and the promoter and coding sequences can still be considered "operationally linked."

[0064] Plasmid vectors are artificially constructed based on natural plasmids to facilitate laboratory operations. Compared to natural plasmids, plasmid vectors typically carry one or more selective marker genes (such as antibiotic resistance genes) and a synthetically produced multiple cloning site sequence containing multiple restriction endonuclease recognition sites. Most non-essential sequences have been removed to minimize the molecular weight for easier genetic engineering manipulation. Commonly used plasmid vectors are generally between 1 kb and 10 kb in size, such as PBR322, PUC series, PGEM series, PET series, and pBluescript (pBS).

[0065] Type 1 diabetes is a type of diabetes in which insufficient insulin is produced, leading to elevated blood sugar levels. Symptoms of type 1 diabetes include frequent urination, increased thirst, increased hunger, weight loss, blurred vision, fatigue, and poor healing. While the exact cause of type 1 diabetes is not fully understood, its underlying mechanism involves the autoimmune destruction of beta cells in the pancreas that produce insulin.

[0066] In the following examples and effects, all reagents are reagent grade or higher. IL-2 in this document may be abbreviated as IL2.

[0067] Example 1: Construction of engineered GLP-1-IL2-Fc fusion protein Experimental materials: Transfection reagent: linear polyethyleneimine (PEI) MW40000 (Yeasen); Culture medium: SMM 293-TII medium (SinoBiological).

[0068] Experimental steps: 1. Constructing the engineered GLP-1-IL2-Fc fusion protein gene sequence like Figure 1 As shown, the fusion protein gene GLP-1-IL2-Fc, containing GLP-1 and human IL2 mutant protein, comprises, in sequence: a signal peptide, glucagon-like peptide-1 (GLP-1), human IL2 mutant protein, and a mutant Fc fragment of IgG1. The Fc fragment of IgG1 undergoes amino acid mutations at L234A, L235A, and P329G, reducing the effector function of the Fc.

[0069] The nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; The nucleotide sequence of glucagon-like peptide-1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; The linker nucleotide sequence connecting GLP-1 and IL2 is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6; The nucleotide sequence of the human IL2 mutant protein is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. The nucleotide sequence of the mutant Fc fragment of IgG1 is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10. The nucleotide sequence of the fusion protein GLP-1-IL2-Fc is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12.

[0070] SEQ ID NO: 1: ACCATGGACTGGACCTGGCGGATCCTGTTTCTGGTCGCCGCCGCCACCGGCGCCCAT; SEQ ID NO: 2: MDWTWRILFLVAAATGAHS; SEQ ID NO: 3: CACGGCGAGGGCACCTTCACCAGCGACGTGTCTAGCTACCTGGAAGAGCAGGCCGCTAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGA; SEQ ID NO: 4: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG; SEQ ID NO: 5: GGAGGCGGCGGCAGCGGCGGAGGTGGCAGCGGCGGCGGCGGATCT; SEQ ID NO: 6: GGGGSGGGGSGGGGS; SEQ ID NO: 7: AGTGCTCCAACATCTTCCAGCACCAAAAAGACCCAGCTGCAGCTGGAGCACCTGCTGCTGGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAGAACCCTAAGCTGACCAGAATGCTGACTTTCAAGTTCTACATGCCCAAAAAAGCGACCGAGCTGAAGCACCTGCAGTGTCTGGAAGAGGAGCTGAAGCCTCTGGAGGAAGTGCTCAATCTGGCCCAGAGCAAGAACTTCCACCTGAGACCTCGGGACCTGATCAGCCGGATCAACGTGATCGTGCTGGAACTTAAAGGCAGCGAGACTACCTTTATGTGTGAATACGCCGATGAGACAGCCACAATCGTGGAATTTCTGAATAGATGGATCACATTTTCTCAGAGCATCATCTCAACACTG; SEQ ID NO: 8: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT; SEQ ID NO: 9: ACCGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCGTGCCCTGCTCCTGAAGCCGCTGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAACCTAAGGACACCCTGATGATCTCCAGAACACCCGAGGTGACATGCGTGGTCGTGGACGTCAGCCACGAAGATCCTGAGGTCAAATTCAACTGGTACGTGGATGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAACCAAGAGAACCTCAGGTGTACACACTGCCTCCAAGCCGCGACGAGCTGACAAAGAATCAAGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTATCCTTCTGACATCGCCGTGGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACAACCCCCCCCGTGCTGGACAGCGATGGCTCCTTCTTCCTTTATTCTAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTTATGCACGAGGCCCTGCATAATCACTACACCCAGAAGAGCCTCAGCCTGAGCCCCGGCAAG; SEQ ID NO: 10: EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; SEQ ID NO: 11: SEQ ID NO: 12: MDWTWRILFLLVAAATGAHSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTEPKSCDKTHTCPPCPAP EAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0071] 2. Transfection of host cells with GLP-1-IL2-Fc fusion protein gene plasmid (1) HEK293 cells (Thermo Fisher, A14527) in logarithmic growth phase were transferred to 1L cell shake flasks for culture at a cell density of 2.0×10⁻⁶. 6 -4.0×10 6 Transfection can be performed when the viability exceeds 90%; (2) For transfection, use linear polyethyleneimine (PEI) 40000 reagent. Dilute 2 mL of 1 mg / mL PEI 40000 with 10 mL of serum-free SMM 293-TII medium and mix thoroughly to form solution I. (3): Dilute the GLP-1-IL2-Fc plasmid with SMM 293-TII medium. Add 600 μg of the plasmid carrying the GLP-1-IL2-Fc fusion protein gene to 10 mL of serum-free SMM 293-TII medium and mix thoroughly to form Solution II. (4) Mix solutions I and II thoroughly to obtain solution III. After incubating at room temperature for 15 min, transfer solution III to a cell shake flask and add 70 mL of SMM 293-TII medium containing 1 wt% penicillin-streptomycin mixture and 0.5 wt% FBS. After transfection, place the cells in a shaker incubator for routine culture.

[0072] (5) Four days after plasmid transfection, centrifuge the cell suspension at 4500×g for 20 min at 4℃ and collect the supernatant.

[0073] Example 2: Purification and Identification of Engineered GLP-1-IL2-Fc Fusion Protein The method is as follows: (a) The supernatant obtained in Example 1 was filtered through 0.45 μm and 0.22 μm polycarbonate membranes using a vacuum filtration device.

[0074] (b) The above filtrate was placed on ice and the fusion protein in the filtrate was adsorbed by loading it onto a MabSelect PrismA gel column (cytiva, 17549801). The fusion protein was then eluted with 50 mM sodium citrate elution buffer at pH 3.2.

[0075] (c) The final protein eluent was concentrated by ultrafiltration through a 30 kDa ultrafiltration tube, and the eluent was replaced with PBS. The protein concentration was determined by a Nanodrop micro spectrophotometer.

[0076] (d) Add Loading Buffer to the fusion protein sample, heat in a 95°C metal bath for 5 min, and then cool on ice.

[0077] (e) Place the precast gel into the electrophoresis tank and add electrophoresis buffer to cover the gel. Remove the comb and add 5 μg of protein sample and 5 μL of marker vertically into the precast gel.

[0078] (f) Run at 200 V for 35 min until electrophoresis is complete. At the end, remove the gel and stain with Coomassie Brilliant Blue on a shaker at room temperature for 30 min.

[0079] (g) Remove the gel from the staining solution and place it in the destaining solution. Let it stand overnight until the background is clear. Take pictures and observe using a gel imaging system.

[0080] Example 3: Construction of engineered GLP-1-IL2 fusion protein Experimental materials: Transfection reagent: linear polyethyleneimine (PEI) MW40000 (Yeasen); Culture medium: SMM 293-TII medium (SinoBiological).

[0081] Experimental steps: 1. Constructing an engineered GLP-1-IL2 fusion protein gene sequence like Figure 2 As shown, the fusion protein gene GLP-1-IL2, which contains GLP-1 and human IL2 mutant protein, includes the following components connected in sequence: signal peptide, glucagon-like peptide-1 (GLP-1), human IL2 mutant protein, and FLAG® epitope tag.

[0082] The FLAG® epitope tag nucleotide sequence is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14.

[0083] SEQ ID NO: 13: GATTACAAGGACGACGATGACAAG; SEQ ID NO: 14: DYKDDDDK.

[0084] The signal peptide nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the glucagon-like peptide-1 nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; the linker nucleotide sequence linking GLP-1 and IL2 is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6; the human IL2 mutant protein nucleotide sequence is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8; the fusion protein GLP-1-IL2 nucleotide sequence is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16.

[0085] SEQ ID NO: 15: ; SEQ ID NO: 16: MDWTWRILFLLVAAATGAHSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTDYKDDDDK.

[0086] 2. Transfection of host cells with GLP-1-IL2 fusion protein gene plasmid (1) HEK 293 cells (Thermo Fisher, A14527) in logarithmic growth phase were transferred to 1L cell shake flasks for culture at a cell density of 2.0×10⁻⁶.6 -4.0×10 6 Transfection can be performed when the viability exceeds 90%; (2) For transfection, use linear polyethyleneimine (PEI) 40000 reagent. Dilute 2 mL of 1 mg / mL PEI 40000 with 10 mL of serum-free SMM 293-TII medium and mix thoroughly to form solution I. (3) Dilute the GLP-1-IL2 plasmid with SMM 293-TII medium. Add 600 μg of the plasmid carrying the GLP-1-IL2 fusion protein gene to 10 mL of serum-free SMM 293-TII medium and mix thoroughly to form Solution II. (4) Mix solutions I and II thoroughly to obtain solution III. After incubating at room temperature for 15 min, transfer solution III to a cell shake flask and add 70 mL of SMM 293-TII medium containing 1 wt% penicillin-streptomycin mixture and 0.5 wt% FBS. After transfection, place the cells in a shaker incubator for routine culture.

[0087] (5) Four days after plasmid transfection, centrifuge the cell suspension at 4500×g for 20 min at 4℃ and collect the supernatant.

[0088] Example 4: Purification and Identification of Engineered GLP-1-IL2 Fusion Protein The method is as follows: (a) The supernatant obtained in Example 3 was filtered through 0.45 μm and 0.22 μm polycarbonate membranes using a vacuum filtration device.

[0089] (b) The above filtrate was placed on ice and the fusion protein in the filtrate was adsorbed by loading it onto an anti-FLAG-tagged M1 Agar Affinity Gel column (Millipore Sigma, A4596). The fusion protein was then eluted with 0.1M glycine elution buffer at pH 3.2.

[0090] (c) The final protein eluent was concentrated by ultrafiltration through a 10 kDa ultrafiltration tube, and the eluent was replaced with PBS. The protein concentration was determined by Nanodrop micro spectrophotometer.

[0091] (d) Add Loading Buffer to the fusion protein sample, heat in a 95°C metal bath for 5 min, and then cool on ice.

[0092] (e) Place the precast gel into the electrophoresis tank and add electrophoresis buffer to cover the gel. Remove the comb and add 5 μg of protein sample and 5 μL of marker vertically into the precast gel.

[0093] (f) Run at 200 V for 35 min until electrophoresis is complete. After electrophoresis, remove the gel and stain with Coomassie Brilliant Blue on a shaker at room temperature for 30 min.

[0094] (g) Remove the gel from the staining solution and place it in the destaining solution. Let it stand overnight until the background is clear. Take pictures and observe using a gel imaging system.

[0095] Example 5: Construction of engineered IL2-Fc fusion protein Experimental materials: Transfection reagent: linear polyethyleneimine (PEI) MW40000 (Yeasen); Culture medium: SMM 293-TII medium (SinoBiological).

[0096] Experimental steps: 1. Constructing the engineered IL2-Fc fusion protein gene sequence like Figure 3 As shown, the fusion protein gene IL2-Fc, which contains a human IL2 mutant protein, includes, in sequence: a signal peptide, a human IL2 mutant protein, and a mutant Fc fragment of IgG1.

[0097] The nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the nucleotide sequence of the human IL2 mutant protein is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8; the nucleotide sequence of the IgG1Fc fragment is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10; the nucleotide sequence of the fusion protein IL2-Fc is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18.

[0098] SEQ ID NO: 17: SEQ ID NO: 18: MDWTWRILFLLVAAATGAHSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0099] 2. Transfection of host cells with IL2-Fc fusion protein gene plasmid (1) HEK293 cells (Thermo Fisher, A14527) in logarithmic growth phase were transferred to 1L cell shake flasks for culture at a cell density of 2.0×10⁻⁶. 6 -4.0×10 6 Transfection can be performed when the viability exceeds 90%; (2) Transfection was performed using linear polyethyleneimine (PEI) 40000 reagent. 2 mL of 1 mg / mL PEI 40000 was diluted with 10 mL of serum-free SMM 293-TII medium and mixed thoroughly to form solution I. (3) Dilute the IL2-Fc plasmid with SMM 293-TII medium. Add 600 μg of the plasmid carrying the IL2-Fc fusion protein and ampicillin resistance gene to 10 mL of serum-free SMM 293-TII medium and mix thoroughly to form Solution II. (4) Mix solutions I and II thoroughly to obtain solution III. After incubating at room temperature for 15 min, transfer solution III to a cell shake flask and add 70 mL of SMM 293-TII medium containing 1 wt% penicillin-streptomycin mixture and 0.5 wt% FBS. After transfection, place the cells in a shaker incubator for routine culture.

[0100] (5) Four days after plasmid transfection, centrifuge the cell suspension at 4500×g for 20 min at 4℃ and collect the supernatant.

[0101] Example 6: Purification and Identification of Engineered IL2-Fc Fusion Protein The method is as follows: (a) The supernatant obtained in Example 5 was filtered through 0.45 μm and 0.22 μm polycarbonate membranes using a vacuum filtration device.

[0102] Steps (b)-(g) are the same as in Example 2 above.

[0103] Example 7: Construction of engineered GLP-1-Fc fusion protein Experimental materials: Transfection reagent: linear polyethyleneimine (PEI) MW40000 (Yeasen); Culture medium: SMM 293-TII medium (SinoBiological).

[0104] Experimental steps: 1. Constructing an engineered GLP-1-Fc fusion protein gene sequence like Figure 4 As shown, the fusion protein gene containing GLP-1 includes, in sequence: signal peptide, GLP-1, and a mutant Fc fragment of IgG1.

[0105] The nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the nucleotide sequence of GLP-1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; the nucleotide sequence of the IgG1Fc fragment is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10; the nucleotide sequence of the fusion protein GLP-1-Fc is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 20.

[0106] SEQ ID NO: 19: GCCACCATGGACTGGACCTGGCGGATCCTGTTCCTGGTGGCCGCCGCCACCGGCGCTCACAGCCACGGCGAGGGCACATTCACATCTGATGTTAGCTCATATCTGGAAGAGCAGGCCGCAAAAGAGTTCATCGCCTGGCTCGTCAAGGGCGGCGGAGGCGGAGGTGGCTCTGGAGGCGGTGGATCTGGTGGTGGCGGATCAGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGCCCCGCTCCTGAGGCCGCCGGAGGTCCTTCCGTGTTCCTGTTTCCTCCAAAACCTAAGGACACCCTGATGATCAGCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTCAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTTTCCGTGCTGACTGTGCTGCATCAAGACTGGCTGAACGGCAAAGAGTACAAGTGTAAAGTGTCCAACAAGGCCCTGGGCGCCCCTATCGAGAAGACCATCAGCAAAGCCAAGGGCCAACCTAGGGAACCCCAGGTGTACACCCTGCCTCCAAGCAGAGATGAGCTGACCAAGAACCAGGTGAGCCTGACATGCCTGGTGAAGGGATTCTACCCCAGCGATATTGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAAAACAACTACAAGACAACACCTCCTGTGCTGGACTCTGACGGCAGCTTTTTCCTGTACAGCAAGCTGACAGTGGACAAGTCTAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCTCTGCACAATCACTACACCCAGAAGAGCCTGTCTCTGAGCCCCGGCAAGTAA; SEQ ID NO: 20: MDWTWRILFLLVAAATGAHSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0107] 2. Transfection of host cells with GLP-1-Fc fusion protein gene plasmid (1) HEK293 cells (Thermo Fisher, A14527) in logarithmic growth phase were transferred to 1L cell shake flasks for culture at a cell density of 2.0×10⁻⁶. 6 -4.0×10 6 Transfection can be performed when the viability exceeds 90%; (2) For transfection, use linear polyethyleneimine (PEI) 40000 reagent. Dilute 2 mL of 1 mg / mL PEI 40000 with 10 mL of serum-free SMM 293-TII medium and mix thoroughly to form solution I. (3) Dilute the GLP-1-Fc plasmid with SMM 293-TII medium. Add 600 μg of the plasmid carrying the GLP-1-Fc fusion protein gene to 10 mL of serum-free SMM 293-TII medium and mix thoroughly to form Solution II. (4) Mix solutions I and II thoroughly to obtain solution III. After incubating at room temperature for 15 min, transfer solution III to a cell shake flask and add 70 mL of SMM 293-TII medium containing 1 wt% antibiotics and 0.5 wt% FBS. After transfection, place the cells in a shaker incubator for routine culture.

[0108] (5) Four days after plasmid transfection, centrifuge the cell suspension at 4500×g for 20 min at 4℃ and collect the supernatant.

[0109] Example 8: Purification and Identification of Engineered GLP-1-Fc Fusion Protein (a) The supernatant obtained in Example 7 was filtered through 0.45 μm and 0.22 μm polycarbonate membranes using a vacuum filtration device.

[0110] Steps (b)-(g) are the same as in Example 2 above.

[0111] Example 9: Construction of engineered GLP-1-Fc-IL2 fusion protein Experimental materials: Transfection reagent: linear polyethyleneimine (PEI) MW40000 (Yeasen); Culture medium: SMM 293-TII medium (SinoBiological).

[0112] Experimental steps: 1. Constructing an engineered GLP-1-Fc-IL2 fusion protein gene sequence like Figure 5 As shown, the fusion protein gene containing GLP-1-Fc-IL2 includes, in sequence: a signal peptide, a mutant Fc fragment of IL2-IgG1, and GLP-1.

[0113] The nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the nucleotide sequence of GLP-1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; the nucleotide sequence of the IgG1Fc fragment is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10; the nucleotide sequence of the fusion protein GLP-1-Fc is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 20; the nucleotide sequence of the fusion protein GLP-1-Fc-IL2 is shown in SEQ ID NO: 21, and the amino acid sequence is shown in SEQ ID NO: 22.

[0114] SEQ ID NO: 21: SEQ ID NO: 22: MDWTWRILFLLVAAATGAHSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISR INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG.

[0115] 2. Transfection of host cells with GLP-1-Fc-IL2 fusion protein gene plasmid Steps (1)-(5) are the same as in Example 3 above.

[0116] Example of effect 1. Stimulation test of pSTAT5 in spleen Treg cells by GLP-1-IL2-Fc, GLP-1-IL2, GLP-1-Fc and IL2-Fc Test subjects: fusion proteins prepared according to Examples 1, 3, 5, and 7; Testing equipment: FACS flow cytometer; Test Procedure: Spleen cells were obtained from NOD mouse spleens after grinding, cleavage, and washing twice. Cells were resuspended in 1640 T cell culture medium (cytokine-free) at 2 million cells / well in U-bottom culture dishes. Fusion proteins prepared according to Examples 1, 3, 5, and 7 were formulated into 10... -2 10 -1 1, 10, 10 2 10 3 10 4 and 10 5Add 200 μL of the above-mentioned medium to pM 1640 medium and incubate in a cell culture incubator for 15 minutes. Immediately add 100 μL of TFP Fix / Perm Buffer, and divide each well (300 μL of cells) into three aliquots of 100 μL each; add 100 μL of TFP Fix / Perm Buffer to each well and incubate at 4°C for 50 minutes. Wash twice with Perm / wash buffer, centrifuge at 600×g for 5 minutes at 4°C. Add 200 μL of Perm Buffer III to each well, mix well, and incubate on ice for 20 minutes. Wash three times with TFP Fix / Perm Buffer. Perform antibody staining, incubate at 4°C in the dark for 45 minutes, wash once with TFP Fix / Perm Buffer, and add 120 μL of Cell Staining Buffer for analysis.

[0117] Test results: such as Figure 6 As shown, the details are as follows: like Figure 6 As shown, the four fusion proteins did not significantly stimulate pSTAT5 in CD8+ T cells and CD4+ Tconv cells. However, the stimulation of pSTAT5 in CD4+ Treg cells showed a gradient increase. At a fusion protein concentration of 100 nM, GLP-1-IL2-Fc from Example 1 had the weakest stimulatory effect on pSTAT5, GLP-1-IL2 from Example 3 had the strongest stimulatory effect, and IL2-Fc from Example 5 had the second strongest effect.

[0118] 2. Type 1 diabetes prevention test for GLP-1-IL2-Fc, GLP-1-IL2, GLP-1-Fc and IL2-Fc Test subjects: Four fusion proteins prepared according to the schemes of Example 1, Example 3, Example 5 and Example 7.

[0119] Test Procedure: Female NOD mice (purchased from Beijing Huafukang) were used as a spontaneous diabetes animal model. Twelve-week-old NOD mice were randomly divided into five groups, designated as groups I-V: Group I (untreated, n=10), Group II (GLP-1-Fc, n=10), Group III (IL2-Fc, n=10), Group IV (GLP-1-IL2, n=10), and Group V (GLP-1-IL2-Fc, n=10). NOD mice in groups II-V were intraperitoneally injected with GLP-1-IL2-Fc, GLP-1-IL2, GLP-1-Fc, and IL2-Fc, respectively, at a dose of 60 nmol / mouse (calculated based on protein molecular weight). NOD mice in group I were intraperitoneally injected with 100 μL of PBS buffer. Injections were administered every 3 days for a total of 10 injections. Blood glucose levels and body weight were monitored using a Roche glucometer and a weighing scale during the treatment period. If a mouse loses 15%-20% of its body weight during treatment, it should be euthanized immediately. After treatment, the mouse should be euthanized again for further analysis.

[0120] Test results: such as Figure 7 As shown, compared with control groups I, II, and III, GLP-1-IL2 and GLP-1-IL2-Fc in groups IV and V effectively prevented diabetes in NOD mice, maintaining their blood glucose levels within the normal range. The treatment effect in group V was superior to that in group IV. Within 120 days of treatment, in group V, 9 out of 10 NOD mice maintained normal blood glucose levels; in group IV, 8 out of 10 NOD mice maintained normal blood glucose levels; in group I, 7 out of 10 NOD mice developed the disease and eventually progressed to hyperglycemia (>600 mg / dL); in group II, 3 out of 10 NOD mice developed the disease and became hyperglycemic; and in group III, 4 out of 10 NOD mice developed the disease and became hyperglycemic.

[0121] like Figure 8 As shown, treatment with GLP-1-IL2-Fc and GLP-1-IL2 fusion proteins did not significantly reduce the body weight of mice, indicating that the fusion protein treatment did not cause significant biotoxicity. However, the progression of diabetes caused a decrease in the body weight of mice. In the experiment, mice with diabetes were euthanized immediately when their body weight decreased by 15%-20%.

[0122] 3. Imaging of the in vivo distribution of fusion proteins Test subjects: NOD mice at different time points after treatment; Test content: Following the above treatment and grouping method, female NOD mice in groups II-V were euthanized 2 h and 6 h after treatment. NOD mice given GLP-1-Fc were used as a positive control group. Organs such as heart, liver, spleen, lung, kidney and pancreas were removed and imaged under a small animal imaging system.

[0123] Test results: such as Figure 9 neutralization Figure 10 As shown, at 2 h of treatment, GLP-1-Fc exhibited strong fluorescence in the pancreas of NOD mice, and two NOD mice in the GLP-1-IL2-Fc group also showed strong fluorescence in their pancreas. The IL2-Fc and GLP-1-IL2 groups showed strong fluorescence in the liver. At 6 h of treatment, GLP-1-Fc still showed strong fluorescence in the pancreas of NOD mice, while the other three groups showed strong fluorescence in the liver.

[0124] 4. Tests of fusion protein on blood biochemistry in NOD mice Test Procedure: After 50 days of treatment, female NOD mice in group IV of test 2 above underwent orbital blood collection. The blood was incubated in coagulation-promoting tubes at room temperature for 1 hour, then centrifuged at 4000 rpm for 10 minutes. The supernatant was collected, and the plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CREA-S), urea (UREA), and albumin (ALB II) were measured.

[0125] Test results: such as Figure 11 As shown, there were no significant differences in various blood biochemical indicators (including ALT, AST, ALP, CREA-S, UREA, and ALB II) in NOD mice in group IV, indicating that GLP-1-IL2-Fc, GLP-1-IL2, GLP-1-Fc and IL2-Fc have good biocompatibility and do not cause significant organ damage.

[0126] 5. Immunofluorescence assay of mouse tissues after treatment Test subjects: Pancreatic tissue of mice after the completion of the above two treatments; Test Procedure: After treatment of female NOD mice in groups II-IV as described above, female NOD mice, untreated NOD mice after the onset of the disease, and healthy mice were euthanized, and the pancreas and spleen of the mice were collected and fixed with paraformaldehyde for 24 hours to embed in paraffin. The pancreas and spleen were cut using a microtome and mounted on glass slides. The sections were dehydrated with xylene, anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol, incubated in 3% H2O2 for 10-20 min, heat-retarded with EDTA for 15-20 min, and sealed in 3% BSA with 0.2% Triton-X 100 for 30 min. Then, they were stained with different primary antibodies: glucagon antibody (Abcam, CAT.Ab92517) and insulin antibody (Abcam, CAT.Ab6995) and incubated overnight at 4°C according to the manufacturer's instructions. After incubation for 30 min with fluorescently labeled secondary antibodies Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor® 488 Conjugate) (CST, CAT. No. 4412); and Anti-rabbit IgG (H+L), (Alexa Fluor® 647 Conjugate) (CST, CAT. No. 4418), the slides were stained with DAPI and analyzed using a digital slide scanner (Olympus VS200).

[0127] Test results: such as Figure 12 The image shows immunofluorescence staining of the pancreas. Green fluorescence represents labeled glucagon, i.e., the islets of Langerhans, while red fluorescence represents labeled insulin. In NOD mice with diabetes, the red fluorescence area was significantly reduced in the untreated group and in groups II and III, indicating that pancreatic lesions had occurred and insulin production was low. Group IV showed a significantly larger red fluorescence area, indicating that the islets were still producing sufficient insulin normally. Therefore, treatment in group IV can alleviate pancreatic lesions and is beneficial in protecting pancreatic β cells.

[0128] 6. Hematoxylin-eosin (H&E) staining assay of tissues Test Procedure: Female NOD mice in group IV were euthanized after treatment, and major organs, including pancreas, heart, liver, spleen, lungs, and kidneys, were collected. These organs were fixed in paraformaldehyde for 24 hours and embedded in paraffin. Sections were then stained with Hematoxylin and eosin (H&E). Analysis was performed using a digital slide scanner (Olympus VS200). Untreated healthy female NOD mice served as the control group, and the same procedures were performed. Test results: such as Figure 13As shown, compared with the untreated group, the pancreatic tissue of mice in groups II-IV showed obvious islet morphology after treatment. However, mice in groups II and III showed significant immune cell infiltration, while the islets in groups IV and V retained their complete shape and showed no significant immune cell infiltration. This indicates that treatment with GLP1-IL2 and GLP1-IL2-Fc fusion protein helps preserve islets and treat diabetes.

[0129] like Figure 14 As shown, there were no significant differences in the major organs (including heart, liver, spleen, lungs, and kidneys) of mice in groups I-IV, indicating that the GLP1-Fc, IL2-Fc, and GLP1-IL2-Fc fusion proteins have good biocompatibility and do not cause significant organ damage.

[0130] 7. Tunnel staining experiment of mouse pancreatic tissue Test subjects: Pancreatic tissue of mice treated with the two treatment regimens for 10 days; Test Procedure: After treatment of female NOD mice in groups II-IV, euthanized female NOD mice, untreated NOD mice, and healthy mice were collected. The pancreas and spleen were fixed in paraformaldehyde for 24 hours and embedded in paraffin. The pancreas and spleen were dissected using a microtome and mounted on slides. The sections were dehydrated using xylene, anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol, then permeabilized and blocked, stained with TUNEL working solution, and finally stained with DAPI. The sections were then analyzed using a digital slide scanner (Olympus VS200).

[0131] Test results: such as Figure 15 The image shows TUNEL staining of the pancreas, where green fluorescence represents apoptotic cells and blue fluorescence represents cell nuclei. The results show that there were obvious apoptotic cells in the PBS treatment group. In groups II-IV, the number of apoptotic cells was significantly reduced after protein treatment. Among them, the treatment with GLP1-Fc and GLP1-IL2-Fc resulted in the fewest apoptotic cells, which is beneficial for reducing apoptosis in pancreatic cells.

[0132] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

[0133] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A bifunctional fusion protein, characterized in that, The fusion protein A consists of glucagon-like peptide-1 (GLP-1), human interleukin-2 (IL2), and an IgG1 Fc fragment (Fc) in the order from the N-terminus to the C-terminus of amino acids. Alternatively, it could be a fusion protein B consisting of GLP-1 and IL2, arranged in the order of amino acids from the N-terminus to the C-terminus.

2. The bifunctional fusion protein according to claim 1, characterized in that, The fusion protein A and fusion protein B also include a signal peptide.

3. The bifunctional fusion protein according to claim 2, characterized in that, The amino acid sequence of the GLP-1 is shown in SEQ ID NO: 4; the amino acid sequence of the IL2 is shown in SEQ ID NO: 8; the amino acid sequence of the Fc is shown in SEQ ID NO: 10; and the amino acid sequence of the signal peptide is shown in SEQ ID NO:

2.

4. The bifunctional fusion protein according to claim 3, characterized in that, The GLP-1 and IL2 are connected via a Linker.

5. The bifunctional fusion protein according to claim 4, characterized in that, The Linker is (GGGGS)n, where n = 1-4.

6. The bifunctional fusion protein according to claim 5, characterized in that, The amino acid sequence of the linker is shown in SEQ ID NO:

6.

7. The bifunctional fusion protein according to any one of claims 1-6, characterized in that, The amino acid sequence of fusion protein A is shown in SEQ ID NO: 12; the amino acid sequence of fusion protein B is shown in SEQ ID NO:

16.

8. A nucleic acid encoding the bifunctional fusion protein according to any one of claims 1-7.

9. The nucleic acid according to claim 8, characterized in that, The nucleotide sequence of the GLP-1 is shown in SEQ ID NO: 3; the nucleotide sequence of the IL2 is shown in SEQ ID NO: 7; the nucleotide sequence of the Fc is shown in SEQ ID NO: 9; the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1; and the nucleotide sequence of the linker is shown in SEQ ID NO:

5.

10. The nucleic acid according to claim 8 or 9, characterized in that, The nucleotide sequence of fusion protein A is shown in SEQ ID NO: 11; the nucleotide sequence of fusion protein B is shown in SEQ ID NO:

15.

11. An expression vector comprising the nucleic acid of claims 8-10.

12. The expression vector according to claim 11, characterized in that, The expression vector is a plasmid, bacteriophage, virus, artificial chromosome, transposon, Cos plasmid vector, integrative vector, or free vector.

13. A host cell comprising the nucleic acid of claims 8-10 or the expression vector of any one of claims 11-12.

14. The host cell according to claim 13, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a bifunctional fusion protein according to any one of claims 1-7, a nucleic acid according to claims 8-10, an expression vector according to any one of claims 11-12, or a host cell according to any one of claims 13-14.

16. The pharmaceutical composition according to claim 15, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

17. A method for preparing the bifunctional fusion protein according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Culturing the host cells according to any one of claims 13-14, centrifuging the cells, and collecting the culture medium; S2. Pass the collected culture medium through polycarbonate membranes with different pore sizes; S3. Pass the filtered culture medium through an adsorption column to adsorb the bifunctional fusion protein; S4. Elution buffer is used to elute the bifunctional fusion protein adsorbed on the adsorption column to obtain a bifunctional fusion protein solution. S5. Ultrafiltration of the bifunctional fusion protein solution obtained by ultrafiltration with an ultrafiltration tube yields a high-concentration bifunctional fusion protein solution.

18. A reagent kit, characterized in that, The kit comprises a bifunctional fusion protein according to any one of claims 1-7, a nucleic acid according to claims 8-10, an expression vector according to any one of claims 11-12, or a host cell according to any one of claims 13-14.

19. The use of the bifunctional fusion protein of any one of claims 1-7, or the nucleic acid of any one of claims 8-10, or the expression vector of any one of claims 11-12, or the host cell of any one of claims 13-14, or the pharmaceutical composition of any one of claims 15-16, or the kit of claim 18, or the use of the fusion protein GLP-1-Fc in the preparation of products for the prevention and / or treatment of autoimmune diseases.

20. The application according to claim 19, characterized in that, The amino acid sequence of the fusion protein GLP-1-Fc is shown in SEQ ID NO:

19.

21. The application according to claim 19 or 20, characterized in that, The autoimmune disease mentioned is type 1 diabetes.

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