Fusion protein based on staphylokinase and glucagon-like peptide and application thereof

Abstract

The invention provides a fusion protein based on staphylokinase and glucagon-like peptide-1 and application of the fusion protein. The amino acid sequence of the fusion protein is shown as any one of SEQ ID No. 7 or SEQ ID No. 11. The fusion protein provided by the invention can reduce the blood sugar level of diabetes and dissolve thrombus, and provides a new direction for treatment of diabetes and complications thereof.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fusion protein based on glucagon and glucagon-like peptide and its applications. Background Technology

[0002] GLP-1 (glucagon-like peptide-1) is an incretin hormone secreted by L cells in the intestine. It primarily regulates blood glucose by stimulating insulin secretion and inhibiting glucagon release. Its action is glucose-dependent, with a relatively low risk of hypoglycemia. Furthermore, GLP-1 can delay gastric emptying and suppress appetite, aiding in weight management. Natural GLP-1 has a half-life of only 2 minutes and is easily degraded by DPP-4 enzymes. Therefore, clinically, GLP-1 receptor agonists (such as semaglutide and liraglutide) or DPP-4 inhibitors (such as sitagliptin) are commonly used for diabetes treatment. These drugs not only effectively lower blood glucose but also improve cardiovascular outcomes, and have become an important treatment option for type 2 diabetes and obesity.

[0003] Staphylokinase (SaK) is a plasminogen activator secreted by Staphylococcus aureus. It specifically activates plasminogen in thrombi, converting it into plasmin, which then dissolves fibrin and exerts a thrombolytic effect. Compared to streptokinase (SK), SaK has a higher affinity for fibrin, resulting in greater thrombolytic efficiency and less likelihood of inducing systemic bleeding. SaK shows potential in treating thrombotic diseases such as acute myocardial infarction and pulmonary embolism, but its potential to induce immune responses limits its clinical application. Currently, recombinant gene technology is used to produce SaK variants with low immunogenicity to improve its safety and efficacy.

[0004] Thrombosis is a common complication of diabetes, especially type 2 diabetes. Patients with diabetes (especially type 2 diabetes) have a significantly increased risk of thrombosis, mainly due to the following mechanisms: 1. Hyperglycemia promotes thrombosis: Long-term hyperglycemia leads to endothelial damage, promotes platelet activation and adhesion, and enhances coagulation function. Elevated blood glucose increases fibrinogen levels, increases blood viscosity, slows blood flow, and facilitates thrombosis. 2. Inflammation and oxidative stress: Diabetes is accompanied by chronic low-grade inflammation. Inflammatory factors (such as IL-6 and TNF-α) promote coagulation and inhibit the fibrinolytic system. Oxidative stress increases free radical production, further damaging the blood vessel wall and promoting thrombosis. 3. Platelet dysfunction: Platelets in diabetic patients are overactivated, making them more prone to aggregation and thrombosis. 4. Impaired fibrinolytic system: Diabetic patients have elevated levels of plasminogen activator inhibitor-1 (PAI-1), which inhibits fibrinolysis and increases the risk of thrombosis. Therefore, it is crucial to address thrombus dissolution while treating diabetic patients. Summary of the Invention

[0005] This invention provides a fusion protein based on glucagon and glucagon-like peptide and its applications.

[0006] On one hand, the present invention provides a fusion protein comprising Staphylokinase (Sak) and glucagon-like peptide-1 (GLP-1).

[0007] In one embodiment, the amino acid sequence of the glucokine is shown in SEQ ID No. 1.

[0008] In one embodiment, the amino acid sequence of the glucagon-like peptide-1 is shown in SEQ ID No. 2.

[0009] In one embodiment, the N-terminus or C-terminus of the staphylokinase is linked to glucagon-like peptide-1.

[0010] In one embodiment, the staphylokinase and the glucagon-like peptide-1 are linked by a linker protein. Preferably, the linker protein includes an enzyme cleavage site; preferably, the linker protein includes a linker; preferably, the enzyme cleavage site includes an enterokinase cleavage site and a TEV protease cleavage site; preferably, the linker protein includes an enterokinase cleavage site, a TEV protease cleavage site, and a linker.

[0011] In one embodiment, the N-terminus or C-terminus of the staphylokinase is linked to a connexin. Preferably, the C-terminus of the staphylokinase is linked to a connexin.

[0012] In one embodiment, the N-terminus or C-terminus of the glucagon-like peptide-1 is linked to a connexin. Preferably, the N-terminus of the glucagon-like peptide-1 is linked to a connexin.

[0013] In one embodiment, the C-terminus of the staphylokinase is linked to a connexin, and the N-terminus of glucagon-like peptide-1 is linked to a connexin.

[0014] In one embodiment, the fusion protein further includes an enterokinase cleavage site, a TEV protease cleavage site, and a linker. In one embodiment, the N-terminus or C-terminus of the enterokinase cleavage site is linked to the linker. In one embodiment, the N-terminus or C-terminus of the TEV protease cleavage site is linked to the linker.

[0015] In one embodiment, the amino acid sequence of the enterokinase cleavage site is shown in SEQ ID No. 3.

[0016] In one embodiment, the amino acid sequence of the TEV protease cleavage site is shown in SEQ ID No. 5.

[0017] In one embodiment, the amino acid sequence of the linker is shown in SEQ ID No. 4.

[0018] In one embodiment, the fusion protein comprises glucagon-like peptide-1, enterokinase cleavage site, TEV protease cleavage site, and linker.

[0019] In one embodiment, the fusion protein is composed of a staphylokinase, a TEV protease cleavage site, a linker, an enterokinase cleavage site, and a glucagon-like peptide-1 linked sequentially.

[0020] In one embodiment, the Staphylokinase, TEV protease cleavage site, linker, enterokinase cleavage site, and glucagon-like peptide-1 are directly linked.

[0021] In one embodiment, the fusion protein is composed of a staphylokinase, an enterokinase cleavage site, a linker, a TEV protease cleavage site, and a glucagon-like peptide-1 linked together in sequence.

[0022] In one embodiment, the Staphylokinase, enterokinase cleavage site, linker, TEV protease cleavage site, and glucagon-like peptide-1 are directly linked.

[0023] In one embodiment, the amino acid sequence of the fusion protein is as shown in either SEQ ID No. 7 or SEQ ID No. 11.

[0024] The fusion protein of the present invention is not limited by its production method. For example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0025] On the other hand, the present invention provides an isolated polynucleotide that encodes the fusion protein.

[0026] In one embodiment, the polynucleotide sequence is as shown in any one of SEQ ID No. 6, SEQ ID No. 9, or SEQ ID No. 10.

[0027] In one embodiment, the polynucleotide sequence is codon-optimized for expression in eukaryotic cells. In another embodiment, the polynucleotide sequence is codon-optimized for expression in prokaryotic cells.

[0028] In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

[0029] On the other hand, the present invention provides an expression vector comprising the aforementioned polynucleotide.

[0030] Preferably, the expression vector further includes a regulatory element operatively connected thereto. In one embodiment, the regulatory element is selected from one or more of the following: enhancers, transposons, promoters, terminators, leader sequences, polyadenylated nucleotide sequences, and marker genes.

[0031] In one embodiment, the expression vector is a type of plasmid, virus, granule, bacteriophage, etc., which are well known to those skilled in the art. Preferably, the expression vector is pICZ1Aα.

[0032] On the other hand, the present invention provides a host cell comprising the fusion protein, or the polynucleotide, or the expression vector.

[0033] In one embodiment, the host cell is transformed by the expression vector.

[0034] In one embodiment, the host cell is a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferably, the host cell is an *Escherichia coli* (e.g., *E. coli* DH5α) cell or a *Pichia pastoris* cell.

[0035] On the other hand, the present invention provides the use of the fusion protein in the preparation of reagents or drugs for treating diabetes and its complications.

[0036] In one implementation, the complications include thrombosis and obesity.

[0037] On the other hand, the present invention provides the use of the fusion protein, wherein the fusion protein is used for:

[0038] (i) Regulate or lower blood sugar;

[0039] (ii) Adjusting or reducing weight;

[0040] (iii) Dissolve blood clots;

[0041] (iv) Prolong clotting time or improve clotting;

[0042] (v) Reduce fibrin content.

[0043] On the other hand, the present invention provides a method for preparing the fusion protein, characterized in that the method includes the following steps:

[0044] 1) Obtain the target gene fusion fragment;

[0045] 2) Insert the target gene fusion fragment into the expression vector to construct a recombinant expression plasmid;

[0046] 3) Transform E. coli with the recombinant expression plasmid to amplify the number of plasmids;

[0047] 4) Extract the plasmid, transform it into Pichia pastoris, and express it in Pichia pastoris;

[0048] 5) Isolation and purification of fusion proteins.

[0049] In one embodiment, the target gene sequence in step 1) is as shown in any one of SEQ ID No. 6, SEQ ID No. 9 or SEQ ID No. 10.

[0050] In one embodiment, the expression vector in step 2) is pICZ1Aα.

[0051] In one embodiment, the specific method of step 3) is as follows: thaw DH5α competent cells on ice; add recombinant expression product, suspend on ice and then heat shock at 42°C, add LB liquid medium and incubate at 37°C, perform LB bleomycin resistance plate plating, and incubate overnight at 37°C upside down.

[0052] In one embodiment, the plasmid extraction in step 4) can be performed using a plasmid extraction kit.

[0053] In one embodiment, step 4) further includes linearizing and concentrating the extracted plasmid.

[0054] In one embodiment, the method further includes a PCR verification step.

[0055] On the other hand, the present invention provides a method for preparing a reagent or drug for treating diabetes and its complications, wherein the reagent or drug contains the fusion protein or the polynucleotide and a pharmaceutically acceptable carrier or excipient.

[0056] The drugs provided by this invention can exist in various dosage forms, such as injections for intravenous injection, transdermal absorption agents for subcutaneous injection and topical application, sprays for nasal, throat, oral, epidermal, and mucous membrane applications, drops for nasal, eye, and ear applications, suppositories, tablets, powders, granules, capsules, oral liquids, ointments, creams, etc., for rectal applications, as well as pulmonary administration preparations and other non-enteric compositions. All of the above-mentioned dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0057] The fusion protein, reagent, or drug described herein may be used clinically in mammals, including humans and animals, and may be administered via intravenous injection or oral, nasal, skin, or pulmonary inhalation routes. The preferred weekly dose is 0.1-20 mg / kg (preferably 0.13-3.51 mg / kg, e.g., 0.13 mg / kg, 0.39 mg / kg, 1.17 mg / kg, 3.51 mg / kg; more preferably, not less than 0.13 mg / kg, not less than 0.39 mg / kg, not less than 0.39 mg / kg, not less than 3.51 mg / kg), and the preferred treatment duration is 10 to 30 days. It may be administered as a single dose or in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment.

[0058] Beneficial effects

[0059] This invention provides a fusion protein comprising glucagon and glucagon-like peptide-1, which can lower blood sugar levels in diabetes, improve weight, and dissolve blood clots, providing a new treatment method for diabetes and thrombotic complications. Attached Figure Description

[0060] Figure 1 PCR validation results of SGLP-1-X-33 protein.

[0061] Figure 2 Electrophoresis diagram of SGLP-1-X-33 protein expression.

[0062] Figure 3 Electrophoresis diagram of SGLP-1-X-33-1 protein expression.

[0063] Figure 4PCR validation results of SGLP-1-X-33-2 protein.

[0064] Figure 5 Results of blood glucose monitoring in mice.

[0065] Figure 6 Mouse weight monitoring results.

[0066] Figure 7 Results of thrombin time in mice.

[0067] Figure 8 Results of fibrin content in mice. Detailed Implementation

[0068] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0069] Example 1: Construction of the Pichia pastoris expression system for the fusion protein SGLP-1

[0070] SaK (Staphylokinase, staphylokinase) protein amino acid sequence (SEQ ID No. 1): MSSSEDKGEYKKGDDASYFEPTGPYLMVNVTGVDGKGNELLSPHYVEFPIKPGTTLTKEKIEYYVEWALDATAYKELRVVELDPSAKIEVTYYDKNKKKEETKSFPITEKGFVVPDLSEHIKNPGFNLITKVVIEKK;

[0071] GLP-1 (glucagon-like peptide-1) polypeptide sequence (SEQ ID No. 2): HGEGTFTSNVSSYLEGQAAKEFIAWLVKGRG;

[0072] The protein sequences of both were synthesized according to the codon preference of Pichia pastoris, and enterokinase cleavage sites (cleavage site: DDDDK, SEQ ID No. 3), linker (GGGGS, SEQ ID No. 4), and TEV protease cleavage sites (cleavage site: ENLYFQG, SEQ ID No. 5) were added between the sequences. The synthesized gene sequence was inserted into the vector pICZ1Aα (other cleavage sites can also be selected, as long as they can be cleaved; other linkers or direct ligation can also be selected). After linearization with SGLP-1-pICZ1Aα, the protein was electroporated into Pichia pastoris strain X-33 to construct the SGLP-1-X-33 protein expression strain (the DNA sequence of the SGLP-1-X-33 protein optimized according to codon preference is shown below, SEQ ID No. 6; the amino acid sequence is shown in SEQ ID No. 7).

[0073] ATGTCCTCCTCTGAAGATAAAGGTGAATACAAGAAGGGTGATGACGCTTCTTACTTTGAACCAACTGGTCCATACTTGATGGTTAACGTTACTGGTGTTGATGGTAAGGGTAACGAATTGTTGTCTCCACATTACGTTGAATTTCCGAT TAAGCCAGGTACTACTTTGACTAAGGAAAAGATTGAATACTACGTTGAATGGGCTTTGGATGCTACTGCTTACAAGGAATTGCGCGTTGTTGAATTGGATCCGTCTGCTAAGATTGAAGTTACTTACTACGATAAGAACAAGAAAAAGG AAGAAACTAAGTCTTTTCCAATTACTGAAAAGGGATTTGTTGTTCCAGATTTGTCTGAACATATTAAGAACCCCGGTTTTAACTTGATTACTAAGGTTGTTATTGAAAAGAAG(Sak)GAAAACTTGTACTTTCAAGGT(TEV protease cleavage site dot)GGTGGTGGTTGGTTCT(linker)GATGATGATGATAAG(enterokinase cleavage site)CATGGTGAAGGTACTTTTACTTCTAACGTTTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAATTTATTGCTTGGTTGGTTAAGGGTCGGGGT(GLP-1);

[0074] Amino acid sequence (SEQ ID No. 7):

[0075] MSSSEDKGEYKKGDDASYFEPTGPYLMVNVTGVDGKGNELLSPHYVEFPIKPGTTLTKEKIEYYVEWALDATAYKELRVVELDPSAKIEVTYYDKNKKKEETKSFPITEKGFVVPDLSEHIKNPGFNLITKVVIEKKENLYFQGGGGGSDDDDKHGEGTFTSNVSSYLEGQAAKEFIAWLVKGRG;

[0076] Vector construction and Pichia pastoris transformation:

[0077] The synthetic gene was homologously recombinated with pICZ1Aα.

[0078] ① PCR linearization vector.

[0079] ②10 μL recombinase; 1.5 μL vector fragment; 2.5 μL inserted gene; 6 μL water; 50℃ for 5 min.

[0080] ③ The number of vectors amplified by DH5α transformation of the recombinant product:

[0081] Thaw DH5α competent cells on ice; add 10 μL of the recombinant product from step ②; stand on ice for 30 min; heat shock at 42℃ for 45 s; add 900 μL of LB liquid medium and incubate at 37℃ and 200 rpm for 1.5 h; take 100 μL for LB bleomycin resistance plate plating; incubate overnight at 37℃ upside down.

[0082] ④ The SGLP-1-pICZ1Aα plasmid was extracted from the transformed strain DH5α in ③. This process was completed using the Tiangen Biopharmaceutical Plasmid Extraction Kit.

[0083] ⑤ Linearize the extracted plasmid: Add SacI nuclease according to the plasmid concentration and digest at 37℃ for 3 hours.

[0084] ⑥ After linearization by enzyme digestion, concentration is performed:

[0085] Add 15 μL of 3M sodium acetate and 375 μL of anhydrous ethanol, gently shake and let stand on ice for 15 min; centrifuge at 12000 rpm for 15 min and discard the supernatant; dry the ethanol; add 20 μL of sterile water to dissolve.

[0086] ⑦ After the linearization and concentration of the SGLP-1-pICZ1Aα plasmid, Pichia pastoris transformation was performed:

[0087] Pichia pastoris was cultured in YPD medium in shake flasks until OD. 6001.3-1.6 Preparation of competent cells: Collect 20 mL of Pichia pastoris culture, centrifuge at 12000 rpm for 2 min, and discard the supernatant; resuspend the cells in an equal volume of sterile water and centrifuge, repeating three times; add 4 mL of pre-cooled 1M sorbitol solution and centrifuge; take 100 μL of 1M sorbitol solution to resuspend the cells, take 90 μL of the resuspended cells, add 10 μL of concentrated linearized plasmid, and let stand for 5 min; transfer to an electroporation cuvette and transform using an electroporator; immediately add 900 μL of 1M sorbitol solution and incubate at 30℃ and 200 rpm for one hour; take 100 μL of the transformation solution and plate on YPD bleomycin-resistant plates, incubate upside down at 30℃ to complete the transformation.

[0088] ⑧ Verification of successful transformation: The genome of the transformed Pichia pastoris was extracted and verified by PCR. The results are as follows: Figure 1 As shown. Figure 1 In the image, lanes 1-4 are shown from left to right. Lane 1 contains a 1kb full-length gold DNA maker, lane 2 contains wild-type Pichia pastoris X-33, lane 3 contains the SGLP-1-pICZ1Aα plasmid, and lane 4 contains SGLP-1-X-33. Figure 1 It can be seen that two bands appeared in lane 4. One band is consistent with lane 2, which is the self-AOX gene of Pichia pastoris X-33 obtained by PCR using AOX universal primers. The other band is consistent with lane 3, which is the foreign gene obtained by PCR using AOX universal primers after the foreign gene was integrated into the Pichia pastoris genome. This indicates that the foreign gene was successfully transformed into the Pichia pastoris genome.

[0089] In addition, a protein expression strain with GLP-1 first and Sak last was constructed (the sequence of which is SEQ ID No. 8 below), but experiments showed that the strain could not successfully express the protein.

[0090] CATGGTGAAGGTACTTTTACTTCTAACGTTTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAATTTATTGCTTGGTTGGTTAAGGGTAGA(GLP-)GAAAACTTGTACTTTCAAGGT(TEV protease cleavage site)GGTGGTGGTGGGTTCT(lin ker)GATGATGATGATAAG (enterokinase cleavage site)ATGTCCTCCTGAAGATAAGGGTGAATACAAGAAGGGTGATGATGCTTCTTACTTTGAACCAACTGGTCCATACTTGATGGTTAACGTTACTGGTGTTGATGGTAAGGGTAACGAATTG TTGTCTCCACATTACGTTGAATTTCCAATTAAGCCAGGTACTACTTTGACTAAGGAAAAGATTGAATACTACGTTGAATGGGCTTTGGATGCTACTGCTTACAAGGAATTGAGAGTTGTTGAATTGGATCCATCTGCTAAGATTGAAG TTACTTACTACGATAAGAACAAGAAGAAGGAAGAAACTAAGTCTTTTCCAATTACTGAAAAGGGATTTGTTGTTCCAGATTTGTCTGAACATATTAAGAACCCAGGTTTTAACTTGATTACTAAGGTTGTTATTGAAAAGAAG(Sak);

[0091] The experimental setup steps are the same as those described above.

[0092] Figure 2 This is an electrophoresis diagram of SGLP-1-X-33 protein expression. Figure 2 In the image, lanes 1-7 are shown from left to right. Lane 1 contains the SMOBIO protein maker PM2510, lane 2 contains the supernatant of the fermentation broth before the addition of the inducer, and lanes 3-7 contain the supernatant of the fermentation broth from days 1 to 5, respectively. The results showed that on day 5 of fermentation, the protein concentration was less than 0.1 mg / mL, indicating low protein yield and that the target protein band was not uniform and showed signs of degradation. In other words, the SGLP-1-X-33 protein expression strain could successfully express the SGLP-1 fusion protein, but the yield was low and protein degradation occurred.

[0093] To optimize the expression of the fusion protein, further codon optimization was performed on the nucleic acid sequence of the SGLP-1-X-33 protein (mainly optimizing the nucleic acid sequences of SaK and GLP-1). The optimized sequence (SGLP-1-X-33-1, SEQ ID No. 9, amino acid sequence as shown in SEQ ID No. 7) is as follows: ATGTCCTCCTCTGAAGATAAGGGTGAATACAAGAAGGGTGATGATGCTTCTTACTTTGAACCAACTGGTCCATACTTGATGGTTAACGTTACTGGTGTTGATGGTAAGGGTAACGAATTGTTGTCTCCACATTACGTTGAATTTCCAATTAAGCCAGGTACTACTTTGACTAAGGAAAAGATTGAATACTACGTTGAATGGGCTTTGGATGCTACTGCTTACAAGGAATTGAGAGTTGTTGAATTGGATCCATCTGCTAAGATTGAAGTTACTTACTACGATAAGAACAAGAA GAAGGAAGAAACTAAGTCTTTTCCAATTACTGAAAAGGGATTTGTTGTTCCAGATTTGTCTGAACATATTAAGAACCCAGGTTTTAACTTGATTACTAAGGTTGTTATTGAAAAGAAG(Sak)GAAAACTTGTACTTTCAAGGT(TEV Protease Cleavage site) GGTGGTGTTGGTTCT (linker) GATGATGATGATAAG (enterokinase cleavage site) CATGGTGAAGGTACTTTTACTTCTAACGTTTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAATTTATTGCTTGGTTGGTTAAGGGTAGAGGT (GLP-1);

[0094] The optimized sequence was expressed and purified according to the steps described above, and the results are as follows: Figure 3 As shown. Figure 3 In the image, lanes 1-7 are shown from left to right. Lane 1 contains the SMOBIO protein maker PM2510, lane 2 contains the supernatant of the fermentation broth before the addition of the inducer, and lanes 3-7 contain the supernatant of the fermentation broth from day 1 to day 5 of induced fermentation, respectively. The results indicate that the codon-optimized SGLP-1-X-33-1 protein reached a concentration of 0.35 mg / mL, and no protein band degradation was observed during expression from day 1 to day 5.

[0095] In addition, the order of adding the restriction enzyme sites was adjusted, with two different linking methods: TEVp-linker-EK (i.e., SGLP-1-X-33-1) and EK-linker-TEVp (SGLP-1-X-33-2, nucleic acid sequence as shown in SEQ ID No. 10, amino acid sequence as shown in SEQ ID No. 11).

[0096] The nucleic acid sequence of SGLP-1-X-33-2 is shown below (SEQ ID No. 10):

[0097] ATGTCCTCCTCTGAAGATAAGGGTGAATACAAGAAGGGTGATGATGCTTCTTACTTTGAACCAACTGGTCCATACTTGATGGTTAACGTTACTGGTGTTGATGGTAAGGGTAACGAATTGTTGTCTCCACATTACGTTGAATTTCCAAT TAAGCCAGGTACTACTTTGACTAAGGAAAAGATTGAATACTACGTTGAATGGGCTTTGGATGCTACTGCTTACAAGGAATTGAGAGTTGTTGAATTGGATCCATCTGCTAAGATTGAAGTTACTTACTACGATAAGAACAAGAAGAAGG AAGAAACTAAGTCTTTTCCAATTACTGAAAAGGGATTTGTTGTTCCAGATTTGTCTGAACATATTAAGAACCCAGGTTTTAACTTGATTACTAAGTTGTTATTGAAAAGAAG(Sak)GATGATGATGATAAG(enterokinase cleavage site)GGTGGTG GTGGTTCT(linker)GAAAACTTGTACTTTCAAGGT(TEV protease cleavage site)CATGGTGAAGGTACTTTTACTTCTAACGTTTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAATTTATTGCTTGGTTGGTTAAGGGTAGAGGT(GLP-1);

[0098] The amino acid sequence is (SEQ ID No. 11):

[0099] MSSSEDKGEYKKGDDASYFEPTGPYLMVNVTGVDGKGNELLSPHYVEFPIKPGTTLTKEKIEYYVEWALDATAYKELRVVELDPSAKIEVTYYDKNKKKEETKSFPITEKGFVVPDLSEHIKNPGFNLITKVVIEKKDDDDKGGGGSENLYFQGHGEGTFTSNVSSYLEGQAAKEFIAWLVKGRG;

[0100] The optimized sequence was expressed and purified according to the steps described above, and the results are as follows: Figure 4 As shown, Figure 4 In the image, lanes 1-3 are shown from left to right. Lane 1 contains the SMOBIO protein maker PM2510, lane 2 contains the supernatant from the fermentation broth on day 5 of EK-linker-TEVp (SGLP-1-X-33-2) induced expression, and lane 3 contains the supernatant from the fermentation broth on day 5 of TEVp-linker-EK (SGLP-1-X-33-1) induced expression. The results showed that the fusion protein (i.e., SGLP-1-X-33-1) yielded the highest amount when using TEVp-linker-EK ligation. This represents the first time that ultra-efficient expression of SGLP-1 (0.35 g / L) in Pichia pastoris has been achieved.

[0101] Example 2: Mouse experiment to verify the effect of the fusion protein SGLP-1 (SGLP-1-X-33-1 in Example 1).

[0102] Mouse experimental methods:

[0103] Five-week-old male C57BL / 6J mice (purchased from Vital Rivers, Beijing) were housed in an SPF-grade animal facility (temperature 23±2℃, humidity 45-55%, 12h light-dark cycle). After one week of acclimatization, they were randomly divided into: (i) normal diet group (ND): 20% protein, 70% carbohydrate, 10% fat; (ii) high-fat, high-sugar diet group (HFSD): 20% protein, 35% carbohydrate, 45% fat.

[0104] Establishment of a diabetes model:

[0105] After 4 weeks of HFSD feeding, mice of suitable weight were selected and injected with streptozotocin (STZ) to induce diabetes. Blood glucose was measured multiple times to confirm successful modeling.

[0106] Establishment of a model for thrombotic complications:

[0107] Treating the femoral artery of diabetic mice with FeCl3 solution for 20 minutes allows iron ions to enter the blood vessel lumen via endocytosis / efflux. Oxidation of high-valence iron ions leads to endothelial damage, triggering platelet activation, adhesion, and aggregation, ultimately resulting in thrombus formation.

[0108] Experimental group design (n=6 per group): normal control group (NC); model control group: 0.012 mg / kg semaglutide group (PC-1; semaglutide is a mature GLP-1 analog that can lower blood sugar and reduce weight), 0.054 mg / kg Sak group (PC-2); treatment group (4 dose gradients): SGLP-1 group (0.13 / 0.39 / 1.17 / 3.51 mg / kg).

[0109] Note: All doses were calculated based on body weight (BW), and all treatments were administered after the thrombosis model was established.

[0110] Blood glucose results for each group of mice are as follows Figure 5 As shown, there were significant differences in fasting blood glucose levels among the groups of mice. The disease control group (NC) maintained a stable hyperglycemic level throughout the experiment, while the diabetic model control groups (PC-1 and PC-2) showed a decrease in blood glucose. The SGLP-1 treatment groups showed a significant dose-dependent hypoglycemic effect. The hypoglycemic effect of the 0.13 mg / kg dose group was limited, with blood glucose levels only slightly lower than those of the model control group; the 0.39 mg / kg and 1.17 mg / kg dose groups showed a progressively increasing hypoglycemic effect; the highest dose group (3.51 mg / kg) had the most significant hypoglycemic effect (after 30 days of treatment, the fasting blood glucose of diabetic thrombotic mice decreased from 8.5 mmol / L to 5.5 mmol / L at SGLP-1 of 3.51 mg / kg), and its blood glucose level was close to that of the normal control group. It is noteworthy that the blood glucose levels of all treatment groups showed a rapid downward trend at the beginning of administration, and then gradually stabilized. Compared with the model control group, all dose groups of SGLP-1 improved the hyperglycemic state of diabetic mice to varying degrees, with the high-dose group showing the most significant therapeutic effect, suggesting that SGLP-1 has a significant dose-dependent hypoglycemic effect. These results provide important evidence for the application of SGLP-1 in the treatment of diabetes.

[0111] Mouse body weight results as follows Figure 6 As shown, compared with the NC group, the body weight of all four SGLP-1 groups was significantly reduced after 30 days of treatment (p<0.0001). The decrease in body weight was greater with increasing drug dosage. Compared with the NC group, the most significant difference in body weight was observed in mice at SGLP-1 at 3.5 mg / kg (from 35g to 25g).

[0112] The results of thrombin time in mice are as follows: Figure 7As shown, diabetic thrombosis model mice (NC group) exhibited significant coagulation dysfunction, with their thrombin time (TT) significantly shortened compared to normal values ​​(approximately 5-10 seconds), confirming a marked hypercoagulable state in the model. Regarding therapeutic intervention, SGLP-1 doses of 0.39, 1.17, and 3.51 mg / mL all prolonged the thrombin time in affected mice, with the 1.17 mg / mL dose showing the most significant prolongation (15-18 seconds).

[0113] The results of fibrin content in mice are as follows Figure 8 As shown, diabetic thrombosis model mice (NC group) exhibited high fibrin levels (5-8 g / L) caused by thrombosis. SGLP-1 was able to reduce fibrin levels, with the most significant reductions observed at doses of 1.17 and 3.51 mg / mL, which reduced fibrin levels in the affected mice to (3-3.5 g / L).

[0114] In summary, SGLP-1 has the potential to improve blood glucose levels and dissolve thrombi in diabetic thrombotic mice, and can reduce the body weight of diabetic mice, providing a new direction for the future treatment of diabetes and thrombotic complications.

[0115] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A fusion protein, characterized in that, The fusion protein includes Staphylokinase and glucagon-like peptide-1.

2. The fusion protein according to claim 1, characterized in that, The fusion protein also includes an enterokinase cleavage site, a TEV protease cleavage site, and a linker.

3. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in either SEQ ID No. 7 or SEQ ID No.

11.

4. An isolated polynucleotide, characterized in that, The polynucleotide encodes the fusion protein according to any one of claims 1-3.

5. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 4.

6. A host cell, characterized in that, The host cell comprises the fusion protein of any one of claims 1-3, or the polynucleotide of claim 4, or the expression vector of claim 5.

7. The use of the fusion protein according to any one of claims 1-3 in the preparation of reagents or drugs for treating diabetes and its complications.

8. Use of the fusion protein according to any one of claims 1-3, characterized in that, The fusion protein is used for: (i) Regulate or lower blood sugar; (ii) Adjusting or reducing weight; (iii) Dissolve blood clots; (iv) Prolong clotting time or improve clotting; (v) Reduce fibrin content.

9. A method for preparing the fusion protein according to any one of claims 1-3, characterized in that, The method includes the following steps: 1) Obtain the target gene fusion fragment; 2) Insert the target gene fusion fragment into the expression vector to construct a recombinant expression plasmid; 3) Transform E. coli with the recombinant expression plasmid to amplify the number of plasmids; 4) Extract the plasmid, transform it into Pichia pastoris, and express it in Pichia pastoris; 5) Isolation and purification of fusion proteins.

10. A method for preparing a reagent or drug for treating diabetes and its complications, characterized in that, The reagent or drug contains the fusion protein of any one of claims 1-3 or the polynucleotide of claim 4, and a pharmaceutically acceptable carrier or excipient.

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