FGF19 mutant protein and its application

Abstract

This invention relates to FGF19 mutant protein and its applications. Compared with FGF19, the mutant protein of this invention retains the functions of FGF19 in upregulating high-density lipoprotein levels and downregulating blood glucose, triglycerides, bile acids, and total cholesterol, while alleviating the side effects of FGF19 such as liver damage and hepatocyte proliferation caused by upregulating low-density lipoprotein, total bilirubin, and direct bilirubin. Furthermore, consistent with FGF19, the mutant protein of this invention can improve hepatocellular lesions.

Description

Technical Field

[0001] This invention relates to the field of functional genes, and in particular to a mutant protein that reduces drug side effects. Background Technology

[0002] FGF19, a member of the endocrine hormone-like subfamily of the fibroblast growth factor (FGF) family, possesses pleiotropic metabolic functions, including precise control over bile acid synthesis. When bile acid levels rise, FGF19 expression increases, thereby inhibiting bile acid synthesis; conversely, when bile acid levels decrease, FGF19 expression decreases, relieving the inhibition of bile acid synthesis. This finely regulated mechanism maintains the dynamic homeostasis of bile acid metabolism. In terms of glucose and lipid metabolism, FGF19 can promote hepatic glycogen synthesis, inhibit gluconeogenesis, and improve insulin sensitivity, thereby lowering blood glucose. Furthermore, FGF19 can inhibit the synthesis of triglycerides and fatty acids, increase fatty acid oxidation, and improve hepatic steatosis.

[0003] Based on its multiple regulatory effects on bile acid and glucose-lipid metabolism, FGF19 can be considered as a candidate drug for treating metabolic diseases such as diabetes, obesity, elevated bile acid levels, and liver disease. However, FGF19 has significant side effects, including upregulating low-density lipoprotein, promoting hepatocyte proliferation leading to liver function damage, and even liver cancer. Therefore, it is necessary to mitigate the side effects of FGF19. Summary of the Invention

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

[0005] The second invention provides a nucleic acid encoding a mutant protein as described in the first invention.

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

[0007] The third invention provides the use of the mutant protein described in the first invention or the nucleic acid described in the second invention in the preparation of a medicament for treating at least one of elevated blood glucose, elevated triglycerides, elevated bile acids, diabetes, and obesity.

[0008] In one specific implementation, the diabetes is type 2 diabetes.

[0009] The beneficial effects of this invention are as follows: Compared with FGF19, the mutant protein of this invention retains the functions of FGF19 in upregulating high-density lipoprotein levels and downregulating blood glucose, triglycerides, bile acids, and total cholesterol, while alleviating the side effects of FGF19 such as liver damage and hepatocyte proliferation caused by upregulating low-density lipoprotein, total bilirubin, and direct bilirubin. Furthermore, consistent with FGF19, the mutant protein of this invention can improve hepatocellular lesions. Attached Figure Description

[0010] Figure 1 The levels of high-density lipoprotein in the serum of each treatment group are shown.

[0011] Figure 2 The levels of low-density lipoprotein in the serum of each treatment group are shown.

[0012] Figure 3 The total cholesterol content in the serum of each treatment group is shown.

[0013] Figure 4 The levels of bile acids in the serum of each treatment group are shown.

[0014] Figure 5 The serum glucose levels in each treatment group are shown in mmol / L.

[0015] Figure 6 The levels of triglycerides in the serum of each treatment group are shown.

[0016] Figure 7 The serum albumin levels in each treatment group are shown.

[0017] Figure 8 The levels of alkaline phosphatase in the serum of each treatment group were shown.

[0018] Figure 9 The total bilirubin content in the serum of each treatment group is shown.

[0019] Figure 10 The levels of direct bilirubin in the serum of each treatment group are shown.

[0020] Figure 11 Immunohistochemical fluorescence micrographs of Ki-67 from each treatment group are shown.

[0021] Figure 12 The images show HE-stained fluorescence micrographs of liver tissue from each treatment group.

[0022] Figure 13 The glucose concentration curves in the culture medium for each treatment group are shown.

[0023] Figure 14 The comparison of glucose concentrations in the culture medium for each treatment group is shown.

[0024] In the above diagram, ns represents p >0.05, no statistically significant difference; * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 indicates a statistically significant difference. Detailed Implementation

[0025] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0026] Unless otherwise specified, the reagents and instruments used in the embodiments of the present invention can be purchased commercially.

[0027] Laboratory Animals and Their Care: The laboratory animals used were sourced from Jiangsu Jicui Biotechnology Co., Ltd. The mouse strain number / name was T002407 / BKS-db, genotype (Lepr)KO / KO, a mouse model used to study type 2 diabetes and obesity. The mice were 10 to 12 weeks old, male, and all animal experiments were conducted in accordance with animal ethics. The laboratory animals were housed at the Animal Center of Wenzhou Medical University.

[0028] HL-7702 cells were normal human hepatocytes, purchased from Shanghai Yaji Biotechnology Co., Ltd.

[0029] Example 1: Preparation of FGF19 protein and FGF19 mutant protein

[0030] The accession number for the nucleic acid encoding the FGF19 protein is GenBank: AF110400.1.

[0031] FGF19 was modified to become FGF19M, the amino acid sequence of which is shown in SEQ ID No. 1, and the nucleic acid sequence encoding it is shown in SEQ ID No. 2.

[0032] Using conventional molecular cloning techniques, the 5' end of FGF19 was fused with bases encoding a small ubiquitin-like modifier (SUMO), a base encoding Gly, and a base encoding a 6×His histidine tag, resulting in a nucleic acid encoding 6×His-Gly-SUMO-FGF19. Specifically, the Gly base lies between the bases encoding the 6-histidine tag and the base encoding SUMO, and the SUMO base lies between the bases encoding Gly and the bases encoding FGF19.

[0033] Using conventional molecular cloning techniques, the 5' end of FGF19M was fused with bases encoding SUMO, Gly, and a six-histidine tag, yielding a nucleic acid encoding 6×His-Gly-SUMO-FGF19M. Specifically, the Gly base is located between the bases encoding the six-histidine tag and the base encoding SUMO, and the SUMO base is located between the bases encoding Gly and the bases encoding FGF19M.

[0034] The nucleic acids encoding 6×His-Gly-SUMO-FGF19 and 6×His-Gly-SUMO-FGF19M were cloned into the expression vector pET24a, respectively, to obtain the recombinant plasmids pET24a-6×His-Gly-SUMO-FGF19 and pET24a-6×His-Gly-SUMO-FGF19M. The recombinant plasmids pET24a-6×His-Gly-SUMO-FGF19 and pET24a-6×His-Gly-SUMO-FGF19M were then transformed into *Escherichia coli* BL21(DE3) using a heat shock method, yielding the recombinant expression strains BL21(DE3) / 6×His-Gly-SUMO-FGF19 and BL21(DE3) / 6×His-Gly-SUMO-FGF19M.

[0035] BL21(DE3) / 6×His-Gly-SUMO-FGF19 cells were cultured in LB liquid medium at 37°C to the logarithmic growth phase. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mmol / L, and the cells were cultured at 16°C for another 14 h. Cells were collected, and the supernatant from cell lysis was used for protein separation using a Ni column. After washing away contaminating proteins, the fusion protein 6×His-Gly-SUMO-FGF19 was eluted with imidazole to obtain the 6×His-Gly-SUMO-FGF19 eluent. SUMOase was added to the 6×His-Gly-SUMO-FGF19 eluent, and hydrolysis was performed at 4°C for 4 h. After complete hydrolysis, the hydrolyzed protein solution (containing the 6×His-Gly-SUMO hydrolysate and FGF19 protein) was obtained. The hydrolyzed protein solution was separated again using a Ni column. The separation of FGF19 protein was achieved by adsorbing 6×His-Gly-SUMO hydrolyzed fragments and trace amounts of unhydrolyzed 6×His-Gly-SUMO-FGF19 onto the Ni column, but not adsorbing FGF19 protein. Specifically, the unadsorbed eluent from the Ni column was collected and loaded onto a hydrophobic column (HiTrapPhenyl FF, Cytiva). The column was then eluted with Tris buffer (25 mmol / L Tris, 2 mmol / L EDTA-2Na, pH 8.0), eluting the FGF19 protein from the column to obtain the FGF19 protein eluent. This eluent was then dialyzed against PBS buffer to obtain a PBS-soluble FGF19 protein solution. Western blot analysis was used to confirm the target protein, and the concentration of the FGF19 protein solution was determined using the Bradford assay.

[0036] Following the same procedures described above, the BL21(DE3) / 6×His-Gly-SUMO-FGF19M recombinant expression strain was cultured, and the FGF19M protein solution was isolated.

[0037] Example 2: Effects of FGF19 protein and FGF19 mutant protein on high-density lipoprotein, low-density lipoprotein and total cholesterol in mice

[0038] Mice were randomly divided into a control group (PBS buffer), an FGF19 group, and an FGF19M group, with five mice in each group. Mice received an intraperitoneal injection once daily at a dose of 1 mg / kg body weight, while the control group received the same volume of PBS buffer. Mice were allowed free access to food and water throughout the experiment, and their diet and weight were monitored. The administration continued for one week.

[0039] Blood was collected from the eyeballs, allowed to stand at room temperature for 2 hours, and centrifuged at 3,000 rpm for 10 minutes. The supernatant was the serum. The high-density lipoprotein (HDL) content in serum was measured using a high-density lipoprotein (HDL) assay kit (surfactant removal method, Shenzhen Raydox Biosciences Co., Ltd.), and the low-density lipoprotein (LDL) content was measured using a low-density lipoprotein (LDL) assay kit (inhibition method, Shenzhen Raydox Biosciences Co., Ltd.). The instrument used was a Chemray 800 fully automated biochemical analyzer (Shenzhen Raydox Biosciences Co., Ltd.). Statistical analysis was then performed. The results are as follows: Figure 1 and Figure 2 As shown, compared with the control group, FGF19 significantly upregulated the levels of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in mouse serum; FGF19M also significantly upregulated HDL levels, but the LDL levels in this group were comparable to those in the control group, meaning that FGF19M did not show an upregulation effect on LDL levels. It is well known to those skilled in the art that the main function of HDL is to reverse the flow of excess and metabolically renewed cholesterol from cells back to the liver for conversion and excretion, earning it the reputation of a "vascular cleanser" and protecting cardiovascular health. LDL, on the other hand, transports cholesterol from the liver to the arterial walls, forming plaques and increasing the risk of atherosclerosis and heart disease. Therefore, elevated HDL levels have certain benefits for the human body, while elevated LDL levels increase the risk of atherosclerosis and cardiovascular disease. The results of this invention show that FGF19M not only retains the function of FGF19 in upregulating high-density lipoprotein levels, but more importantly, FGF19M reduces the side effects of FGF19 upregulating low-density lipoprotein. Therefore, FGF19M has superior lipoprotein regulatory activity.

[0040] Blood was collected from the eyeballs, allowed to stand at room temperature for 2 hours, and centrifuged at 3,000 rpm for 10 minutes. The supernatant was the serum. The total cholesterol content in the serum was determined using a total cholesterol assay kit (CHOD-PAP method, Shenzhen RaydLife Science Co., Ltd.). The instrument used was a Chemray 800 fully automated biochemical analyzer (Shenzhen RaydLife Science Co., Ltd.). Statistical analysis was then performed. The results are as follows: Figure 3 As shown, compared with the control group, both FGF19 and FGF19M significantly downregulated the total cholesterol content in mouse serum, and the downregulation effects of FGF19 and FGF19M were comparable, indicating that FGF19M retained the function of FGF19 in downregulating total cholesterol content.

[0041] Example 3: Effects of FGF19 protein and FGF19 mutant protein on bile acids in mice

[0042] The treatment of the mouse control group, FGF19 group and FGF19M group was the same as in Example 2.

[0043] Blood was collected from the eyeballs, allowed to stand at room temperature for 2 hours, and centrifuged at 3,000 rpm for 10 minutes. The supernatant was the serum. The bile acid content in the serum was detected using a bile acid assay kit (V-generation enzymatic method, Shenzhen Raydox Biosciences Co., Ltd.). The instrument used was a Chemray 800 fully automated biochemical analyzer (Shenzhen Raydox Biosciences Co., Ltd.). Statistical analysis was then performed, and the results are as follows: Figure 4 As shown in the figure. The results showed that the serum bile acid levels in the FGF19M group and the FGF19 group were comparable, and compared with the control group, both the FGF19M group and the FGF19 group significantly reduced the serum bile acid levels, indicating that FGF19M retained the function of FGF19 in downregulating bile acids.

[0044] Example 4: Effects of FGF19 protein and FGF19 mutant protein on blood glucose in diabetic and obese mice

[0045] Mice were randomly divided into an FGF19 group and an FGF19M group, with 5 mice in each group. Blood glucose levels were measured before administration: Mice were fixed in place, and their tails were disinfected with an alcohol swab. A lancet was inserted 3-5 cm into the tail, and the first 1-2 drops of blood were wiped away with a cotton swab. A blood glucose test strip was attached to the front of the glucometer, and the mouse was gently pinched from the base to the tip of its tail. Blood was collected at the tail tip, and the blood glucose level was recorded using the siphon effect of the test strip. Then, either FGF19 protein solution or FGF19M protein solution was injected intraperitoneally into the mice at a dose of 1 mg / kg body weight. Blood glucose levels in different groups were measured 4 hours after administration, following the same procedure. Results are shown below. Figure 5 .

[0046] Compared with the pre-administration blood glucose levels in mice, both FGF19 and FGF19M exhibited hypoglycemic effects, indicating that FGF19M retained the hypoglycemic function of FGF19.

[0047] Example 5: Effects of FGF19 protein and FGF19 mutant protein on triglyceride levels in diabetic and obese mice

[0048] The treatment of the mouse control group, FGF19 group and FGF19M group was the same as in Example 2.

[0049] Blood was collected from the eyeballs, allowed to stand at room temperature for 2 hours, and centrifuged at 3,000 rpm for 10 minutes. The supernatant was the serum. The serum triglyceride content was detected using a triglyceride assay kit (GPO-PAP method, Shenzhen Raydox Biosciences Co., Ltd.). The instrument used was a Chemray 800 fully automated biochemical analyzer (Shenzhen Raydox Biosciences Co., Ltd.). Statistical analysis was then performed, and the results are as follows: Figure 6As shown in the figure. The results showed that, compared with the control group, both FGF19 and FGF19M could downregulate triglyceride levels, and the downregulation levels of the two were similar, indicating that FGF19M retained the function of FGF19 in downregulating triglycerides.

[0050] Example 6: Effects of FGF19 protein and FGF19 mutant protein on albumin and alkaline phosphatase in diabetic and obese mice

[0051] The treatment of the mouse control group, FGF19 group and FGF19M group was the same as in Example 2.

[0052] Blood was collected from the eyeballs, allowed to stand at room temperature for 2 hours, and centrifuged at 3,000 rpm for 10 minutes. The supernatant was the serum. The albumin content in serum was detected using an albumin assay kit (bromocresol green method, Shenzhen Raydox Biosciences Co., Ltd.), and the alkaline phosphatase content in serum was detected using an alkaline phosphatase assay kit (NPP substrate-AMP buffer method, Shenzhen Raydox Biosciences Co., Ltd.). The instrument used was a Chemray 800 fully automated biochemical analyzer (Shenzhen Raydox Biosciences Co., Ltd.). Statistical analysis was then performed, and the results are as follows: Figure 7 and Figure 8 As shown in the figure. The results showed that, compared with the control group, both FGF19 and FGF19M could increase the levels of albumin and alkaline phosphatase in mouse serum, and the effects of the two were comparable. This indicates that FGF19M retains the activity of FGF19 in improving liver function by increasing the levels of albumin and alkaline phosphatase.

[0053] Example 7: Effects of FGF19 protein and FGF19 mutant protein on total bilirubin and direct bilirubin in diabetic and obese mice

[0054] The treatment of the mouse control group, FGF19 group and FGF19M group was the same as in Example 2.

[0055] Blood was collected from the eyeballs, allowed to stand at room temperature for 2 hours, and centrifuged at 3,000 rpm for 10 minutes. The supernatant was the serum. The total bilirubin and direct bilirubin levels in the serum of mice from different groups were detected using a total bilirubin assay kit (endpoint method / C120, Changchun Huili Biotechnology Co., Ltd.) and a direct bilirubin assay kit (endpoint method / C119, Changchun Huili Biotechnology Co., Ltd.). The instrument used was a Chemray 800 fully automated biochemical analyzer (Shenzhen Leidu Life Science Co., Ltd.). Statistical analysis was then performed, and the results are shown below. Figure 9 and Figure 10As shown in the figure. The results showed that, compared with the control group, FGF19 significantly increased the levels of total bilirubin and direct bilirubin in serum, indicating that hepatocytes were damaged; while FGF19M was comparable to the control group, indicating that FGF19M eliminated the liver-damaging activity of FGF19.

[0056] Example 8: Effects of FGF19 protein and FGF19 mutant protein on mouse hepatocytes

[0057] The treatment of the mouse control group, FGF19 group and FGF19M group was the same as in Example 2.

[0058] Mouse liver tissue was collected and analyzed using Ki-67 immunohistochemistry (Ki-67 immunohistochemistry is an immunohistochemical staining method used to detect cell proliferation activity). Results were obtained using an upright fluorescence microscope (Nikon, Japan). Figure 11 As shown, compared with the control group, the Ki-67 positivity rate of the FGF19 group was significantly increased, while the Ki-67 positivity rate of the FGF19M group was significantly lower than that of the FGF19 group. This indicates that FGF19M significantly weakens the hepatocyte proliferation-promoting activity of FGF19 in vivo, reduces the side effect of FGF19 causing liver cancer through hepatocyte proliferation, and improves the safety of medication.

[0059] Example 9: Effects of FGF19 protein and FGF19 mutant protein on liver tissue morphology in diabetic and obese mice

[0060] The treatment of the mouse control group, FGF19 group and FGF19M group was the same as in Example 2.

[0061] Mouse liver tissue was collected and stained with hematoxylin-eosin (HE) using a hematoxylin-eosin staining kit. The staining was observed using an upright fluorescence microscope (Nikon, Japan). Results are shown in the figure below. Figure 12 The results showed that the control group mice had round, tense vacuoles of varying sizes in the cytoplasm of their hepatocytes, indicating hepatocellular lesions. In contrast, the hepatocytes of mice in the FGF19M and FGF19 groups had normal cytoplasm, and the presence of hepatocyte vacuoles was significantly reduced, suggesting that FGF19M and FGF19 improved the hepatocellular lesions in mice to some extent.

[0062] Example 10: Effects of FGF19 protein and FGF19 mutant protein on glucose uptake in HL-7702 cells

[0063] HL-7702 cell lines were revived, passaged to the second generation, and cultured in complete medium at 37°C and 5% CO2 for 8 h. The medium was then removed, and the cells were digested with trypsin at 37°C for 5 min. The cell pellet was obtained by centrifugation and resuspended in complete medium at a density of (1.6-1.8) × 10⁻⁶ cells / year. 5 Cells / mL. The cell suspension was then inoculated into 96-well plates at a rate of 100 μL / well and incubated for 24 hours at 37°C, 5% CO2. An equal volume of maintenance medium containing 0.5% serum was then added, and the plates were cultured for another 24 hours under the same conditions. Serial concentrations of FGF19 or FGF19M were added, with six replicates per group, and the plates were cultured at 37°C, 5% CO2 for 48 hours. An equal volume of PBS buffer containing FGF19 or FGF19M protein solution was added to the maintenance medium as a control.

[0064] The glucose oxidase method was used. After treatment with a glucose assay kit (Shenyang Wanlei Biotechnology Co., Ltd.), the absorbance was measured at a wavelength of 510 nm using an ELISA reader. The concentration of glucose in the culture medium was calculated. The results are shown below. Figure 13 The results showed that the glucose concentration in the culture medium of both the FGF19 and FGF19M groups decreased with increasing concentrations of FGF19 or FGF19M. Both FGF19 and FGF19M promoted glucose uptake by HL-7702 hepatocytes. Statistical analysis was performed on different groups at a concentration of 100 μg / mL for FGF19 and FGF19M. The results are shown in [the table below]. Figure 14 Analysis revealed that the glucose concentration in the culture medium of both the FGF19 group and the FGF19M group was significantly lower than that of the control group. Furthermore, the glucose concentration in the culture medium of the FGF19M group was significantly lower than that of the FGF19 group. This indicates that FGF19M is more effective than FGF19 in promoting glucose uptake by hepatocytes and is more helpful in lowering blood sugar.

Claims

1. The use of a mutant protein or nucleic acid in the preparation of at least one medicament for treating diabetes and obesity, wherein the amino acid sequence of the mutant protein is shown in SEQ ID No. 1, and the nucleic acid encodes the mutant protein.

2. The application according to claim 1, characterized in that, The diabetes mentioned is type 2 diabetes.

3. The application according to claim 1 or 2, characterized in that, The base sequence of the nucleic acid is shown in SEQ ID No. 2.

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