Long-acting fibroblast growth factor 21 fusion protein and application thereof

Abstract

The present application relates to a long-acting fibroblast growth factor 21 fusion protein and application thereof. The amino acid sequence of the fusion protein of the present application is shown in SEQ ID No. 1, which can effectively prolong the half-life of fibroblast growth factor 21, and then make fibroblast growth factor 21 have long-acting property, and on this basis, the fusion protein retains the known functions of fibroblast growth factor 21. In addition, the present application also finds that the fusion protein Nb-FGF21 can significantly improve skin stains, and can be used to treat skin stains.

Description

Technical Field

[0001] This invention relates to the field of functional proteins, and particularly to fusion proteins containing fibroblast growth factor 21 and their applications. Background Technology

[0002] Melasma is a chronic, acquired facial hyperpigmentation disorder, clinically manifested as symmetrically distributed light or dark brown patches of varying shades with indistinct borders on the cheeks, forehead, and jawline. Currently, numerous treatment options are available clinically; however, it remains a refractory pigmentary disorder.

[0003] Fibroblast growth factor 21 (FGF21) belongs to the FGF19 subfamily and is an endocrine growth factor. Current technology suggests that it plays a wide range of roles in regulating bile acid, lipid, glucose, and energy metabolism homeostasis. However, FGF21 has a molecular weight of only 19.5 kilodaltons (kDa) and a half-life in vivo of only 0.5 to 2 hours, limiting its clinical application. Summary of the Invention

[0004] One aspect of the present invention provides a fusion protein, the amino acid sequence of which is shown in SEQ ID No. 1.

[0005] One invention provides a nucleic acid encoding a fusion protein as described in one invention.

[0006] In one specific embodiment, the sequence of the nucleic acid is shown in SEQ ID No. 2.

[0007] The third invention provides the use of the fusion protein as described in the first invention or the nucleic acid as described in the second invention in the preparation of a medicament for treating at least one of the following diseases: type 2 diabetes, fatty liver disease, cardiovascular disease caused by lipid metabolism disorders, and skin pigmentation.

[0008] In one specific embodiment, the skin pigmentation includes at least one of melasma, sunspots, and age spots.

[0009] The beneficial effects of this invention are as follows: The fusion protein of this invention can bind to human serum albumin, thereby effectively prolonging the half-life of fibroblast growth factor 21 by increasing its molecular weight, thus giving fibroblast growth factor 21 a long-lasting effect. Furthermore, the fusion protein retains the known functions of fibroblast growth factor 21, such as its ability to treat type 2 diabetes, fatty liver disease, and cardiovascular diseases caused by lipid metabolism disorders. In addition, this invention has found that the fusion protein Nb-FGF21 can significantly improve skin pigmentation (e.g., melasma), and can be used to treat melasma, sunspots, or age spots. Attached Figure Description

[0010] Figure 1 The SDS-PAGE assay of methanol-induced GS115 / pPICZαA-Nb-FGF21 is shown. In the diagram, M represents the protein molecular weight marker; lane 1: sample before induction; lane 2: 0.5% methanol induced for 24 hours; lane 3: 0.5% methanol induced for 48 hours; lane 4: 0.5% methanol induced for 72 hours; lane 5: 0.5% methanol induced for 96 hours; lane 6: 1.0% methanol induced for 24 hours; lane 7: 1.0% methanol induced for 48 hours; lane 8: 1.0% methanol induced for 72 hours; lane 9: 1.0% methanol induced for 96 hours; kDa represents molecular weight in kilodaltons; the arrow indicates the fusion protein Nb-FGF21.

[0011] Figure 2 The Western blot analysis of GS115 / pPICZαA-Nb-FGF21 induced with 0.5% methanol is shown. In the figure, M represents the protein molecular weight marker; lane 1: sample before induction; lane 2: 0.5% methanol induced for 24 hours; lane 3: 0.5% methanol induced for 48 hours; lane 4: 0.5% methanol induced for 72 hours; lane 5: 0.5% methanol induced for 96 hours; 35 and 40 on the left side of the figure represent 35 and 40 kilodaltons (kDa), respectively.

[0012] Figure 3 The following is displayed: M: protein molecular weight marker; lane 1: sample before induction; lane 2: protein lysate; lane 3: desalting elution buffer; lane 4: sample loaded onto Q chromatography column (desalting elution buffer); lane 5: Q chromatography column exit solution; lane 6: gradient elution buffer containing 0.3 mol / L sodium chloride.

[0013] Figure 4 The binding affinity of the fusion protein Nb-FGF21 to human serum albumin was shown.

[0014] Figure 5Optical micrographs of 3T3-L1 cells at three induction stages are shown. × indicates magnification.

[0015] Figure 6 Analysis of glucose uptake capacity in 3T3-L1 cells after differentiation was demonstrated by the fusion protein Nb-FGF21.

[0016] Figure 7 This study demonstrates how the fusion protein Nb-FGF21 inhibits lipid accumulation in 3T3-L1 cells undergoing adipogenic differentiation. A: Morphological observation of the inhibition of adipogenic differentiation in 3T3-L1 cells by different concentrations of the fusion protein Nb-FGF21; B: Relative area analysis of lipid staining in 3T3-L1 cells. The * in the bar chart indicates a significant difference between the lipid area in the treatment group and the lipid area in the negative control group (Con), with *P<0.05, **P<0.01, and ***P<0.001.

[0017] Figure 8 The results show the levels of triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol in the blood of diabetic mice after different treatments. In the figures, # represents comparisons between treatment groups and the model group (#P<0.05, ##P<0.01); * represents comparisons between treatment groups (*P<0.05, *P<0.01).

[0018] Figure 9 The results show the levels of alkaline phosphatase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the blood of diabetic mice after different treatments. In the figures, # represents comparisons between treatment groups and the model group (#P<0.05, ##P<0.01); * represents comparisons between treatment groups (*P<0.05, *P<0.01).

[0019] Figure 10 The results of H&E staining and Oil Red O staining of mouse liver tissue are shown.

[0020] Figure 11 This study presents a before-and-after comparison of the clinical treatment of melasma with the fusion protein Nb-FGF21. Photo A shows the image before treatment, and photo B shows the image 90 days after treatment.

[0021] Figure 12 This study presents a before-and-after comparison of clinical treatment of melasma with the fusion protein Nb-FGF19. Photo A shows the image before treatment, and photo B shows the image 90 days after treatment. Detailed Implementation

[0022] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0023] Unless otherwise specified, the biochemical reagents, proteases, etc. used in the embodiments of this invention can be purchased commercially.

[0024] Unless otherwise specified, the molecular biology manipulation methods used in the embodiments of the present invention are based on "Molecular Cloning: A Laboratory Manual" (Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0025] The Pichia pastoris expression vector pPICZαA (catalog number: V19520), the Pichia pastoris expression strain GS115 (catalog number: C18100), and the Escherichia coli competent cells TOP10F (catalog number: C404003) were purchased from Thermo Fisher Scientific Co., Ltd.

[0026] Restriction endonucleases Eco RI (Item No.: 1040A) Sac I (item number: 1078A) and S PE Product I (item number: 1086A) was purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0027] The ion chromatography gels (Sephadex G-25, catalog number: 17003301; QSepharose Fast Flow, catalog number: 17051001) used for purifying the fusion protein were purchased from Cytiva Biotechnology (Hangzhou) Co., Ltd.

[0028] Human serum albumin (catalog number: A1653) was purchased from Sigma.

[0029] The CM5 chip (item number: BR100399) was purchased from Cytiva Biotechnology (Hangzhou) Co., Ltd.

[0030] FGF21 protein lyophilized powder was purchased from Beijing Baipusaisi Biotechnology Co., Ltd., product number: FG1-H5243.

[0031] Dulbecco's Modified Eagle Medium (DMEM; catalog number: 41401ES76) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0032] 3T3-L1 cells (catalog number: CL-173) were purchased from the American Type Culture Collection (ATCC).

[0033] The following kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.: Aspartate aminotransferase (AST) Assay Kit (Catalog No.: C010-1-1), Alanine aminotransferase (ALT) Assay Kit (Catalog No.: C009-1-1), Alkaline phosphatase Assay Kit (Catalog No.: A059-1-1), Total Cholesterol Assay Kit (Catalog No.: A111-1-1), Triglyceride Assay Kit (Catalog No.: A110-2-1), Low-Density Lipoprotein Cholesterol Assay Kit (Catalog No.: A113-2-1), and High-Density Lipoprotein Cholesterol Assay Kit (Catalog No.: A112-2-1).

[0034] Oil Red O staining kit (catalog number: 60720ES60) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0035] Basic phosphate buffer: 5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, pH=7.4.

[0036] Example 1: Construction and Induction of Expression of the Fusion Protein Nb-FGF21 Plasmid

[0037] The amino acid sequence of the fusion protein Nb-FGF21 is shown in SEQ ID No. 1, and the nucleic acid sequence encoding it is shown in SEQ ID No. 2. The nucleic acid sequence shown in SEQ ID No. 2 was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0038] The nucleic acid fragment shown in SEQ ID No. 2 was cloned into the Pichia pastoris expression vector pPICZαA using genetic engineering methods, resulting in the recombinant plasmid pPICZαA-Nb-FGF21. The correctly sequenced recombinant plasmid was then used in S... PE After linearization, the cells were electroporated into the Pichia pastoris GS115 host cell, and positive bacteria were screened on bleomycin-containing solid medium plates (final concentration 100 μg / mL) and named GS115 / pPICZαA-Nb-FGF21.

[0039] Single colonies of GS115 / pPICZαA-Nb-FGF21 were picked from resistance plates and inoculated into 5 mL of Yeast Extract Peptone Dextrose Medium (YPD). The culture was incubated at 30°C and 250 rpm for 24 hours with shaking. Then, the colonies were transferred at a volume ratio of 0.2% to 50 mL of Buffered Glycerol-Complex Medium (BMGY), and incubated at 30°C and 250 rpm for 24 hours with shaking. The cells were then collected by centrifugation at 3000×g for 5 minutes. The cells were then resuspended in 300 mL of Buffered Methanol-Complex Medium (BMMY) containing 0.5% and 1.0% methanol, and incubated at 30°C and 250 rpm. Every 24 hours, 100% methanol was added to the medium to a final concentration of 0.5% or 1.0%, and the culture was continued for 96 hours. Samples were taken before methanol induction and every 24 hours after methanol induction. The fermentation supernatant was collected by centrifuging at 12,000 rpm for 20 minutes and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (See attached image.) Figure 1 . Figure 1 The results showed that after 96 hours of cultivation, both treatment methods using different concentrations of methanol could induce the expression of the fusion protein in recombinant yeast. The molecular weight of the fusion protein Nb-FGF21 was approximately 35.5 kilodaltons (kDa), consistent with its theoretical molecular weight.

[0040] After SDS-PAGE of the fermentation supernatant induced by 0.5% methanol, protein hybridization analysis (Western blot) was performed using FGF21 monoclonal antibody (Selleck Chemicals LLC, catalog number: F1541) as the primary antibody and horseradish peroxidase-labeled goat anti-rabbit antibody (Beyond Biotech, Shanghai, catalog number: A0208) as the secondary antibody. The results are shown in the figure below. Figure 2 . Figure 2 The results showed that the fusion protein Nb-FGF21 could react positively with the FGF21 antibody.

[0041] Example 2: Isolation and purification of fusion protein Nb-FGF21

[0042] After inducing GS115 / pPICZαA-Nb-FGF21 fermentation for 96 hours using 0.5% methanol solution, the fermentation supernatant was obtained as in Example 1. Then, a protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), was immediately added to the fermentation supernatant to a final concentration of 1 mmol / L. Subsequently, while stirring the fermentation supernatant with a magnetic stirrer, ammonium sulfate powder was slowly added until the ammonium sulfate concentration reached 30% saturation (approximately 164 g / L). The mixture was centrifuged at 4°C and 12,000 rpm for 30 minutes, and the precipitate was collected and weighed. The precipitate was redissolved in basal phosphate buffer at a ratio of 1 mg: 50 mL to obtain a protein lysate.

[0043] The protein lysis buffer was loaded onto a Sephadex G-25 column at a flow rate of 10 mL / min, and eluted with basal phosphate buffer to remove salts. The desalted eluent was collected. The desalted eluent was then processed using a Q Sepharose Fast chromatography system. Samples were loaded onto a Flow chromatography column at a flow rate of 10 mL / min. Gradient elution was then performed sequentially using elution buffers containing different sodium chloride concentrations: low sodium chloride concentration elution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.3 mol / L sodium chloride, pH=7.4), medium sodium chloride concentration elution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.6 mol / L sodium chloride, pH=7.4), and high sodium chloride concentration elution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 1.0 mol / L sodium chloride, pH=7.4). Each sodium chloride gradient elution was collected. Samples from each stage were analyzed by SDS-PAGE electrophoresis. Results are shown in [Figure 1]. Figure 3 . Figure 3 The results showed that the target protein was eluted in a gradient elution buffer containing 0.3 mol / L sodium chloride, which was the Nb-FGF21 protein solution. Quantitative analysis revealed that the concentration of the Nb-FGF21 protein solution was 1.2 mg / mL.

[0044] Example 3: Construction, induction of expression, and purification of the Nb-FGF19 fusion protein expression plasmid

[0045] The amino acid sequence of the fusion protein Nb-FGF19 is shown in SEQ ID No. 3, and the nucleic acid sequence encoding it is shown in SEQ ID No. 4. The nucleic acid sequence shown in SEQ ID No. 4 was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0046] The nucleic acid fragment shown in SEQ ID No. 2 in Example 1 was replaced with the nucleic acid fragment shown in SEQ ID No. 4 to obtain the recombinant plasmid pPICZαA-Nb-FGF19. The positive bacteria screened were GS115 / pPICZαA-Nb-FGF19. FGF19 monoclonal antibody (Selleck Chemicals LLC, catalog number: A2653, USA) was used as the primary antibody for protein hybridization analysis.

[0047] Other operations are the same as in Examples 1 and 2.

[0048] Finally, an Nb-FGF19 protein solution was obtained, wherein the target protein Nb-FGF19 was eluted in a gradient elution buffer containing 0.3 mol / L sodium chloride.

[0049] Quantitative analysis revealed that the concentration of the Nb-FGF19 protein solution was 0.6 mg / mL.

[0050] Example 4: Binding activity analysis of fusion protein Nb-FGF21 with human serum albumin

[0051] Human serum albumin was diluted to 10 μg / mL with 20 mmol / L pH 5.0 sodium acetate solution. Following the operating procedure in the instrument manual, human serum albumin was coupled to the CM5 chip using the Biacore T200 instrument to obtain a ligand-coupled chip.

[0052] The Nb-FGF21 protein solution was serially diluted with dilution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.15 mol / L sodium chloride, pH=7.4) to obtain Nb-FGF21 protein diluents of different concentrations: 200 μg / mL, 100 μg / mL, 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL.

[0053] The FGF21 protein was serially diluted with dilution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.15 mol / L sodium chloride, pH=7.4) to obtain different concentrations of FGF21 protein diluents: 280 μg / mL, 140 μg / mL, 70 μg / mL, 35 μg / mL, 17 μg / mL, 9 μg / mL, 5 μg / mL, and 3 μg / mL.

[0054] The ligand-coupled chip was placed into the Biacore T200 instrument. Then, different concentrations of Nb-FGF21 protein dilutions or FGF21 protein dilutions were placed on the sample tube rack, and then placed into the Biacore T200 instrument. The measurement was started according to the instrument's operating instructions. The parameters for the test solution entering the ligand-coupled chip were set as follows: flow rate 30 μL / min, sample binding time 180 s, and dissociation time 300 s. Results are shown below. Figure 4 . Figure 4 The results showed that the affinity of the fusion protein Nb-FGF21 for human serum albumin reached 6.03 × 10⁻⁶. -9 The moles per liter; however, the affinity value of FGF21 protein for human serum albumin was negative, indicating that FGF21 has no affinity for serum albumin. This further illustrates that Nb-FGF21 has a longer-lasting effect compared to FGF21.

[0055] Example 5: Analysis of Nb-FGF21's effect on glucose uptake activity in 3T3-L1 adipocytes

[0056] 3T3-L1 cells were cultured in complete medium (Duborough modified Eagle Medium containing 10% fetal bovine serum) at a rate of 2 × 10⁶ cells / year. 5 The cells were seeded at a density of / wells into 6-well plates and cultured at 37 degrees Celsius until the cell coverage reached 100%. Then, the cells were cultured for another 48 hours to enter the induction phase.

[0057] Determination of glucose content in culture supernatant: Induction phase 1: Discard the original culture medium and add fresh adipogenic induction medium I (Dupuyet modified Eagle's medium, 10% fetal bovine serum, 1 μmol / L dexamethasone solution, 100 μg / mL 3-isobutyl-1-methylxanthine and 10 μg / mL insulin) and culture continuously for 4 days. Induction phase 2: Replace with adipogenic induction medium II (Dupuyet modified Eagle's medium, 10% fetal bovine serum and 10 μg / mL insulin) and culture continuously for 4 days. Induction phase 3: Replace adipogenic induction medium II in the well plate with complete culture medium and culture continuously until obvious lipid droplets appear in the cells (about 6 days). Optical microscope images of 3T3-L1 cells at different induction phases are shown below. Figure 5 After the induction process, the testing phase began: the culture medium was discarded, and fresh starvation medium (DuPont modified Eagle's medium, 0.5% fetal bovine serum, glucose concentration of 25 mmol / L) was added, and the medium was incubated at 37°C for 4 hours; the medium was then discarded again, and starvation medium containing different final concentrations of Nb-FGF21 protein or FGF21 was added, and the medium was continuously incubated for 24 hours. The glucose content in the supernatant of the culture medium was determined using a commercial glucose assay kit (Beijing Solarbio Science & Technology Co., Ltd., catalog number: BC2500). The results are shown in the figure below. Figure 6 The experimental results showed that both the fusion proteins Nb-FGF21 and FGF21 exhibited dose-dependent promotion of glucose uptake activity in 3T3-L1 cells from the culture medium, and there was no statistically significant difference between the two.

[0058] Oil Red O staining: Induction phase 1: Discard the original culture medium and add fresh adipogenic induction medium I (Dupuyet modified Eagle's medium, 10% fetal bovine serum, 1 μmol / L dexamethasone solution, 100 μg / mL 3-isobutyl-1-methylxanthine and 10 μg / mL insulin). At the same time, add the fusion protein Nb-FGF21 at final concentrations of 10 nmol / L, 100 nmol / L, 500 nmol / L, 1000 nmol / L and 2000 nmol / L respectively, and culture continuously for 4 days; Induction phase 2: Change to adipogenic induction medium II (Dupuyet modified Eagle's medium, 10% fetal bovine serum and 10 μg / mL insulin), and add the fusion protein Nb-FGF21 at final concentrations of 10 nmol / L, 100 nmol / L, 500 nmol / L, 1000 nmol / L and 2000 nmol / L again, and culture continuously for 4 days. Induction Phase 3: Replace the adipogenic induction medium II in the well plates with complete medium containing 10% fetal bovine serum, and culture continuously until lipid droplets form in the cells (approximately 6 days). A negative control (Con) was prepared by adding the same volume of dilution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.15 mol / L sodium chloride, pH=7.4) to the cells at a final concentration of 2000 nmol / L for the fusion protein Nb-FGF21. Lipid droplet formation in the cells was detected using an Oil Red O staining kit. Results are shown in [Figure 1]. Figure 7 Experimental results showed that the fusion protein could inhibit the abnormal accumulation of intracellular lipid droplets in a concentration-dependent manner.

[0059] Example 6: Fusion protein Nb-FGF21 alleviates liver damage in diabetic rat model

[0060] db / db mice are a spontaneous type 2 diabetes model animal with a point mutation in the leptin receptor leading to leptin signaling pathway disorder. They often exhibit metabolic disorder symptoms such as obesity, insulin resistance, hyperglycemia, and fatty liver.

[0061] db / db mice were randomly divided into a model group, an FGF21 treatment group (0.5 mg / kg, equivalent to the molar dosage used in the high-dose fusion protein group), a low-dose fusion protein group (0.5 mg / kg), and a high-dose fusion protein group (1.0 mg / kg). During the experiment, all four groups of mice were fed a standard maintenance diet and had free access to water. Drug administration was performed via tail vein injection, while the model group mice received saline injections. The drug was administered every two days for 15 consecutive days. Day 0 was defined as the date of the first injection. Mice were sacrificed two days after the last injection, and were fasted for 12 hours prior to sacrifice. Blood was collected after sacrifice. After cardiac perfusion, the liver was rapidly dissected and removed.

[0062] The levels of triglycerides in blood samples were determined using a triglyceride assay kit, the levels of total cholesterol in blood samples were determined using a total cholesterol assay kit, the levels of high-density lipoprotein cholesterol (HDL-C) in blood samples were determined using a high-density lipoprotein cholesterol (HDL-C) assay kit, and the levels of low-density lipoprotein cholesterol (LDL-C) in blood samples were determined using a low-density lipoprotein cholesterol (LDL-C) assay kit. Results are shown in [Figure number missing]. Figure 8 The results showed that, compared with model animals, the fusion protein (especially the high-dose group) significantly improved biochemical indicators such as triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the blood of experimental mice.

[0063] The levels of alkaline phosphatase (ALP) in blood samples were determined using an alkaline phosphatase assay kit, the levels of alanine aminotransferase (ALT) in blood samples were determined using an alanine aminotransferase (ALT) assay kit, and the levels of aspartate aminotransferase (AST) in blood samples were determined using an aspartate aminotransferase (AST) assay kit. Results are shown in [Figure number missing]. Figure 9 Blood biochemistry results showed that, compared to the model group, both protein treatments significantly downregulated the levels of alkaline phosphatase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Compared to the FGF21 treatment group, the high-dose fusion protein group (i.e., the same molar concentration of the fusion protein) showed a greater advantage in inhibiting AST levels. These results indicate that the fusion protein can effectively alleviate liver damage in diabetic model animals.

[0064] A portion of liver tissue was fixed in 4% paraformaldehyde solution and used for hematoxylin and eosin staining (H&E staining). The results are shown in the figure. Figure 10 . Figure 10H&E staining results showed that the liver cells of the model group mice were loosely arranged, with vacuoles of varying sizes inside the cells, and some showed macrovesicular fatty degeneration. The cells in the low-dose fusion protein treatment group and the FGF21 treatment group were relatively tightly arranged, with a small number of fatty vacuoles; while the cells in the high-dose fusion protein treatment group were tightly arranged, and the improvement effect was the most obvious.

[0065] Another portion of liver tissue was used for Oil Red O staining, and the results are shown below. Figure 10 . Figure 10 Oil Red O staining results showed that large areas of red lipid droplets were deposited in the liver tissue of the model group mice, and larger lipid droplets could be observed. Both the fusion protein and FGF21 treatment groups significantly reduced abnormal lipid accumulation in the liver.

[0066] In summary, both in vitro and in vivo experimental results indicate that the fusion protein can improve symptoms of metabolic disorders and alleviate liver damage caused by metabolic disorders.

[0067] Example 7: Subcutaneous injection of fusion protein improves melasma.

[0068] The Nb-FGF21 protein solution obtained from the separation and purification in Example 2 was prepared into a final concentration of 100 μg / mL using dilution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.15 mol / L sodium chloride, pH=7.4).

[0069] The Nb-FGF19 protein solution obtained from the separation and purification in Example 3 was prepared into a final concentration of 100 μg / mL using dilution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.15 mol / L sodium chloride, pH=7.4).

[0070] Twenty volunteers with melasma on their faces were recruited. In 10 randomly selected participants, a 100 μg / mL Nb-FGF21 protein solution was injected into the melasma area between the epidermis and dermis using a syringe. In another 10 randomly selected participants, a 100 μg / mL Nb-FGF19 protein solution was injected into the same area. Injections were given every two weeks for six consecutive weeks. Photos were taken before injection (day 0) and after six injections (day 90) using the same parameters. One participant's before-and-after photos with Nb-FGF21 protein injection are shown below. Figure 11 Photos of one of the subjects before and after injection of Nb-FGF19 protein are shown below. Figure 12The results showed that Nb-FGF21 effectively promoted the fading of age spots on the face and significantly reduced the area of ​​pigmentation; while Nb-FGF19 did not significantly reduce age spots.

Claims

1. A fusion protein having the amino acid sequence shown in SEQ ID No.

1.

2. The nucleic acid encoding the fusion protein as described in claim 1.

3. The nucleic acid according to claim 2, characterized in that, The sequence of the nucleic acid is shown in SEQ ID No.

2.

4. The use of the fusion protein as described in claim 1 or the nucleic acid as described in claim 2 or 3 in the preparation of a drug for improving melasma.

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