FGF21, GLP-1 and Glucagon three-receptor agonist protein and application thereof

Abstract

The invention discloses an FGF21 (Fibroblast Growth Factor 21), GLP-1 (Glucagon) and Glucagon three-receptor agonist protein. The amino acid sequence of the three-receptor agonist protein is shown as SEQ ID NO: 1 or SEQ ID NO: 2. The invention also provides nucleic acid for coding the tri-receptor stimulant protein, a recombinant vector, a host cell and a method for producing the tri-receptor stimulant protein, and also provides application of the tri-receptor stimulant protein in preparation of drugs for treating NAFLD, lipid-lowering drugs, drugs for treating lipid metabolism disorder, weight-reducing drugs and drugs for reducing blood sugar. After db / db mice are administered for a long time, the hypoglycemic activity is higher, the sugar tolerance is improved, weight gain can be inhibited, polydipsia and polyphagia are improved, the liver is protected, liver cell fat vacuoles are reduced, hepatic fibrosis is inhibited, the contents of TG, TC and LDL in blood fat can be reduced, lipid metabolism disorder can be regulated, and the overall efficacy is superior to that of FGF21.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology. More specifically, this invention relates to an FGF21, GLP-1, and Glucagon triple receptor agonist protein and its applications. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with a global prevalence of 25%. It encompasses a wide range of conditions, from simple fat accumulation to steatohepatitis, fibrosis, and cirrhosis. NAFLD has a bidirectional association with metabolic syndrome; diabetes increases the risk of NAFLD, and NAFLD patients often also have type 2 diabetes and obesity. While existing medications do offer some relief for NAFLD, they all have limitations and cannot be considered cures. For example, metformin, an insulin sensitizer, has good glycemic control, but its effect on lipid homeostasis is not particularly effective; statins, lipid-lowering drugs, have good cholesterol-regulating effects, but they have no beneficial impact on liver histology in NASH patients. As NAFLD is increasingly recognized as a component of metabolic syndrome, designing and developing drugs that improve NAFLD in multiple ways, including maintaining lipid homeostasis, regulating glycemic levels, and reducing weight, holds potential advantages.

[0003] Fibroblast growth factor 21 (FGF21, NCBI ID NP_061986.1) participates in various mechanisms of inhibiting NASH development, such as stimulating glucose uptake, improving insulin resistance, regulating and maintaining lipid homeostasis to reduce obesity, β-cell protection, reducing lipotoxicity, and alleviating liver fibrosis. However, it does not lower blood glucose. Glucagon-like peptide-1 (GLP-1), a novel and promising drug for treating type 2 diabetes, has highly effective hypoglycemic and insulin-sensitizing effects. It can improve the histological condition of NAFLD by enhancing fatty acid oxidation. Glucagon has various beneficial effects on energy and lipid metabolism, inhibiting lipogenesis and stimulating lipolysis, and reducing weight by decreasing food intake and stimulating energy expenditure.

[0004] Therefore, designing and developing a triple receptor agonist fusion protein that can simultaneously leverage the advantages of FGF21, GLP-1, and Glucagon would be beneficial for the preparation of NAFLD therapeutics. Summary of the Invention

[0005] One object of the present invention is to provide a triple receptor agonist protein of FGF21, GLP-1 and Glucagon and its applications, which can help in the preparation of NAFLD therapeutic drugs.

[0006] To achieve these objectives and other advantages of the present invention, according to one aspect of the present invention, the present invention provides a triple receptor agonist protein of FGF21, GLP-1 and Glucagon, the amino acid sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0007] The present invention also provides nucleic acids encoding the aforementioned FGF21, GLP-1 and Glucagon triple receptor agonist proteins.

[0008] The present invention also provides a recombinant vector containing the aforementioned nucleic acid.

[0009] The present invention also provides a host cell containing the recombinant vector described above.

[0010] The present invention also provides a method for producing the aforementioned FGF21, GLP-1, and Glucagon triple receptor agonist proteins, comprising: culturing the aforementioned host cells; and recovering the triple receptor agonist proteins from the aforementioned host cells or cultures.

[0011] The present invention also provides the use of the aforementioned FGF21, GLP-1 and Glucagon triple receptor agonist proteins in the preparation of drugs for the treatment of NAFLD.

[0012] The present invention also provides the application of the aforementioned FGF21, GLP-1 and Glucagon triple receptor agonist proteins in the preparation of lipid-lowering drugs.

[0013] The present invention also provides the use of the aforementioned FGF21, GLP-1 and Glucagon triple receptor agonist proteins in the preparation of drugs for treating lipid metabolism disorders.

[0014] The present invention also provides the application of the aforementioned FGF21, GLP-1 and Glucagon triple receptor agonist proteins in the preparation of weight-loss drugs.

[0015] The present invention also provides the application of the aforementioned FGF21, GLP-1 and Glucagon triple receptor agonist proteins in the preparation of hypoglycemic drugs.

[0016] The present invention has at least the following beneficial effects: This invention can simultaneously activate three receptors, achieving a synergistic effect in improving NAFLD, blood glucose control, and weight loss, overcoming the shortcomings of existing drugs (such as metformin's weak lipid-regulating effect and statins' lack of benefit to liver histology). In mouse liver AML12 cells induced by NAFLD, this invention significantly promotes glucose absorption and its effect in alleviating lipid accumulation far surpasses that of FGF21, effectively regulating hepatic cell lipid homeostasis. Long-term administration of this invention to db / db mice demonstrates stronger hypoglycemic activity, maintaining normal blood glucose levels for one day and improving glucose tolerance. It can also inhibit weight gain, improve polydipsia and polyphagia, protect the liver, reduce hepatic cell fat vacuoles, inhibit liver fibrosis, and reduce the levels of TG, TC, and LDL in blood lipids, regulating lipid metabolism disorders. Overall, its efficacy is superior to FGF21, providing a highly effective treatment option for NAFLD.

[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 The expression and purification analysis results of two FGF21, GLP-1, and Glucagon triple receptor agonists (FGG1 and FGG2) are presented: A is the SDS-PAGE electrophoresis image of FGG1, M is the protein molecular weight standard, and lanes 1-6 correspond to the samples before induction, after induction, supernatant, precipitate, after purification with HisTrap™ HP column, and after purification with molecular sieve, respectively. The target protein FGG1 is approximately 40 kDa, and the target band gradually becomes clearer and richer in subsequent purification steps after induction. B is the SDS-PAGE electrophoresis image of FGG2, with the same lane settings as A. The target protein FGG2 is approximately 40 kDa, showing the same band enrichment trend as A, indicating that FGG2 is also effectively expressed. C is a comparison of the SDS-PAGE electrophoresis images of the purified proteins, M is the molecular weight standard, and lanes 1-3 are FGF21, FGG1, and FGG2, respectively. Each target protein band is single, demonstrating good purification effect. D is the elution curve of FGG1 purified by HisTrap™ HP column. The horizontal axis represents the elution volume (ml), and the vertical axis represents the absorbance at 280 nm (mAU). A clear target protein peak appears around 1600 ml, indicating that FGG1 was effectively eluted. E is the molecular sieve purification curve of FGG1. The horizontal axis represents the volume (ml), and the vertical axis represents mAU (280 nm). A concentrated target protein peak appears around 40 ml. F is the HisTrap™ HP column elution curve of FGG2. The horizontal and vertical axes are the same as D. A target protein peak appears around 1600 ml, and the elution characteristics are similar to FGG1. G is the molecular sieve purification curve of FGG2. The horizontal and vertical axes are the same as E. A single concentrated target protein peak appears around 40 ml, indicating that the molecular sieve step further improves protein purity.

[0019] Figure 2 The graph shows the results of two triadic receptor agonist proteins promoting glucose uptake in mouse liver AML12 cells. The horizontal axis represents the logarithm of the sample concentration (log[sample]), and the vertical axis represents the relative glucose uptake. The groups include a blank control (Control), an FGF21 treatment group, and FGG1 and FGG2 treatment groups. The data trend shows that glucose uptake in all treatment groups increases in a dose-dependent manner with increasing concentration. At the same concentration, the glucose uptake in the FGG1 and FGG2 groups is significantly higher than that in the Control and FGF21 groups. Among them, FGG2 has a slightly better promoting effect, indicating that triadic receptor agonist proteins have a stronger promoting effect on glucose uptake in liver cells.

[0020] Figure 3 This image shows the results of Oil Red staining experiments on mouse liver AML12 cells, used to simulate the regulatory effects of FGF21, GLP-1, and Glucagon triple receptor agonists on lipid homeostasis in NAFLD liver cells in vitro. Sub-figure A shows the Oil Red staining morphology of cells in different treatment groups, displaying the lipid droplet distribution in the Control, FFA (free fatty acids, i.e., oleic acid and palmitic acid), FFA+FGF21, FFA+FGG1, and FFA+FGG2 groups. Sub-figure B is a bar chart of the average lipid droplet area, with the x-axis representing the different treatment groups and the y-axis representing the average lipid droplet area (unit: ×10). -5 mm 2 In terms of data trends, lipid droplets in the FFA group were deeply stained and numerous, while lipid droplets in the FFA+FGF21 group were reduced, and the reduction in lipid droplets in the FFA+FGG1 and FFA+FGG2 groups was more significant. The average area of ​​lipid droplets in the FFA group was much higher than that in the Control group, the area in the FFA+FGF21 group was reduced, and the area in the FFA+FGG1 and FFA+FGG2 groups was further reduced with statistically significant differences.

[0021] Figure 4The following figures illustrate the experimental results of the effects of FGF21, GLP-1, and Glucagon triple receptor agonists on glucose tolerance and hypoglycemic effects in NAFLD model db / db mice: Subfigure A is a schematic diagram of the experimental procedure, with the horizontal axis representing weeks, showing the experimental period for 10-week-old db / db mice, the drug administration phase from weeks 10 to 13, and the LIGO detection time points, clearly defining the sequence of operations; Subfigure B is a graph showing the dynamic changes in blood glucose after a single drug administration, with the horizontal axis representing time (day) and the vertical axis representing blood glucose (blood). The trend shows that blood glucose remained high in the PBS group, slightly decreased in the FGF21 group, and decreased more significantly in the FGG1 and FGG2 groups, with the FGG2 group showing the most significant decrease. Subfigure C is a bar chart of blood glucose AUC after a single dose, with the horizontal axis representing the PBS, FGF21, FGG1, and FGG2 groups and the vertical axis representing AUC (area under the curve). The results show that the AUC of the FGG1 and FGG2 groups was significantly lower than that of the PBS and FGF21 groups, and the difference was statistically significant. Subfigure D is a graph of blood glucose changes after 30 days of continuous administration, with the horizontal axis representing time (day) and the vertical axis representing blood glucose (mmol / L). Blood glucose remained high in the PBS group, slightly decreased in the FGF21 group, and continued to decrease in the FGG1 and FGG2 groups, with the FGG2 group reaching and maintaining the normal range. Subfigure E is a graph of blood glucose changes over long-term administration, with the horizontal axis representing time (day) and the vertical axis representing blood glucose change (mmol / L). (mmol / L), the blood glucose changes in the FGG1 and FGG2 groups were greater than those in the FGF21 group, and the glucose-lowering effect was more sustained; Subfigure F is a bar chart of AUC statistics for long-term administration, with the horizontal axis representing each treatment group and the vertical axis representing AUC. The AUC of the FGG1 and FGG2 groups was significantly reduced; Figure G is a graph of blood glucose changes in the glucose tolerance test, with the horizontal axis representing time (day) and the vertical axis representing blood glucose changes. The FGG2 group was able to control blood glucose at a low level and quickly return to normal; Subfigure H is a bar chart of AUC statistics for glucose tolerance, with the horizontal axis representing each treatment group and the vertical axis representing AUC. The AUC of the FGG2 group was significantly reduced, and the glucose tolerance improvement was better. Overall, the three receptor agonist proteins showed better glucose-lowering and glucose tolerance-regulating effects than FGF21, and the FGG2 activity was more prominent.

[0022] Figure 5The following figures illustrate the effects of FGF21, GLP-1, and Glucagon triple receptor agonists on body weight and food and water intake in NAFLD model db / db mice: Subfigure A shows the changes in mouse body weight after long-term administration, with the horizontal axis representing time (day) and the vertical axis representing body weight (g). The trend shows that the body weight in the PBS group continued to increase, the body weight gain in the FGF21 group slowed slightly, while the body weight gain in the FGG1 and FGG2 groups was significantly inhibited. The body weight in the FGG2 group even showed a slight downward trend, with the weight control effect being significantly better than that in the FGF21 group. Subfigure B is a bar chart showing the statistical changes in water intake in each group, with the horizontal axis representing the PBS, FGF21, FGG1, and FGG2 treatment groups, and the vertical axis representing water change (g). The results showed that the PBS group had a positive change in water intake, the FGF21 group had a slight decrease in water intake, and the FGG1 and FGG2 groups had a significant negative decrease in water intake. The differences between the groups were statistically significant, reflecting the improvement of polydipsia symptoms. Subplot C is a graph of cumulative food intake changes, with the horizontal axis representing time (day) and the vertical axis representing cumulative food intake (g). The trend is that the cumulative food intake in the PBS group increased rapidly, the growth rate in the FGF21 group slowed down, and the growth rate of cumulative food intake in the FGG1 and FGG2 groups was even slower, with the total food intake significantly lower than the first two groups. Subplot D is a graph of cumulative water intake changes, with the horizontal axis representing time (day) and the vertical axis representing cumulative water intake (g). The cumulative water intake in the PBS group increased the fastest, the growth rate in the FGF21 group slowed down, the growth rate of cumulative water intake in the FGG1 and FGG2 groups further decreased, and the cumulative water intake in the FGG2 group was the lowest. Overall trends indicate that the triple receptor agonist protein is superior to FGF21 in inhibiting body weight and improving polydipsia and polyphagia in db / db mice.

[0023] Figure 6The experimental results show the therapeutic effects of FGF21, GLP-1, and Glucagon triple receptor agonists on liver injury in NAFLD model db / db mice: Subfigure A shows liver tissue staining images of different treatment groups (Vehicle (solvent control group), FGF21, FGG1, FGG2), including H&E staining (showing hepatocyte structure), Picrosirius Red staining (showing fibrosis), and Oil Red staining (showing lipid droplets). Morphological trends show that hepatocytes in the FGG1 and FGG2 groups are tightly and orderly arranged, with only small, dispersed lipid droplets, and fibrosis is significantly reduced, with the structural repair and pathological improvement being more prominent in the FGG1 group. Subfigure B is a bar chart of liver fibrosis area, with the horizontal axis representing the above treatment groups and the vertical axis representing fibrosis area (Fibrotic area, %). The data trend shows that the fibrosis area is highest in the Vehicle group, slightly lower in the FGF21 group, and significantly reduced in the FGG1 and FGG2 groups. The differences between groups are statistically significant, demonstrating a stronger inhibitory effect on fibrosis. Figure C is a bar chart showing the statistical significance of the average lipid droplet area. The horizontal axis represents each treatment group, and the vertical axis represents the average lipid droplet area (×10). -5 mm 2 The trend shows that the lipid droplet area is the largest in the Vehicle group, the lipid droplet area is reduced in the FGF21 group, the lipid droplet area is significantly reduced in the FGG1 and FGG2 groups, and the lipid droplet improvement effect is the best in the FGG1 group.

[0024] Figure 7 The following figures show the experimental results of the effects of FGF21, GLP-1, and Glucagon triple receptor agonists on lipid metabolism disorders in NAFLD model db / db mice: Subfigure A is a bar chart of triglyceride (TG) content, with the horizontal axis representing the PBS, FGF21, FGG1, and FGG2 treatment groups, and the vertical axis representing TG concentration (mM). The trend shows that the PBS group had a higher TG level, the FGF21 group had a slight decrease, and the FGG1 and FGG2 groups had a significant decrease in TG, with the regulatory effect being better than that of FGF21; Subfigure B is a bar chart of total cholesterol (TC) content, with the horizontal axis the same as above. The ordinate of the graph represents total cholesterol (TC) concentration (mM). The PBS group had the highest TC level, the FGF21 group showed a slight decrease, and the FGG1 and FGG2 groups showed a significant decrease in TC, with statistically significant differences between groups. Subgraph C is a bar chart of low-density lipoprotein (LDL) content, with the same ordinate as before. The PBS group had a higher LDL level, the FGF21 group showed a slight decrease, and the FGG1 and FGG2 groups showed a significant decrease in LDL, indicating a more pronounced improvement. Figure D is a bar chart of high-density lipoprotein (HDL) content, with the same ordinate as before. Overall, the data indicate that triad receptor agonists are more effective than FGF21 in treating lipid metabolism disorders. Detailed Implementation

[0025] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of components in a specific posture. If the specific posture changes, the directional indication will also change accordingly. When an element is referred to as "fixed to" or "set on" another element, it can be directly on the other element or may have an intervening element present. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. Descriptions involving "first," "second," etc., in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features.

[0027] It should be noted that the technical solutions of the various embodiments of this application can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this application.

[0028] The embodiments of this application provide FGF21, GLP-1 and Glucagon triple receptor agonist proteins, with amino acid sequences of SEQ ID NO:1 (hereinafter also referred to as FGG1) or SEQ ID NO:2 (hereinafter also referred to as FGG2).

[0029] The amino acid sequence of SEQ ID NO: 1 (FGG1) is as follows: HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLH LPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASGTGSAGSAAGSGGVDHGQGTFTSDYSKYLDSRRAQDFVEWLKNGGPSSGAPPPS The amino acid sequence of SEQ ID NO: 2 (FGG2) is as follows: HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLH LPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASGTGSAGSAAGSGGVDHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Traditional single-receptor agonists have significant limitations: while FGF21 can regulate lipid homeostasis, it cannot lower blood glucose; GLP-1 can effectively lower blood glucose but lacks the ability to improve liver fibrosis; even the GLP-1 / Glucagon dual-receptor agonist peptide lacks the comprehensive regulatory capacity of FGF21 on lipid metabolism. The protein in this embodiment, through specific sequence design, achieves synergistic activity of three receptors. It can regulate lipids and inhibit liver fibrosis like FGF21, effectively control blood glucose like GLP-1, and promote energy expenditure to reduce weight like Glucagon. Ultimately, it achieves a synergistic function of improving NAFLD, controlling blood glucose, and reducing weight, completely overcoming the functional deficiencies of existing agonists.

[0030] The embodiments of this application provide nucleic acids, which are essentially genetic material encoding a triple-receptor agonist protein, specifically encompassing DNA (such as cDNA) and complementary RNA. As can be seen from the specific implementation process, this nucleic acid can be obtained by amplifying a cDNA fragment using PCR technology. Its base sequence matches the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2. Although synonymous codon substitution based on codon degeneracy is allowed, it is essential to ensure that the final expressed protein retains the activation activity of all three receptors. The uniqueness of this nucleic acid lies in the fact that its encoded protein can simultaneously activate three receptors, and the sequence design ensures the correct spatial structure of the expression product. This is a core prerequisite for achieving synergistic triple-receptor activity and also provides a crucial template for subsequent construction of recombinant vectors and expression of the target protein in host cells.

[0031] The embodiments of this application also provide recombinant vectors, the core components of which are nucleic acids encoding triple-receptor agonist proteins and expression regulatory sequences (such as promoters, terminators, selection marker genes, etc.) operably linked to these nucleic acids. These vectors can stably replicate in host cells and drive the expression of target proteins. Examples include prokaryotic recombinant vectors, such as the commonly used pET-28a(+) vector, and eukaryotic recombinant vectors, such as the pcDNA3.1 vector. Regardless of the vector, the operable linking of the nucleic acid and the regulatory sequence must be ensured, i.e., the promoter can precisely regulate nucleic acid transcription to ensure the correct synthesis of the target protein, providing a foundation for subsequent expression in host cells.

[0032] The embodiments of this application also provide host cells, the core of which contains a recombinant vector or encodes nucleic acid capable of expressing the target triadic receptor agonist protein. Examples include prokaryotic host cells, such as competent *Escherichia coli* BL-21(DE3), and eukaryotic host cells, such as Chinese hamster ovary cells. All host cells must be screened to ensure that only positive cells are used for subsequent culture, avoiding contamination by other cells that could affect protein production efficiency.

[0033] Embodiments of this application also provide a production method, including culturing host cells and recovering the target protein from the host cells / culture. For example, the first step is to culture host cells: competent Escherichia coli BL-21(DE3) containing the recombinant vector is inoculated into LB liquid medium containing 2% glucose and 30 mg / mL kanamycin, and cultured at 37°C and 200 rpm until OD600 = 0.8-1.0. IPTG is added to induce expression for 4 hours to promote the expression of the target protein. The second step is to recover and purify the protein: bacteria are collected using a large-capacity homogenizer, lysed in 25 mM phosphate buffer (pH 7.5) containing 300 mM NaCl, sonicated, and centrifuged at 4°C and 15000 rpm to collect the precipitate. The precipitate is washed with water for injection, treated with Tris buffer, EDTA, 8 M guanidine hydrochloride, and DTT, and centrifuged and filtered. The supernatant is dialyzed and refolded for 36 hours. The precipitate is then purified using a HisTrap™ HP chromatography column (0-2 M imidazole linear gradient elution), and finally concentrated using a Superdex™-75 gel filtration column to obtain the target protein with a purity >98%. This method is adapted to prokaryotic hosts, has clear steps and high purification efficiency, and can effectively preserve the three receptor activation activity of the protein.

[0034] The embodiments of this application also provide applications of triadic receptor agonist proteins for the preparation of drugs for treating NAFLD, lipid-lowering drugs, drugs for treating lipid metabolism disorders, weight-loss drugs, and hypoglycemic drugs. For example, subcutaneous injection formulations: purified FGG1 / FGG2 protein is mixed with stabilizers (such as sucrose, mannitol) and buffers (such as phosphate buffer) to prepare a subcutaneous injection with a concentration of 1 mg / mL. Long-acting microsphere formulations: using polylactic-coated glycolic acid copolymer (PLGA) as a carrier, the target protein is encapsulated into microspheres with a diameter of 5-10 μm for intramuscular injection. Oral formulations: the protein is modified by enteric coating (to avoid destruction by gastric acid) and permeability enhancers (such as bile salts) to prepare oral capsules.

[0035] The following is a description of a specific embodiment.

[0036] Example 1: Genetic engineering expression and purification of two FGF21, GLP-1 and Glucagon triple receptor agonists (SEQ ID NO:1, SEQ ID NO:2).

[0037] The cDNA fragments encoding the FGF21, GLP-1, and Glucagon triple receptor agonist proteins were amplified by PCR and cloned into an *E. coli* expression vector. The expression vector was added to competent *E. coli* BL-21(DE3) in LB broth containing 2% glucose and 30 mg / mL kanamycin and cultured at 37°C and 200 rpm for amplification. When the OD was measured at 600 nm to be 0.8–1.0, 1 ml of bacterial culture was aspirated to prepare a pre-induction sample with a final concentration of 1 mM. IPTG (1 M IPTG, 1:1000 ratio) was added to induce recombinant protein expression, and the sample was cultured at 37°C for 4 hours to prepare a post-induction sample. Bacteria were collected using a high-capacity homogenizer and lysed in 25 mM Na+ / K+ phosphate buffer (pH 7.5) containing 300 mM NaCl, using 40 ml of phosphate buffer per 800 ml of bacterial culture. Cells were disrupted using a cell sonicator, and the lysate was centrifuged at 15,000 rpm for 30 minutes at 4°C to obtain a precipitate. The supernatant was discarded. The collected precipitate was washed with 4.7 ml of water for injection, and 0.5 ml of Tris buffer (pH=8.2) and 1.2 ml of 0.5 M EDTA were added. The mixture was stirred on a magnetic stirrer, and then 23 ml of 8 M guanidine hydrochloride was added and stirred for 1 h. Finally, 600 μl of 1 M DTT was added and stirred for 1 h. The mixture was centrifuged at 12,000 rpm at 4°C. The supernatant was filtered through a 0.22 μm filter and added to a dialysis bag containing 600 ml of 8 M guanidine hydrochloride, 30 ml of Tris buffer (pH=8.2), and 1170 ml of water for injection. The dialysis bag was placed in a refolding solution containing 135 ml Tris buffer (pH 8.2), 78.9 g NaCl, 900 ml glycerol, 7965 ml water for injection, and 1.575 g cysteine. The NaCl concentration in the refolding solution was gradually decreased every 12 h to allow the proteins in the dialysis bag to regain their three-dimensional structure. After 36 h, the refolded protein solution in the dialysis bag was centrifuged at 4000 rpm for 20 min at 4°C, filtered through a 0.22 μm microporous membrane, and then used. HisTrap TM The HP chromatography column was pre-equilibrated in buffer A (25 mM Tris-HCl, pH 8.0) and then subjected to HisTrap. TMProteins were purified using HP chromatography columns. The bound FGF21, GLP-1, and Glucagon triple receptor agonists were eluted with a linear gradient of 0–2 M imidazole in 25 mM Tris-HCl (pH 8.0). In this example, 25 mM Tris-HCl (pH 8.0) containing 2 M imidazole was used for linear gradient elution, with the eluent concentration in buffer A gradually increasing from 0% to 100% over 40 min. The purity of the purified protein was assessed using SDS-PAGE electrophoresis, and the results are shown below. Figure 1 As shown. The protein was concentrated in buffer C (1 M NaCl, 25 mM Tris-HCl, pH 8.0) using a gel filtration column (Superdex™-75GE Healthcare, Piscataway, NJ) to obtain FGF21, GLP-1 and Glucagon triple receptor agonists with a purity >98%.

[0038] This embodiment yielded FGG1 and FGG2 triple receptor agonist proteins with a purity >98%, providing core raw materials for drug preparation. These proteins can be used to prepare drugs for treating NAFLD, as well as lipid-lowering, lipid metabolism disorder treatment, weight loss, and blood sugar-lowering drugs, forming a crucial foundation for the development of multifunctional metabolism-related drugs.

[0039] Example 2: In an in vitro experiment on glucose uptake in mouse liver AML12 cells, the effects of FGF21, GLP-1 and Glucagon triad receptor agonists (SEQ ID NO: 1, SEQ ID NO: 2) on glucose uptake in liver cells were simulated to be stronger.

[0040] Mouse liver AML12 cells were counted and seeded in equal amounts in 96-well plates, and cultured in a cell culture incubator until the cells adhered and merged into a monolayer. After 12 hours of starvation culture, AML12 cells were added to a medium containing FGF21, GLP-1, and Glucagon triple receptor agonists FGG1, FGG2, and FGF21 at concentration gradients of 0.001, 0.01, 0.1, 1, 10, 100, and 1000 ng / ml (AML12 cell-specific basal medium + 1% FBS + 1% penicillin and streptomycin). After 24 hours of culture, the medium from the corresponding wells was transferred to 1.5 ml EP tubes, centrifuged at 12000 rpm to precipitate the remaining suspended cells, and 10 μl of the supernatant was transferred to a new 96-well plate. 100 μl of working solution was added, along with blank control wells (working solution, 10 μl of medium) and standard wells (working solution, 10 μl of standard concentration solution). The mixture was thoroughly mixed, incubated at 37 ℃ for 10 min, and the absorbance of each well was measured at 505 nm. The relative glucose absorbance of AML12 cells was obtained by calculating the remaining glucose content from the absorbance. The experimental results are as follows: Figure 2 As shown, after administration of gradient concentrations, the hypoglycemic activity of FGG1 and FGG2 was significantly better than that of FGF21, and the differences in glucose uptake were statistically significant, indicating that FGF21, GLP-1 and Glucagon triple receptor agonists have a stronger promoting effect on glucose uptake by liver cells.

[0041] This embodiment confirms that FGG1 and FGG2 can more effectively promote glucose uptake by liver cells, indicating that they can be used to prepare hypoglycemic drugs; improving liver glucose metabolism is beneficial for NAFLD treatment, so they can also be used to prepare drugs for treating NAFLD.

[0042] Example 3: Oil Red staining experiments on mouse liver AML12 cells demonstrated that FGF21, GLP-1, and Glucagon triad receptor agonists (SEQ ID NO: 1, SEQ ID NO: 2) exert a stronger regulatory effect on lipid homeostasis in liver cells of NAFLD patients.

[0043] Mouse liver AML12 cells were counted and seeded in equal volumes into 6-well plates. Cells were cultured in a cell culture incubator until they adhered and reached 80%-90% confluence. AML12 cells were cultured in a medium containing 250 ng / ml of FGF21, GLP-1, and Glucagon triple receptor agonists FGG1, FGG2, and FGF21 (AML12 cell-specific basal medium + 0.3 mM oleic acid (OA) + 0.3 mM palmitic acid (PA) + 1% FBS + 1% penicillin and streptomycin). PBS was used as a control. After 24 h of culture, the medium was aspirated, and the cells were washed with 60% isopropanol for 2 min. The isopropanol was then aspirated, and the cells were stained with Oil Red O working solution for 10 min. The Oil Red O working solution was aspirated, and the cells were stained with 60% isopropanol until differentiation was complete and the mesenchyme was clear. The cells were then washed with distilled water, and the distilled water was aspirated. Hematoxylin was then stained for 90 s, rinsed with distilled water, and observed under an inverted microscope. The experimental results are as follows: Figure 3 As shown, after treating mouse liver AML12 cells with NAFLD modeling by 250 ng / ml of FGF21, GLP-1 and Glucagon triple receptor agonists for 24 hours, FGG1 and FGG2 exerted a more superior therapeutic effect in alleviating lipid accumulation. The difference in lipid droplet content was statistically significant, indicating that FGF21, GLP-1 and Glucagon triple receptor agonists (SEQ ID NO: 1, SEQ ID NO: 2) have a stronger regulatory effect on lipid homeostasis in liver cells of NAFLD patients.

[0044] In this embodiment, FGG1 and FGG2 significantly alleviated lipid accumulation in NAFLD modeling cells, indicating that they can be used to prepare drugs for treating NAFLD; they can also regulate liver lipid homeostasis, and therefore are also suitable for preparing lipid-lowering drugs and drugs for treating lipid metabolism disorders.

[0045] Example 4: Study on the effects of FGF21, GLP-1 and Glucagon triple receptor agonists (SEQ ID NO: 1, SEQ ID NO: 2) on glucose tolerance and hypoglycemic effects in NAFLD model db / db mice.

[0046] Male db / db NAFLD model mice: 1. Genetic background: C57BL / 6J mice with the leptin receptor gene knocked out are homozygous and infertile; 2. Characteristics: Prone to obesity, rapid weight gain, reduced activity, and high blood lipids; 3. Uses: As a NAFLD model, it is mainly used for research on NAFLD drug screening, physiological and biochemical indicators of obesity, and drug treatment.

[0047] Male db / dbNAFLD model mice (10 weeks old) were randomly divided into four groups: control group (PBS solvent), FGF21 treatment group, FGG1 treatment group, and FGG2 treatment group, with 6 mice in each group. Protein (1.0 mg / kg) or PBS was administered intraperitoneally once daily for 4 weeks. Experimental results are as follows: Figure 4 As shown, after a single administration of FGG1 and FGG2, the hypoglycemic effect was improved compared to FGF21. FGG2 also lowered blood glucose levels in mice to the normal range and maintained this effect for one day. After four weeks of treatment with FGG1 and FGG2, blood glucose levels in db / db mice returned to normal, demonstrating a superior hypoglycemic effect compared to FGF21. Furthermore, glucose tolerance tests showed that injection of FGG2 controlled blood glucose within a lower range and restored it to normal values ​​quickly, significantly improving glucose tolerance. This indicates that FGF21, GLP-1, and Glucagon triad receptor agonists (SEQ ID NO:1, SEQ ID NO:2) exhibit better hypoglycemic activity after long-term administration to the NAFLD model db / db mice, and the hypoglycemic activity of FGG2 is efficiently preserved.

[0048] This embodiment shows that FGG1 and FGG2 can effectively lower blood sugar and improve glucose tolerance, and can be used to prepare hypoglycemic drugs; since NAFLD is often accompanied by hyperglycemia, their hypoglycemic effect helps in the treatment of NAFLD, so they can also be used to prepare drugs for the treatment of NAFLD.

[0049] Example 5: A study on the effects of long-term administration of FGF21, GLP-1 and Glucagon triad receptor agonists (SEQ ID NO: 1, SEQ ID NO: 2) on weight reduction in NAFLD model db / db mice.

[0050] The specific experimental method is the same as in Example 4. The experimental results are as follows: Figure 5 As shown, four weeks of treatment with FGG1 and FGG2 inhibited weight gain in obese NAFLD model mice, demonstrating a superior weight-reduction effect compared to FGF21. Simultaneously, the polydipsia and polyphagia symptoms in db / db mice were significantly improved, and the food and water intake of mice in the FGG1 and FGG2 treatment groups was lower than that in the FGF21 treatment group. This indicates that FGF21, GLP-1, and Glucagon triad receptor agonists have a better weight-reduction effect after long-term administration to the NAFLD model db / db mice.

[0051] In this embodiment, FGG1 and FGG2 inhibit the weight gain of db / db mice and improve polydipsia and polyphagia, which can be used to prepare weight loss drugs; while obesity is an important cause of NAFLD, and controlling weight is beneficial to the treatment of NAFLD, so they can also be used to prepare drugs for the treatment of NAFLD.

[0052] Example 6: A study on the therapeutic effects of long-term administration of FGF21, GLP-1, and Glucagon triad receptor agonists (SEQ ID NO: 1, SEQ ID NO: 2) on liver injury in NAFLD model db / db mice. The specific experimental methods were the same as in Example 4. The experimental results are as follows: Figure 6 As shown, after four weeks of treatment with FGG1 and FGG2, mouse hepatocytes were arranged tightly and orderly, and no large fat vacuoles were observed. The cell nuclei returned to the center of the cell, with only small fat vacuoles scattered around the nuclei. Compared with FGF21, FGG1 showed a better therapeutic effect on liver injury. Meanwhile, Oil Red staining results showed that lipid droplets in db / db mice treated with FGG1 and FGG2 were significantly reduced in number and size, with FGG1 showing the most significant improvement. Furthermore, Sirius Red staining results showed that fibrosis in db / db mice treated with FGG1 and FGG2 was inhibited, with FGG1 showing the most significant inhibitory effect. These results indicate that FGF21, GLP-1, and the Glucagon triad receptor agonist protein have better therapeutic effects on liver injury in the NAFLD model db / db mice after long-term administration.

[0053] This embodiment confirms that FGG1 and FGG2 can improve hepatocyte structure, reduce lipid droplets, and inhibit liver fibrosis, directly targeting the pathological changes of NAFLD. Therefore, they can be used efficiently to prepare drugs for the treatment of NAFLD, providing core pharmacodynamic support for the treatment of NAFLD.

[0054] Example 7: The study on the therapeutic effects of long-term administration of FGF21, GLP-1, and Glucagon triad receptor agonists (SEQ ID NO:1, SEQ ID NO:2) on lipid metabolism disorders in NAFLD model db / db mice followed by FGF21, GLP-1, and Glucagon triad receptor agonists (SEQ ID NO:1, SEQ ID NO:2) was conducted using the same experimental methods as in Example 4. The experimental results are as follows: Figure 7As shown, after four weeks of treatment with FGG1 and FGG2, the levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL) in the blood lipids of mice decreased, demonstrating a superior therapeutic effect on lipid metabolism disorders compared to FGF21. Furthermore, the differences in high-density lipoprotein (HDL) levels were not statistically significant, consistent with previous studies showing that FGF21 had no regulatory effect on HDL. This indicates that FGF21, GLP-1, and the Glucagon triad receptor agonist protein have better therapeutic effects on lipid metabolism disorders after long-term administration to the NAFLD model db / db mice.

[0055] In this embodiment, FGG1 and FGG2 reduce TG, TC, and LDL, and can be used to prepare lipid-lowering drugs and drugs for treating lipid metabolism disorders. Since lipid disorders aggravate NAFLD, their lipid-regulating effect is beneficial to the improvement of NAFLD, so they can also be used to prepare drugs for treating NAFLD.

[0056] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A triple receptor agonist protein of FGF21, GLP-1, and Glucagon, characterized in that, The amino acid sequence of the triad receptor agonist protein is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The nucleic acid encoding the FGF21, GLP-1 and Glucagon triple receptor agonist proteins as described in claim 1.

3. A recombinant vector containing the nucleic acid of claim 2.

4. A host cell containing the recombinant vector of claim 3.

5. A method for producing FGF21, GLP-1, and Glucagon triple receptor agonist proteins, characterized in that, include: Cultivate the host cells as described in claim 4; The triad receptor agonist protein is recovered from the host cell or culture.

6. The use of the FGF21, GLP-1 and Glucagon triple receptor agonist proteins as described in claim 1 in the preparation of a medicament for treating NAFLD.

7. The use of the FGF21, GLP-1 and Glucagon triple receptor agonist proteins as described in claim 1 in the preparation of lipid-lowering drugs.

8. The use of the FGF21, GLP-1 and Glucagon triple receptor agonist proteins as described in claim 1 in the preparation of drugs for treating lipid metabolism disorders.

9. The use of the FGF21, GLP-1 and Glucagon triple receptor agonist proteins as described in claim 1 in the preparation of weight-loss drugs.

10. The use of the FGF21, GLP-1 and Glucagon triple receptor agonist proteins as described in claim 1 in the preparation of hypoglycemic drugs.

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