rhFGF21 fusion proteins, polynucleotides encoding rhFGF21 fusion proteins, compositions containing rhFGF21 fusion proteins, and uses of rhFGF21 fusion proteins

Abstract

Recombinant human fibroblast growth factor 21 (rhFGF21) fusion proteins, polynucleotides encoding the rhFGF21 fusion proteins, recombinant expression vectors, recombinant cells, pharmaceutical compositions and kits containing the rhFGF21 fusion proteins, uses of the rhFGF21 fusion proteins, and methods for producing the rhFGF21 fusion proteins. Efficient and soluble expression is achieved by the synthesis of rhFGF21-(VPGXG) n This was achieved using genetic engineering methods to obtain a fusion protein, where rhFGF21 and an elastin-like protein (VPGXG) with an appropriate cycle number n were synthesized. n By means of fusion with rhFGF21, the resulting fusion protein has better biological activity than wild-type rhFGF21. Further experimental results indicate that rhFGF21-(VPGXG) n The fusion protein has high expression efficiency and can effectively reduce blood glucose levels in diabetic animals, and has the advantage of longer duration of efficacy in reducing blood glucose levels compared with wild-type hFGF21.

Description

[Technical Field]

[0001] The present application is in the field of biomedicine, and in particular, relates to recombinant human fibroblast growth factor 21 (rhFGF21) and elastin-like protein (VPGXG) fused together. n the present invention relates to fusion proteins comprising the above-mentioned nucleotides, polynucleotides encoding the fusion proteins, recombinant expression vectors and recombinant cells, compositions and kits comprising the fusion proteins, their uses, and methods for producing the fusion proteins. [Background technology]

[0002] Fibroblast growth factor 21 (FGF21) is a metabolic regulator produced primarily by the liver that exerts potent hypoglycemic and hypolipidemic effects in animal models of obesity and type 2 diabetes. The primary sites of FGF21 metabolic action are adipose tissue, liver, and pancreas. Experimental studies have shown improvements in diabetic compensation and dyslipidemia after administration of FGF21 in diabetic mice and primates.

[0003] However, FGF21 has a very short circulatory half-life in vivo, and therefore cannot achieve the desired efficacy. Modification of FGF21 with polyethylene glycol (PEG) can effectively improve its pharmacokinetics, improve drug distribution, and enhance its efficacy. Nevertheless, current PEGylated FGF21 has drawbacks, such as low reaction yields and difficulties in controlling the binding site and coupling stoichiometry, which increase production costs. Therefore, developing a site-specific modification method that is rapid and efficient, with mild reaction conditions and simple procedures, is particularly important to ensure the efficacy of FGF21.

[0004] The pentapeptide repeating unit is a polypeptide with elasticity and temperature sensitivity, consisting of five amino acids, VPGXG. X (i.e., Xaa) can be any amino acid except proline. It has good biocompatibility, but for different proteins, different cycle numbers (i.e., VPGXG unit repeats) will have different effects on the expression level, half-life, and activity. The appropriate number of cycles of elastin to ensure the activity of FGF21 and extend its half-life has not yet been reported and needs to be explored experimentally. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, the development of suitable fusion proteins is necessary to ensure the activity of human fibroblast growth factor 21 (hFGF21) and extend its half-life, while the further development of suitable in vitro expression systems for fusion proteins is also necessary to efficiently obtain active fusion proteins. [Means for solving the problem]

[0006] In this application, in view of the problems in the medical process of rhFGF21, such as the short half-life of rhFGF21 and the inability to guarantee the activity of rhFGF21, the present inventors have provided a novel recombinant human fibroblast growth factor 21 fusion protein through research. Furthermore, the present inventors have also developed a prokaryotic expression system suitable for the fusion protein, which in turn allows the soluble expression of the rhFGF21 fusion protein with desired efficiency.

[0007] In one aspect, the present application relates to a recombinant human fibroblast growth factor 21 fusion protein, wherein the fusion protein comprises rhFGF21 and (VPGXG) fused to each other. n rhFGF21 comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 80% identity to SEQ ID NO: 1, wherein X is A and / or V, and n is an integer selected from 20 to 80.

[0008] In another aspect, the present application relates to a polynucleotide encoding the above-described fusion protein.

[0009] In another aspect, the present application relates to a recombinant expression vector containing a polynucleotide encoding the above-described fusion protein.

[0010] In another aspect, the present application relates to a recombinant cell containing the above-described recombinant expression vector or having the above-described polynucleotide integrated into its genome.

[0011] In another aspect, the present application relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises the fusion protein described above.

[0012] In another aspect, the present application relates to a kit, wherein the kit comprises the fusion protein described above or the pharmaceutical composition described above.

[0013] In another aspect, the present application relates to use of the above-mentioned fusion protein in the preparation of a medicament for preventing or treating a disease associated with abnormal glucose metabolism. Alternatively, the present application relates to a method for preventing or treating a disease associated with abnormal glucose metabolism, comprising administering the above-mentioned fusion protein to a subject in need thereof. Alternatively, the present application relates to a fusion protein for use in the prevention or treatment of a disease associated with abnormal glucose metabolism.

[0014] In yet another aspect, the present application relates to a method of producing the above-described fusion protein of the present application, wherein the method comprises culturing the above-described recombinant cell, then inducing the recombinant cell to express, and purifying the expression product to obtain the fusion protein.

[0015] In this specification, the effect of the fusion protein described in the present application on the treatment of diabetes is evaluated using a diabetic model mouse. The results of animal experiments show that the fusion protein of the present application can efficiently reduce blood glucose levels in diabetic model animals compared to wild-type hFGF21, and has the advantage of, for example, a longer duration of efficacy (i.e., a longer half-life) in reducing blood glucose levels. In addition, the fusion protein described in the present application exhibits good ability in promoting glucose uptake and can achieve desired expression efficiency. [Brief explanation of the drawings]

[0016] [Figure 1] Electropherograms showing the expression levels of SUMO-rhFGF21-(VPGXG)n fusion proteins. Reference symbols represent the following: 1: protein marker, 2: protein expression before IPTG induction, 3: SUMO-rhFGF21, 4: SUMO-rhFGF21-(VPGXG)20, 5: SUMO-rhFGF21-(VPGXG)40, 6: SUMO-rhFGF21-(VPGXG)60, and 7: SUMO-rhFGF21-(VPGXG)80. Grayscale analysis of the results shows that the percentage of SUMO-rhFGF21-(VPGXG)40 expression relative to the total protein expression is higher than that of the other groups, i.e., under the same conditions, SUMO-rhFGF21-(VPGXG)40 is expressed more efficiently: SUMO-rhFGF21-(VPGXG)40, 11.2%; SUMO-rhFGF21-(VPGXG)20, 9.7%; SUMO-rhFGF21-(VPGXG)60, 10.1%; SUMO-rhFGF21-(VPGXG)80, 3.2%. [Figure 2]This is a graph showing changes in glucose uptake in β-Klotho-3T3-L1 adipocytes after stimulation with various test samples. After stimulation with 10 nmol / L SUMO-rhFGF21 (denoted as "rhFGF21"), the glucose uptake level of β-Klotho-3T3-L1 adipocytes was approximately 35%. After stimulation with 10 nmol / L rhFGF21-(VPGXG)20, the glucose uptake level of β-Klotho-3T3-L1 adipocytes increased to approximately 39%. After stimulation with 10 nmol / L rhFGF21-(VPGXG)40, the glucose uptake level of β-Klotho-3T3-L1 adipocytes increased to approximately 46%. After stimulation with 10 nmol / L rhFGF21-(VPGXG)60, the glucose uptake level of β-Klotho-3T3-L1 adipocytes increased to approximately 37%. After stimulation with 10 nmol / L rhFGF21-(VPGXG)80, the glucose uptake level of β-Klotho-3T3-L1 adipocytes increased by approximately 32%. 10 nmol / L rhFGF21-(VPGXG)40 showed a better ability to promote glucose uptake at the cellular level compared to other groups. [Figure 3] 1 is an HPLC spectrogram showing the analysis of the purity of rhFGF21-(VPGXG)40 protein. Throughout the HPLC analysis, a peak of rhFGF21-(VPGXG)40 protein began to appear at a retention time of approximately 6 minutes, and its purity may reach 95% or more. [Figure 4] 1 is a graph showing the blood glucose lowering results after 12 hours of treatment of STZ model mice with various test samples, where the blood glucose lowering effect of the rhFGF21 treatment group gradually decreased 12 hours after administration compared with the model group. The rhFGF21-(VPGXG)20 treatment group, rhFGF21-(VPGXG)40 treatment group, rhFGF21-(VPGXG)60 treatment group, and rhFGF21-(VPGXG)80 treatment groups still showed certain therapeutic effects, with the rhFGF21-(VPGXG)40 treatment group showing the best blood glucose lowering effect. Here, *p<0.05 and **p<0.01 compared with the model group; #p<0.05 and ##p<0.01 compared with the normal group. [Figure 5] This is a graph showing the results of an oral glucose tolerance test (OGTT). 30 minutes after oral administration of glucose, the blood glucose level in the model group increased significantly, and the blood glucose level in the rhFGF21-(VPGXG)40-treated group was significantly lower than that in the model group. Compared with the model group, *p<0.05, **p<0.01; compared with the normal group, #p<0.05, ##p<0.01. [Figure 6] This is a graph showing the changes in blood glucose levels in mice in each group over 58 days. The blood glucose levels of mice in the model group were consistently maintained at relatively high levels. Meanwhile, the blood glucose levels of mice in the rhFGF21-(VPGXG)40-treated group were significantly lower than those of the model group, maintained at relatively low levels, approaching those of mice in the normal group after treatment, and its effectiveness lasted longer than that of rhFGF21. Here, *p<0.05, **p<0.01 compared to the model group; #p<0.05, ##p<0.01 compared to the normal group. [Figure 7] Graph showing serum insulin concentration of mice in each group. The serum insulin concentration of mice in the model group was significantly decreased, while the serum insulin concentration of mice in the rhFGF21-(VPGXG)40 treatment group was significantly increased compared to the model group. Here, *p<0.05, **p<0.01 compared to the model group; #p<0.05, ##p<0.01 compared to the normal group. [Figure 8] 1 is a graph showing the glycated hemoglobin level in mice of each group. After 30 days of administration of the corresponding test sample, the glycated hemoglobin level of the model group mice remained relatively high, while the rhFGF21-(VPGXG)40-treated group mice showed a significant decrease in the glycated hemoglobin level. Here, compared with the model group, *p<0.05, **p<0.01; compared with the normal group, #p<0.05, ##p<0.01. [Figure 9]This is a graph showing the relative expression level of the G6Pase gene in the liver of mice in each group. The expression level of G6Pase in the liver of the model group mice was significantly increased, while the expression level of G6Pase in the liver of the rhFGF21-(VPGXG)40-treated mice was significantly decreased. Here, *p<0.05, **p<0.01 compared to the model group; #p<0.05, ##p<0.01 compared to the normal group. [Figure 10] This is a graph showing the relative expression level of PCK gene in the liver of mice in each group. The expression level of PCK gene in the liver of mice in the model group was significantly increased, while the expression level of PCK gene in the liver of mice in the rhFGF21-(VPGXG)40 treatment group was significantly decreased. Here, *p<0.05, **p<0.01 compared to the model group; #p<0.05, ##p<0.01 compared to the normal group. [Figure 11] This is a graph showing the expression levels of G6Pase and PCK proteins in the livers of mice in each group. The expression levels of G6Pase and PCK proteins in the livers of mice in the model group were significantly increased, whereas the expression levels of G6Pase and PCK proteins in the livers of mice in the rhFGF21-(VPGXG)40 treatment group were significantly decreased. Compared to the model group, *p<0.05, **p<0.01; compared to the normal group, #p<0.05, ##p<0.01. [Figure 12] This is a graph showing the expression levels of G6Pase and PCK proteins in the livers of mice in each group. The expression levels of G6Pase and PCK proteins in the livers of mice in the model group were significantly increased, whereas the expression levels of G6Pase and PCK proteins in the livers of mice in the rhFGF21-(VPGXG)40 treatment group were significantly decreased. Compared to the model group, *p<0.05, **p<0.01; compared to the normal group, #p<0.05, ##p<0.01. [Figure 13] Photographs showing the results of HE staining and immunohistochemistry of the pancreas. The pancreatic islets of the model mice were more severely damaged, and the amount of insulin secreted was significantly reduced, whereas the pancreatic islets of the rhFGF21-(VPGXG)40-treated mice were significantly improved compared to the model group. [Figure 14] FIG. 1 is a spectrogram showing the results of HPLC analysis of rhFGF21-(VPGXG)40 protein after storage at 25°C for 14 days in citrate buffer solutions with different pH values ​​(pH 5.0 and 5.5) and the same protein stored at -80°C. [Figure 15] FIG. 1 is a spectrogram showing the HPLC analysis results of rhFGF21-(VPGXG)40 protein after storage for 25 days at 25°C in aqueous arginine solutions with different concentrations (containing 50 mM, 100 mM, and 150 mM arginine) and the same protein stored at -80°C. [Figure 16] FIG. 1 is a spectrogram showing the results of HPLC analysis of rhFGF21-(VPGXG)40 protein after storage in arginine and citrate buffer solution (pH 5.5) at 25° C. for 35 days and the same protein stored at −80° C. [Figure 17] FIG. 1 is a spectrogram showing the HPLC analysis results of rhFGF21-(VPGXG)40 protein after storage at 4°C for 60 days in arginine + citrate buffer solutions with different pH values ​​(pH 5.0 and 5.5) and the same protein stored at -80°C. [Figure 18] FIG. 1 is a graph showing the results of promoting glucose uptake in HepG2 cells by different doses (10 nmol, 100 nmol and 1000 nmol) of rhFGF21-(VPGXG)40 protein after storage at 25° C. or −80° C. for 35 days. DETAILED DESCRIPTION OF THE INVENTION

[0017] In each of the fusion proteins shown above, X is A and V, and the ratio of A to V is 2:3.

[0018] The present application will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present application will be more clearly understood through the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of the present application. It should be understood by those skilled in the art that modifications or substitutions can be made to the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions shall fall within the protection scope of the present application.

[0019] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs, and can be found, for example, in Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

[0020] In the present disclosure, unless otherwise specified, the terms "model group" and "model control group" can be used interchangeably, and the terms "normal group," "mock group," and "normal control group" can be used interchangeably.

[0021] Unless otherwise specified, the terms "recombinant plasmid" and "recombinant expression vector" can be used interchangeably herein.

[0022] Unless otherwise specified, the term "linker" as used herein refers to the linker between rhFGF21 and (VPGXG) n and (VPGXG) nand the repeating units of the nucleotide sequence can be an amino acid sequence (e.g., can be from about 1 to about 5 amino acids in length) used to link the repeating units of the nucleotide sequence to one another and can be used in identification, and can include, for example, but are not limited to, linker peptides and restriction sites commonly known in the art.

[0023] Unless otherwise stated, singular terms herein include the plural and vice versa.

[0024] Unless otherwise indicated, the terms "comprise," "comprises," and "comprising" or their equivalents ("contain," "containing," "include," and "including") used herein are open-ended expressions meaning that other unspecified elements, ingredients, and steps may be in addition to those already listed.

[0025] Unless otherwise specified, the term "treating" herein includes reversing, alleviating, ameliorating, suppressing, slowing or halting the progression or severity of the associated condition.

[0026] Unless otherwise specified, the term "subject" herein can be used interchangeably with the terms "individual" and "patient" and includes vertebrates, such as birds, fish, and mammals, including, but not limited to, mice, rats, guinea pigs, dogs, pigs, chickens, rabbits, monkeys (e.g., rhesus monkeys), humans, etc.

[0027] Unless otherwise specified, all numbers used herein to express quantities of ingredients, measurements, or reaction conditions are to be understood as being modified in all instances by the term "about." When applied to percentages, the term "about" can represent ±1%.

[0028] In the present disclosure, the percentage of identity (degree of homology) between sequences can be determined by comparing two sequences, for example, using computer programs commonly used for this purpose that are freely accessible on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0029] In one embodiment, the present application provides a recombinant human fibroblast growth factor 21 fusion protein, wherein the fusion protein comprises rhFGF21 and (VPGXG) fused to each other. n rhFGF21 comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 80% identity to SEQ ID NO: 1, wherein X is A and / or V, and n is an integer selected from 20 to 80.

[0030] In a preferred embodiment, rhFGF21 comprises an amino acid sequence having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, 98% or 99% identity to SEQ ID NO:1.

[0031] In a preferred embodiment, rhFGF21 is SEQ ID NO: 1 (SEQ ID NO: 1: [ka] ) has the amino acid sequence described in

[0032] In a preferred embodiment, X is A and V, and the ratio of A to V is (1-3):(1-3), for example, the ratio of A to V is (1-2):(1-3), (1-2):(2-3), (2-3):(1-3), (2-3):(2-3), (2-3):(1-2), 1:(1-3), 2:(1-3), or 3:(1-3). In a more preferred embodiment, X is A and V, and the ratio of A to V is 2:3 (i.e., (VPGXG) n Here, it is [(VPGAG)2(VPGVG)3] m In this case, the value of m satisfies n=m×(2+3).

[0033] In preferred embodiments, n is an integer selected from 30 to 80, for example, n is an integer selected from 30 to 70, 30 to 60, 30 to 50, 40 to 80, 40 to 70, 40 to 60, or 40 to 50. In certain embodiments, n is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75. In certain embodiments, n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. In some more preferred embodiments, n is 40 to 60, for example 40.

[0034] In a preferred embodiment, the fusion protein comprises rhFGF21, (VPGXG) n and optionally a linker.

[0035] In a preferred embodiment, the amino acid sequence of the linker is RS and / or GS.

[0036] In a preferred embodiment, the fusion protein has an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, 98% or 99% identity to SEQ ID NO:2.

[0037] In a preferred embodiment, the fusion protein is SEQ ID NO:2 (SEQ ID NO:2: [ka] ) has the amino acid sequence described in

[0038] In the present disclosure, a fusion protein comprising rhFGF21 having an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or a fusion protein comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 2 has hypoglycemic activity.

[0039] In one embodiment, the present application provides a polynucleotide encoding the above-described fusion protein. Unless otherwise specified, the terms "polynucleotide," "nucleic acid," "nucleic acid sequence," and "gene" are used interchangeably herein.

[0040] In some preferred embodiments, the portion of the polynucleotide encoding rhFGF21 in the above-described fusion protein comprises the nucleotide sequence set forth in SEQ ID NO:3, or a nucleotide sequence having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, 98% or 99% identity to SEQ ID NO:3.

[0041] In a further preferred embodiment, the portion of the polynucleotide encoding rhFGF21 in the above-described fusion protein is SEQ ID NO:3 (SEQ ID NO:3: [ka] )

[0042] In some preferred embodiments, the portion of the polynucleotide encoding the linker in the fusion protein described above comprises a nucleotide sequence set forth in SEQ ID NO:4 and / or SEQ ID NO:5, or a nucleotide sequence having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, 98% or 99% identity to SEQ ID NO:4 and / or SEQ ID NO:5.

[0043] In a further preferred embodiment, the portion of the polynucleotide encoding the linker in the above-mentioned fusion protein has the nucleotide sequence set forth in SEQ ID NO: 4 and / or SEQ ID NO: 5 (SEQ ID NO: 4: AGATCT; SEQ ID NO: 5: GGATCC).

[0044] In some preferred embodiments, the polynucleotide encoding the fusion protein comprises the nucleotide sequence set forth in SEQ ID NO:6, or a nucleotide sequence having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, 98% or 99% identity to SEQ ID NO:6.

[0045] In a preferred embodiment, the polynucleotide encoding the fusion protein is SEQ ID NO: 6 (SEQ ID NO: 6: [ka] TIFF0007729842000005.tif22162).

[0046] In one embodiment, the present application provides a recombinant expression vector containing a polynucleotide encoding the fusion protein described above.

[0047] Although many types of expression vectors are currently available, selecting an appropriate vector for a specific protein is not easy. Many expression systems achieve high protein expression levels, but the expressed proteins are poorly soluble. It is preferable that the fusion proteins of the present application can be expressed in a prokaryotic expression system to facilitate mass production and subsequent purification. Therefore, a prokaryotic expression vector suitable for a prokaryotic expression system is contemplated herein. Considering that molecular chaperones help support protein folding, a prokaryotic expression vector carrying a molecular chaperone can be selected. In this case, the molecular chaperone is preferably a small ubiquitin-like modifier protein (SUMO). SUMO is a small ubiquitin-like modifier protein that can facilitate the correct folding of target proteins, thereby improving the expression and solubility of the target proteins.

[0048] Non-limiting examples of suitable expression vectors suitable for the present application include, but are not limited to, pSUMO vectors, PET-based vectors (eg, PET30a vectors).

[0049] In one embodiment, the present application provides a recombinant cell containing the above-described recombinant expression vector or having the above-described polynucleotide integrated into its genome.

[0050] Considering the convenience of subsequent fermentation and purification, the host cells used to construct the recombinant cells are preferably prokaryotic host cells, including, but not limited to, Escherichia coli (E. coli) (e.g., E. coli Rossetta (DE3)), Agrobacterium tumefaciens, Staphylococcus aureus, Staphylococcus albus, Lactobacillus acidophilus, Bacillus anthracis, Bacillus subtilis, or Bacillus thuringiensis.

[0051] In one embodiment, the present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the fusion protein described above.

[0052] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.The pharmaceutically acceptable excipient can be selected from, but is not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, coloring agents, disintegrants, bulking agents, lubricants, wetting agents, osmotic pressure adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, diluents, etc.For other available pharmaceutically acceptable excipients, see, for example, RC Rowe et al. (mainly translated by Zemin Zheng), Handbook of Pharmaceutical Excipients, 4th ed., Chemical Industry Press, 2005.

[0053] In one embodiment, the present application relates to a kit, wherein the kit comprises the fusion protein described above or the pharmaceutical composition described above.

[0054] In some preferred embodiments, the kit further comprises a citrate buffer solution at pH 5.0 to 5.5.

[0055] In some preferred embodiments, the kit further comprises an aqueous arginine solution, preferably at a concentration of 50 mmol / L to 150 mmol / L.

[0056] In some preferred embodiments, the kit further comprises an arginine and citrate buffer solution at pH 5.0-5.5.

[0057] In some preferred embodiments, the kit further comprises a citrate buffer solution of pH 5.0-5.5 and an arginine aqueous solution, and the fusion protein or pharmaceutical composition, the citrate buffer solution of pH 5.0-5.5, and the arginine aqueous solution are packaged together or separately.

[0058] In one embodiment, the present application relates to the use of the above-described fusion protein in the preparation of a medicament for the prevention or treatment of a disease associated with abnormal glucose metabolism. In an alternative embodiment, the present application relates to a method for preventing or treating a disease associated with abnormal glucose metabolism, comprising administering the above-described fusion protein to a subject in need thereof. In an alternative embodiment, the present application relates to the fusion protein for use in the prevention or treatment of a disease associated with abnormal glucose metabolism.

[0059] In a preferred embodiment, the disease associated with abnormal glucose metabolism is selected from, but is not limited to, diabetes and diabetes-related metabolic diseases. Preferably, the diabetes-related metabolic diseases are selected from, but are not limited to, diabetic nephropathy, dyslipidemia, obesity, cardiovascular disease, metabolic syndrome, lipid metabolism disorders, non-alcoholic fatty liver disease (NAFLD), or nervous system diseases (e.g., epilepsy, depression, etc.).

[0060] In one embodiment, the present application provides a method for producing the above-mentioned fusion protein of the present application, wherein the method comprises: culturing a recombinant cell, then inducing the recombinant cell to express, and purifying the expression product to obtain the fusion protein.

[0061] In a preferred embodiment, the recombinant cell may be a recombinant prokaryotic cell, preferably, examples of recombinant prokaryotic cells include, but are not limited to, recombinant Escherichia coli (e.g., recombinant E. coli Rossetta (DE3)), recombinant Agrobacterium tumefaciens, recombinant Staphylococcus aureus, recombinant Staphylococcus aureus, recombinant Lactobacillus acidophilus, recombinant Bacillus anthracis, recombinant Bacillus subtilis, or recombinant Bacillus thuringiensis.

[0062] Those skilled in the art can culture recombinant cells by selecting conventional culture media and conditions according to the type of recombinant cell (see, for example, records such as Manufacture and Application of Microbial Medium (primarily edited by Tianshou Chen), China Agricultural Press, 1995; Practical Handbook of Microbial Medium (primarily edited by Lehao ​​Ma et al.), Jilin Science and Technology Press, 2005; http: / / www.cgmcc.net / ; and https: / / www.sigmaaldrich.com / china-mainland / technical-documents / articles / biology / microbial-media.html).

[0063] Expression in the recombinant cells can be induced using conventional inducers known to those skilled in the art, and in exemplary embodiments, expression in the recombinant cells is induced using an inducer selected from IPTG and / or lactose.

[0064] The expression product can be purified to obtain the fusion protein using conventional purification means known to those skilled in the art. Conventional purification means can be, for example, but are not limited to, ion exchange chromatography, adsorption chromatography, affinity chromatography, molecular sieve chromatography, hydrophobic chromatography, or any combination thereof. In a preferred embodiment, purification of the expression product includes (i) inducing expression, centrifuging the recombinant cells to collect the bacteria, disrupting the bacteria, and then collecting the supernatant by centrifugation, (ii) subjecting the supernatant to ion exchange chromatography and collecting the eluate, and (iii) subjecting the eluate to affinity chromatography to obtain the fusion protein.

[0065] In some preferred embodiments, the recombinant cells are centrifuged at 10,000-12,000 rpm / min to collect the bacteria.

[0066] In some preferred embodiments, the bacteria are disrupted by adding lysozyme to the bacteria and subjecting them to sonication.

[0067] In some preferred embodiments, the supernatant is collected by centrifugation at 10,000-12,000 rpm / min.

[0068] In some preferred embodiments, ion exchange chromatography is performed using an agarose gel, such as a DEAE agarose gel.

[0069] In some preferred embodiments, the supernatant is eluted using a buffer containing 25 mmol / L Tris-HCl and 0.25 mol / L NaCl at a pH of 8.0 to 8.2.

[0070] In some preferred embodiments, affinity chromatography is performed using Ni·NTA resin.

[0071] In some preferred embodiments, affinity chromatography involves equilibrating an affinity chromatography column with an equilibration buffer, then loading the eluate onto the affinity chromatography column, washing the column with a wash buffer, and then eluting the target protein with an elution buffer.

[0072] In some preferred embodiments, the equilibration buffer contains 50 mmol / L Tris-HCl, 0.5 mol / L NaCl, and 10 mmol / L imidazole at a pH of 8.0 to 8.2.

[0073] In some preferred embodiments, the wash buffer contains 50 mmol / L Tris-HCl, 500 mmol / L NaCl, and 50 mmol / L imidazole at a pH of 8.0 to 8.2.

[0074] In some preferred embodiments, the elution buffer contains 50 mmol / L Tris-HCl, 500 mmol / L NaCl, and 500 mmol / L imidazole at a pH of 8.0 to 8.2.

[0075] In some preferred embodiments, the methods for producing the fusion proteins described above further comprise: (1) synthesizing a polynucleotide encoding rhFGF21; and 20 synthesizing a polynucleotide encoding (2) the above polynucleotide encoding rhFGF21 and (VPGXG) 20 performing enzymatic digestion and ligation on a polynucleotide encoding the fusion protein; and obtaining a polynucleotide encoding the fusion protein by PCR reaction; (3) performing enzymatic digestion and ligation on the polynucleotide encoding the fusion protein and the expression vector to obtain a recombinant expression vector; introducing the recombinant expression vector into a host cell; and screening to obtain a recombinant cell.

[0076] A polynucleotide sequence encoding rhFGF21 can be synthesized using conventional gene synthesis means by codon optimization based on the protein sequence of rhFGF21 or its DNA or RNA sequence known in the art (see, for example, https: / / www.ncbi.nlm.nih.gov / protein / NP_061986.1; https: / / www.ncbi.nlm.nih.gov / nuccore / NG_033945.1; or https: / / www.ncbi.nlm.nih.gov / nuccore / NM_019113.4).

[0077] In a preferred embodiment, in step (1), the polynucleotide sequence encoding rhFGF21 comprises the nucleotide sequence set forth in SEQ ID NO: 3, or a nucleotide sequence having at least 80%, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, 98% or 99% identity to SEQ ID NO: 3.

[0078] In a preferred embodiment, in step (1), the polynucleotide encoding rhFGF21 has the nucleotide sequence set forth in SEQ ID NO:3.

[0079] In a preferred embodiment, in step (1), (VPGXG) 20 The polynucleotide encoding comprises the nucleotide sequence set forth in SEQ ID NO:7 or a nucleotide sequence having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, 98% or 99% identity to SEQ ID NO:7.

[0080] In a preferred embodiment, in step (1), (VPGXG) 20 The polynucleotide encoding the sequence of SEQ ID NO: 7 (SEQ ID NO: 7: [ka] )

[0081] In a preferred embodiment, in step (2), the polynucleotides encoding the linkers selected from RS and / or GS are each (VPGXG) 20 Preferably, the polynucleotides having the nucleotide sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 5 are optionally linked to the 5' and 3' ends of the polynucleotides encoding (VPGXG) 20 are linked to the 5' and 3' ends of a polynucleotide encoding

[0082] In a preferred embodiment, in step (2), the enzymatic digestion is carried out using BglII and BamHI restriction endonucleases.

[0083] Suitable conventional PCR experimental conditions and steps can be selected by those skilled in the art based on general knowledge in the art (see, for example, Molecular Cloning: A Laboratory Manual (4th ed.) (edited by J. Sambrook et al., translated primarily by Fuchu He), Science Press, 2017), or PCR reactions can be performed using commercially available PCR kits according to their instructions. Other primer pairs and other means of gene synthesis known in the art may also be used to obtain polynucleotides encoding the above-mentioned fusion proteins, without limitation.

[0084] The above-mentioned enzymatic digestion and ligation have the meaning well known in the art, and herein, enzymatic digestion and ligation refer to the following: specific cleavage of two target DNA fragments or one target DNA fragment and a vector DNA molecule using a restriction nucleic acid endonuclease, followed by ligation of the ends of the two enzymatically digested target DNA fragments or the end of the enzymatically digested target DNA fragment and a vector DNA molecule using a ligase, thereby obtaining a recombinant molecule or a recombinant vector.

[0085] In a preferred embodiment, in step (3), enzymatic digestion is carried out using BsaI and BamHI restriction endonucleases.

[0086] Regarding the specific means for introducing the recombinant expression vector into the host cell, any conventional means known in the art can be used, such as transformation, transfection, etc. Preferably, the recombinant expression vector is introduced into the host cell by transformation. Transformation refers to a method of treating a host cell using some of the known methods in molecular biology and genetic engineering to make the treated host cell competent, thereby contacting the host cell with exogenous DNA, resulting in the exogenous DNA being introduced into the competent host cell.

[0087] In a preferred embodiment, in step (3), the host cell is a prokaryotic host cell, preferably including, but not limited to, Escherichia coli (e.g., E. coli Rossetta (DE3)), Agrobacterium tumefaciens, Staphylococcus aureus, Staphylococcus aureus, Lactobacillus acidophilus, Bacillus anthracis, Bacillus subtilis, or Bacillus thuringiensis.

[0088] For host cells into which recombinant expression vectors are introduced, positive recombinant cells can be obtained by screening using conventional means, such as resistance screening. Those skilled in the art can culture recombinant cells by selecting conventional culture media and culture conditions according to the type of recombinant cell (see, for example, "Manufacture and Application of Microbial Medium" (mainly edited by Tianshou Chen), China Agricultural Press, 1995; "Practical Handbook of Microbial Medium" (mainly edited by Lehao ​​Ma et al.), Jilin Science and Technology Press, 2005; http: / / www.cgmcc.net / ; and https: / / www.sigmaaldrich.com / china-mainland / technical-documents / articles / biology / microbial-media.html, etc.).

[0089] In a preferred embodiment, in step (3), the expression vector is selected from, but not limited to, a pSUMO vector and a PET-based vector (e.g., a PET30a vector). In a more preferred embodiment, a pSUMO vector carrying a small ubiquitin-like modifier protein (SUMO) as a molecular chaperone is used as the expression vector, and Escherichia coli Rossetta (DE3) is used as the host cell.

[0090] In an exemplary embodiment, a recombinant pSUMO vector containing a polynucleotide encoding the fusion protein is transformed into a host cell, the recombinant cells obtained by screening are cultured, the recombinant cells are induced to express the SUMO fusion protein, and the expression product is purified by ion exchange chromatography and affinity chromatography to obtain a pre-purified SUMO fusion protein. After dialysis, the molecular chaperone SUMO linked to the fusion protein is cleaved using a SUMO protease, and the fusion protein after SUMO cleavage is purified by affinity chromatography to obtain a purified fusion protein.

[0091] The exemplary technical solution of the present application can be illustrated by the following numbered paragraphs: 1. A recombinant human fibroblast growth factor 21 fusion protein, wherein the fusion protein is a recombinant human fibroblast growth factor 21 (rhFGF21) and a recombinant human fibroblast growth factor 21 (VPGXG) fused together. n wherein rhFGF21 comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 80% identity to SEQ ID NO: 1, X is A and / or V, and n is an integer selected from 20 to 80.

[0092] 2. The fusion protein according to paragraph 1, wherein X is A and V, and the ratio of A to V is (1-3):(1-3), preferably 2:3.

[0093] 3. A fusion protein according to paragraph 1 or 2, wherein n is an integer from 40 to 60, preferably 40.

[0094] 4. rhFGF21 fused to each other, (VPGXG) n 4. A fusion protein according to any one of paragraphs 1 to 3, comprising:

[0095] 5. A fusion protein according to paragraph 4, wherein the amino acid sequence of the linker is RS and / or GS.

[0096] 6. A fusion protein according to any one of paragraphs 1 to 5, having an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence having at least 80% identity to SEQ ID NO:2.

[0097] 7. A polynucleotide encoding a fusion protein according to any one of paragraphs 1 to 6.

[0098] 8. The polynucleotide of paragraph 7, wherein the portion of the polynucleotide encoding rhFGF21 in the fusion protein comprises the nucleotide sequence set forth in SEQ ID NO:3, or a nucleotide sequence having at least 80% identity to SEQ ID NO:3.

[0099] 9. The polynucleotide of paragraph 7 or 8, wherein the portion of the polynucleotide encoding the linker in the fusion protein comprises the nucleotide sequence set forth in SEQ ID NO:4 and / or SEQ ID NO:5, or a nucleotide sequence having at least 80% identity to SEQ ID NO:4 and / or SEQ ID NO:5.

[0100] 10. A polynucleotide according to any one of paragraphs 7 to 9, comprising the nucleotide sequence shown in SEQ ID NO:6 or a nucleotide sequence having at least 80% identity to SEQ ID NO:6.

[0101] 11. A recombinant expression vector comprising a polynucleotide according to any one of paragraphs 7 to 10.

[0102] 12. The recombinant expression vector of paragraph 11, which is a prokaryotic recombinant expression vector.

[0103] 13. The recombinant expression vector according to paragraph 12, which is a prokaryotic recombinant expression vector carrying a molecular chaperone.

[0104] 14. The recombinant expression vector of paragraph 13, wherein the molecular chaperone is a small ubiquitin-like modifying protein.

[0105] 15. The recombinant expression vector of paragraph 11 or 12, which is a recombinant pSUMO vector or a recombinant PET-based vector.

[0106] 16. A recombinant cell comprising a recombinant expression vector according to any one of paragraphs 11 to 15 or having integrated into its genome a polynucleotide according to any one of paragraphs 7 to 10.

[0107] 17. A recombinant cell according to paragraph 16, wherein the host cell for constructing the recombinant cell is a prokaryotic host cell.

[0108] 18. The recombinant cell of paragraph 17, wherein the prokaryotic host cell is selected from Escherichia coli, Agrobacterium tumefaciens, Staphylococcus aureus, Staphylococcus aureus, Lactobacillus acidophilus, Bacillus anthracis, Bacillus subtilis, or Bacillus thuringiensis.

[0109] 19. A pharmaceutical composition comprising a fusion protein according to any one of paragraphs 1 to 6.

[0110] 20. The pharmaceutical composition according to paragraph 19, further comprising a pharmaceutically acceptable excipient.

[0111] 21. A kit comprising a fusion protein according to any one of paragraphs 1 to 6 or a pharmaceutical composition according to paragraph 19 or 20.

[0112] 22. The kit of paragraph 21, further comprising a citrate buffer solution having a pH of 5.0 to 5.5.

[0113] 23. The kit according to paragraph 21 or 22, further comprising an aqueous arginine solution, preferably an aqueous arginine solution having a concentration of 50 mmol / L to 150 mmol / L.

[0114] 24. A kit according to any one of paragraphs 21 to 23, further comprising an arginine + citrate buffer solution having a pH of 5.0 to 5.5.

[0115] 25. The kit of paragraph 21, wherein the kit further comprises an aqueous arginine solution and a citrate buffer solution of pH 5.0 to 5.5, and the fusion protein or pharmaceutical composition, the citrate buffer solution of pH 5.0 to 5.5, and the aqueous arginine solution are packaged together or separately.

[0116] 26. Use of a fusion protein according to any one of paragraphs 1 to 6 in the preparation of a medicament for the prevention or treatment of a disease associated with abnormal glucose metabolism.

[0117] 27. The use according to paragraph 26, wherein the disease associated with abnormal glucose metabolism is selected from diabetes and diabetes-related metabolic diseases, preferably the diabetes-related metabolic diseases are selected from diabetic nephropathy, dyslipidemia, obesity, cardiovascular disease, metabolic syndrome, lipid metabolism disorders, non-alcoholic fatty liver disease, or nervous system diseases.

[0118] 28. A method for producing a fusion protein according to any one of paragraphs 1 to 6, comprising culturing a recombinant cell according to any one of paragraphs 16 to 18, then inducing the recombinant cell to express the protein, and purifying the expression product to obtain the fusion protein.

[0119] 29. The method of paragraph 28, wherein expression in the recombinant cells is induced using an inducer selected from IPTG and / or lactose.

[0120] 30. The method of paragraph 28 or 29, wherein purification of the expression product comprises (i) inducing expression, then centrifuging the recombinant cells to collect the bacteria, disrupting the bacteria, and then collecting the supernatant by centrifugation; (ii) subjecting the supernatant to ion exchange chromatography and collecting the eluate; and (iii) subjecting the eluate to affinity chromatography to obtain the fusion protein.

[0121] 31. (1) synthesizing a polynucleotide encoding rhFGF21; and20 synthesizing a polynucleotide encoding (2) a polynucleotide encoding rhFGF21 and (VPGXG) 20 performing enzymatic digestion and ligation on a polynucleotide encoding the fusion protein; and obtaining a polynucleotide encoding the fusion protein by PCR reaction; (3) performing enzymatic digestion and ligation on the polynucleotide encoding the fusion protein and the expression vector to obtain a recombinant expression vector; introducing the recombinant expression vector into a host cell; and screening to obtain a recombinant cell. 31. The method of any one of paragraphs 28 to 30, further comprising:

[0122] 32. The method of paragraph 31, wherein in step (1), the polynucleotide sequence encoding rhFGF21 comprises the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence having at least 80% identity to SEQ ID NO:3.

[0123] 33. In step (1), (VPGXG) 20 33. The method of paragraph 31 or 32, wherein the polynucleotide encoding comprises the nucleotide sequence set forth in SEQ ID NO:7 or a nucleotide sequence having at least 80% identity to SEQ ID NO:7.

[0124] 34. In step (2), the polynucleotide encoding the linker selected from RS and / or GS is (VPGXG), 20 34. The method of any one of paragraphs 31 to 33, wherein the polynucleotide encoding the

[0125] 35. The method of any one of paragraphs 31 to 34, wherein in step (2), the enzymatic digestion is carried out using BglII and BamHI restriction endonucleases.

[0126] 36. The method of any one of paragraphs 31 to 35, wherein in step (3), the enzymatic digestion is carried out using BsaI and BamHI restriction endonucleases.

[0127] 37. The method according to any one of paragraphs 31 to 36, wherein in step (3), the expression vector is selected from a pSUMO vector and a PET-based vector.

[0128] 38. The method of any one of paragraphs 31 to 37, wherein in step (3), the expression vector is a pSUMO vector and the host cell is Escherichia coli Rossetta (DE3).

[0129] It should be noted that the operational steps involved in this application, such as gene design, synthesis and cloning, construction of prokaryotic expression vectors, nucleic acid extraction and sequence analysis and identification, and isolation and purification of expression products, can be performed according to techniques known in the art (see, for example, the archives of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY). Unless otherwise indicated, the technical meanings used in this example have conventional meanings known to those skilled in the art. Unless otherwise indicated, the reagents, materials and equipment used in the following examples are commercially available.

[0130] [Example] [Example 1] Preparation of recombinant human fibroblast growth factor 21 fusion protein gene rhFGF21 gene (VPGXG) 20 The genes were linked via the BglII restriction site, and then PCR was performed to obtain rhFGF21-(VPGXG) 20 The BsaI and BamHI restriction sites were introduced at the 5' and 3' ends of the rhFGF21-(VPGXG) 20 was subjected to BglII and BamHI enzyme digestion using the BamHI restriction site (VPGXG) 20 It is linked to another gene, rhFGF21-(VPGXG) 40PCR was then performed to obtain rhFGF21-(VPGXG) 40 A BsaI restriction site was introduced at the 5' end of rhFGF21-(VPGXG) 40 A stop codon and a BamHI restriction site were introduced at the 3' end of

[0131] 1. Synthesis of rhFGF21 gene with enzyme restriction sites The amino acid sequence of wild-type hFGF21 (SEQ ID NO: 1) was searched on NCBI, and the nucleotide sequence encoding rhFGF21 (SEQ ID NO: 3) was obtained through codon optimization. Synthesis of a gene (SEQ ID NO: 8) containing a BsaI enzyme restriction site (SEQ ID NO: 10: GGTCTCAGGT) and a BglII enzyme restriction site (SEQ ID NO: 4: AGATCT) was outsourced to a gene synthesis company (SEQ ID NO: 8: [ka] ).

[0132] 2. Linker (VPGXG) 20 Preparation of the gene Linker (VPGXG) having the nucleotide sequence set forth in SEQ ID NO: 9 20 The synthesis of the gene was outsourced to a gene synthesis company, and in this case, the gene consisted of: a polynucleotide encoding a linker RS ​​having at its 5' end the nucleotide sequence set forth in SEQ ID NO:4 (i.e., a BglII restriction site), the nucleotide sequence of which is set forth in SEQ ID NO:7 (VPGXG) 20 and a polynucleotide encoding a linker GS (SEQ ID NO: 9) having the nucleotide sequence set forth in SEQ ID NO: 5 (i.e., a BamHI restriction site) at its 3' end. [ka] ).

[0133] 3. rhFGF21-(VPGXG) 40 Preparation of fusion protein genes The gene for rhFGF21 with the restriction sites obtained above and the linker (VPGXG) 20 The gene was used as a template. Enzymatic digestion was performed using BglII restriction endonuclease according to the manufacturer's instructions, and ligation was performed using T4 DNA ligase to generate rhFGF21-(VPGXG) 20 The nucleotide sequence of the polypeptide was obtained.

[0134] rhFGF21-(VPGXG) 40 To obtain the gene for the fusion protein, PCR was performed using primers P1 (SEQ ID NO: 11) and P2 (SEQ ID NO: 12) and rTaq enzyme according to the 50 μL system described below. 20 BsaI and BamHI restriction sites were introduced at the 5' and 3' ends of the nucleotide sequence of the polypeptide. [ka]

[0135] The above nucleotide sequence with introduced restriction sites was ligated into a T vector using Solution I ligase, followed by digestion with BamHI restriction endonuclease. Meanwhile, the linker - (VPGXG) 20 The gene was digested with BglII and BamHI restriction endonucleases according to the manufacturer's instructions, and the digested products of the two enzymes were extracted from the gel using a DNA Fragment Gel Extraction Kit (purchased from Omega, Cat. No.: D6492) and then ligated with T4 DNA ligase.

[0136] The ligation product was then used as a template to perform a PCR reaction (50 μL system) using rTaq enzyme to introduce a stop codon (TCA): 10x PCR buffer 5.0μL dNTP 4.0 μL Template 2.0 μL (10 ng) P1 1.0 μL (10 pmol) P3 1.0 μL (10 pmol) rTaq enzyme 0.5 μL ddH2O 36.5μL Here, the sequences of primer P1 and primer P3 (SEQ ID NO: 13) were as follows: [ka]

[0137] The resulting PCR product was extracted from the gel using a DNA fragment gel extraction kit (purchased from Omega, Cat. No.: D6492) to give rhFGF21-(VPGXG) 40 The gene for the fusion protein was obtained.

[0138] 4. Preparation of Other Fusion Protein Genes rhFGF21-(VPGXG) 20 , rhFGF21-(VPGXG) 60 , and rhFGF21-(VPGXG) 80 The genes for each fusion protein were transformed into rhFGF21-(VPGXG) 40 The fusion protein was obtained according to the same procedures and conditions as described above for the preparation of the gene.

[0139] where rhFGF21-(VPGXG) 20 To prepare the gene for the fusion protein, the obtained rhFGF21-(VPGXG) 20 A stop codon was introduced directly into the 3' end of the nucleotide sequence of the polypeptide via PCR without introducing any additional restriction sites, but in this case the 50 μL PCR system was the same as above.

[0140] [Example 2] Preparation of human fibroblast growth factor 21 fusion protein pSUMO and E. coli Rossetta (DE3) involved in this example were commercially available. 1. rhFGF21-(VPGXG) 40 Construction of recombinant prokaryotic expression vectors for genes Target gene rhFGF21-(VPGXG) obtained in Example 1 40 The fragment and the prokaryotic expression vector pSUMO were double-digested with BsaI and BamHI enzymes according to the manufacturer's instructions, and the digestion products were purified using a DNA fragment gel extraction kit (purchased from Omega, Cat. No.: D6492), followed by ligation overnight at 16°C using T4 DNA ligase in a 10 μL ligation reaction system according to the manufacturer's instructions.

[0141] After double digestion with BsaI and BamHI enzymes and identification of the digestion product by agarose gel electrophoresis, the recombinant plasmid pSUMO-rhFGF21-(VPGXG) containing the correct sequence was obtained. 40 was constructed.

[0142] 2. rhFGF21-(VPGXG) 40 Protein expression and purification (1) Induced expression The recombinant plasmid pSUMO-rhFGF21-(VPGXG) containing the correct sequence 40 The resulting vector was transformed into competent E. coli Rossetta (DE3) expression cells. The transformed competent cells were plated on LB plates containing 100 μg / mL ampicillin and incubated at 37°C for 10–12 hours. A single colony of transformed recombinant bacteria was then selected and inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin. The colony was then incubated at 37°C for 10 hours. The culture was inoculated into 500 mL of LB medium containing 100 μg / mL ampicillin at a ratio of 1:100 and incubated at 37°C for 2 hours. When the A600 was 0.3–0.6, IPTG was added to a final concentration of 0.5 mmol / L for induction. (Prior to induction, the culture was harvested and centrifuged at 12,000 rpm for 30 minutes to obtain the supernatant and precipitate. The supernatant and precipitate were analyzed by 15% SDS-PAGE electrophoresis.) After 3 hours of induction, bacteria were harvested by centrifugation at 12000 rpm / min for 30 minutes.

[0143] (2) Protein purification After collecting the bacteria by centrifugation, lysozyme was added to the bacteria to a final concentration of 1 mg / mL and allowed to stand on ice for 1 hour. The bacteria were then sonicated and, after lysis, centrifuged (12,000 rpm / min, 30 min) to collect the supernatant. The resulting supernatant and precipitate were collected and analyzed by 15% SDS-PAGE electrophoresis. In this case, the soluble expressed target protein may account for more than 70% of the total target protein in the bacteria. The supernatant was purified through a DEAE Sepharose FF chromatography column. Elution was performed with Buffer 1 (25 mmol / L Tris-HCl, 0.25 mol / L NaCl, pH 8.0), and the ion exchange chromatography eluate was collected. The ion exchange chromatography eluate was subjected to affinity chromatography on a Ni·NTA resin column. The Ni·NTA resin column was first equilibrated with equilibration buffer (50 mmol / L Tris-HCl, pH 8.0, 0.5 mol / L NaCl, 10 mmol / L imidazole). The eluate was then loaded onto the column. After loading, washing buffer (50 mmol / L Tris-HCl, pH 8.0, 0.5 mol / L NaCl, 50 mmol / L imidazole) was used for washing. Finally, the target protein was eluted with elution buffer (50 mmol / L Tris-HCl, pH 8.0, 0.5 mol / L NaCl, 500 mmol / L imidazole). The solution from each elution peak was collected to obtain the affinity chromatography eluate, which was then subjected to SDS-PAGE analysis.

[0144] The affinity chromatography eluate was dialyzed overnight at 4°C against PBS buffer (pH 7.4) to remove imidazole, resulting in the fusion protein SUMO-rhFGF21-(VPGXG). 40 To this was added DTT to a final concentration of 2 mmol / L and SUMO Protease-I (with His tag) (cleavage ratio: 1:50, i.e., 1 mg of SUMO Protease-I: SUMO-rhFGF21-(VPGXG) 4050 mg of rhFGF21-(VPGXG) was added, and cleavage was carried out overnight at 4° C. The resulting cleavage product was thoroughly mixed with Ni-NTA agarose, and the flow-through was collected, where nonspecifically adsorbed rhFGF21-(VPGXG) 40 The protein was eluted with PBS buffer (pH 7.4). After HPLC analysis (mobile phase was PBS buffer at a flow rate of 1 mL / min, column: Agilent AdvanceBio SEC 300 Å, 2.7 μm, 7.8 × 300 mm, Cat. No.: PL1180-5301), purified rhFGF21-(VPGXG) 40 The protein (having the amino acid sequence set forth in SEQ ID NO: 2) was shown to have a purity of greater than 95% (FIG. 3).

[0145] rhFGF21-(VPGXG) 20 , rhFGF21-(VPGXG) 60 and rhFGF21-(VPGXG) 80 The expression and purification of each protein was performed using rhFGF21-(VPGXG) 40 The expression and purification procedures and conditions were the same as those described above for the proteins. The expression levels of each protein were analyzed using 15% SDS-PAGE electrophoresis (Figure 1).

[0146] [Example 3] Activity test of human fibroblast growth factor 21 fusion protein 1. rhFGF21-(VPGXG) at the cellular level 40 Protein activity assay β-Klotho was cloned into the retroviral eukaryotic expression vector pBMN-β Klotho-IRES-EGFP, and green fluorescent protein was used as a reporter gene. The recombinant retroviral vector was transfected into 293 packaging cells by liposome transfection, and the viral supernatant was collected by centrifugation and used to infect 3T3-L1 progenitor cells (purchased from ATCC, Cat. No.: CL-173). β-Klotho-3T3-L1 stable cell lines expressing β-Klotho, known as β-Klotho-3T3-L1 adipocytes, were screened using flow cytometry. β-Klotho-3T3-L1 adipocytes were starved for 12 hours with purified rhFGF21-(VPGXG). 40 The proteins were diluted with cell culture medium (DMEM, Gibco, C11965500BT) to final concentrations of 10 nmol / L, 100 nmol / L, and 1000 nmol / L, and the dilutions were added at different concentrations at 1 ml per well to differentiate mature adipocytes. At least three replicate wells were set for each concentration.

[0147] Detection of sugar concentration (glucose concentration): After incubating the cells treated above for 24 hours, 2 μL of the culture supernatant was transferred to 200 μL of glucose assay solution to measure the glucose content. Each concentration was measured at least three times, and after incubation at 37°C for 5-10 minutes, the OD was measured at 500 nm. The cellular glucose consumption rate was calculated as follows, and the experimental results were statistically analyzed.

[0148] The residual glucose concentration in the culture medium (C) is calculated using the formula: Glucose concentration (mmol / L) = OD 試料 / OD 標品 ×5.55mmol / L was calculated as follows. The cellular glucose consumption rate is calculated by the formula: Cellular glucose consumption rate (%) = [(C モック群のグルコース -C 投与群のグルコース ) / C モック群のグルコース ]×100% was calculated as follows.

[0149] Different concentrations of rhFGF21-(VPGXG) 40 After treating the adipocytes with the protein for 24 hours, the glucose content in the culture medium was measured using a microscale glucose assay kit (GOD-POD method). Statistical analysis showed that the rhFGF21-(VPGXG) group significantly increased glucose levels compared to the untreated control group. 40 The uptake and utilization of glucose by the cells treated with the protein was significantly improved, and the content of residual glucose in the culture medium was significantly reduced.

[0150] β-Klotho-3T3-L1 adipocytes were treated with rhFGF21 and the fusion proteins prepared according to the above method. The results showed that the glucose uptake of untreated β-Klotho-3T3-L1 adipocytes was about 27%, and after stimulation with 10 nmol / L rhFGF21, it increased to about 35%, and after stimulation with 10 nmol / L rhFGF21-(VPGXG), it increased to about 35%. 20 After stimulation with 10 nmol / L rhFGF21-(VPGXG), the increase was approximately 39%. 40 After stimulation with 10 nmol / L rhFGF21-(VPGXG), the percentage increased to approximately 46%. 60 After stimulation with 10 nmol / L rhFGF21-(VPGXG), the percentage increased to approximately 37%. 80 The results showed that after stimulation with 10 nmol / L rhFGF21-(VPGXG), the 40 The fusion protein had the highest activity compared with rhFGF21 and other fusion proteins at the same dose (FIG. 2).

[0151] 2. Comparison of the in vivo hypoglycemic activity of rhFGF21 and various fusion proteins in animals Diabetic mice were obtained by inducing STZ (80 mg / kg) in C57BL / 6J mice (Changchun Yisi Experimental Animal Technology Co., Ltd., Animal Quality Certificate No.: SCXK(Ji)-2011-0004). Mice with a consistent blood glucose level above 13.8 mmol / L were considered diabetic.

[0152] The established diabetic model mice were randomly divided into six groups of eight mice per group. Meanwhile, a group of eight normal mice was used as a mock control group. Mice in the model control group were injected with PBS buffer and rhFGF21-(VPGXG) by subcutaneous administration. 40 Mice in the treatment group were injected with the fusion protein at a dose of 1 mg / kg / day, and rhFGF21-(VPGXG) 20 , rhFGF21-(VPGXG) 60 and rhFGF21-(VPGXG) 80 Mice in each treatment group received rhFGF21-(VPGXG) 40 The same dose of fusion protein as in the treatment group was injected. 12 hours after the injection was completed (the mice were allowed to feed freely during this time), the blood glucose levels of the mice in each group were measured. The results showed that 12 hours after administration, the blood glucose lowering effect of the rhFGF21 treatment group was reduced compared with the model group; each fusion protein treatment group showed a certain therapeutic effect, especially rhFGF21-(VPGXG) 40 The blood glucose levels of mice treated with the fusion protein were significantly lower than those of the model group (p<0.01). 40 The treatment group had the highest blood glucose lowering effect compared to the other groups (Figure 4).

[0153] 3. rhFGF21-(VPGXG) 40 In vivo activity testing of proteins (1) rhFGF21-(VPGXG) in a diabetic mouse model 40 Protein activity detection Diabetic mice (blood glucose consistently above 13.8 mmol / L) were obtained by inducing STZ (80 mg / kg) in C57BL / 6J mice (Changchun Yisi Experimental Animal Technology Co., Ltd., Animal Quality Certificate No.: SCXK(Ji)-2011-0004) and randomly divided into 5 groups with 8 mice per group. Meanwhile, 8 normal mice were used as normal controls. Mice in the model control group were injected with PBS buffer and rhFGF21-(VPGXG) by subcutaneous administration. 40 The mice in the treatment group were injected with the fusion protein at a dose of 1 mg / kg / day for 58 consecutive days. The experiment started at 8:00 am (at which point the blood glucose levels of the model animals were relatively high for the day), and the mice were allowed to feed ad libitum throughout the experiment.

[0154] Model control group: Diabetic mice were subcutaneously injected with PBS buffer at approximately 8:00 am, with a single injection volume of 0.15 mL.

[0155] rhFGF21-(VPGXG) 40 Treatment group: rhFGF21-(VPGXG) in diabetic mice 40 The fusion protein (solvent: PBS buffer) was injected subcutaneously every day at approximately 8:00 am, with a single injection dose of rhFGF21-(VPGXG) 40 The fusion protein is 1 mg / kg / day (total protein amount) and the single injection volume is 0.15 mL.

[0156] Normal control group: Healthy mice were injected subcutaneously with PBS buffer every day at approximately 8:00 am, with a single injection volume of 0.15 mL.

[0157] The blood glucose levels of mice in each group were measured every two days, and the results showed that rhFGF21-(VPGXG) 40The blood glucose levels of mice treated with the fusion protein for three days were significantly lower than those of the model group, and the blood glucose levels of the treated mice were maintained at a relatively low level and approached those of the normal group six days after treatment. After 30 consecutive days of treatment, the glucose tolerance levels of mice in each group were measured by OGTT. The results showed that the blood glucose levels of mice in the model group significantly increased within 30 minutes after oral administration of glucose and then slowly decreased 30 minutes later. rhFGF21-(VPGXG) 40 The blood glucose levels of the mice in the treatment group were significantly lower than those of the model group, and within 60 minutes, the blood glucose levels had decreased to approach those of the mice in the normal group (Figure 5). 40 After treatment with the fusion protein, the glucose tolerance of the mice was significantly improved. During the 58-day injection period, the blood glucose levels of the mice in the model group were consistently maintained at relatively high levels. 40 The blood glucose levels of the mice in the treatment group were significantly lower than those of the model group. After treatment, blood glucose levels were maintained at a relatively low level and approached those of the normal group. The effectiveness of rhFGF21 lasted longer than that of rhFGF21 (Figure 6). The serum insulin levels of the mice in the model group were significantly lower than those of the normal group. 40 After treatment with the fusion protein, serum insulin content was significantly elevated compared to the model group (Figure 7). The glycated hemoglobin level of the mice in the model group was significantly elevated compared to the normal group. Compared with the model group, the glycated hemoglobin level was significantly elevated in the rhFGF21-(VPGXG) 40 The expression levels of G6Pase and PCK in the liver were measured by real-time fluorescent quantitative PCR and Western blot. The results showed that the expression levels of G6Pase and PCK in the model group mice were significantly elevated compared with those in the normal group, and that the expression levels of rhFGF21-(VPGXG) 40After treatment with , the expression levels of G6Pase and PCK in the mice were significantly reduced compared to the model group (Figures 9 to 12). In addition, damage to the pancreatic islets was evaluated using HE staining and immunohistochemistry. The results showed that the expression levels of G6Pase and PCK in the model group mice were significantly reduced compared to the model group mice (Figures 9 to 12). Pancreatic islets were severely damaged and the amount of secreted insulin was significantly reduced, whereas rhFGF21-(VPGXG) 40 The pancreatic islet damage in the treated mice was significantly improved, and the amount of secreted insulin was significantly increased (FIG. 13).

[0158] [Example 4] Determination of conditions for stable storage of human fibroblast growth factor 21 fusion proteins 1. rhFGF21-(VPGXG) 40 Determining the buffer solution for proteins The following buffers were prepared: acetate buffer solutions of pH 4.0 and pH 5.0 (prepared by dissolving acetic acid in water); citrate buffer solutions of pH 5.0, pH 5.5, and pH 6.0 (prepared by dissolving citric acid and sodium citrate in water); phosphate buffer solutions of pH 6.5 and pH 7.0 (prepared by dissolving disodium hydrogen phosphate and sodium dihydrogen phosphate in water); and Tris-HCl buffer solutions of pH 7.5 and pH 8.0 (prepared by dissolving Tris-HCl in water). A HiPrep™ 26 / 10 desalting column was connected to an AKTA purifier 100 system. First, the protective solution (20% ethanol) in the desalting column was washed out with 8 to 10 column volumes of distilled water. Then, the desalting column was equilibrated with the target buffer solution of 8 to 10 column volumes. The sample (rhFGF21-(VPGXG) containing a concentration of 2 mg / mL in phosphate buffer at pH 7.4) was added. 40 The UV curve first rose. When the UV curve dropped to the baseline, the conductance curve rose, and a collector was used to collect the protein at the corresponding UV absorption peak. 40 Protein was obtained. Nine groups of rhFGF21-(VPGXG) were exchanged into multiple buffer solutions.40 The proteins were stored in a 25°C incubator protected from light. The appearance of the stored proteins was checked at regular intervals, and the proteins were sampled for 15% SDS-PAGE electrophoresis and HPLC assay (PBS buffer, 1 mL / min, chromatography column: Agilent AdvanceBio SEC 300 Å, 2.7 μm, 7.8 × 300 mm, Cat. No.: PL1180-5301). rhFGF21-(VPGXG) stored at -80°C 40 An aqueous solution of the protein was used as a control.

[0159] The results showed that rhFGF21-(VPGXG) 40 The protein appeared cloudy to the naked eye immediately after exchange into acetate buffer solution at pH 4.0. 40 The protein appeared cloudy at 7 days in acetate buffer at pH 4.5, at 3 days in Tris-HCl buffer at pH 8.0, and at 7 days in Tris-HCl buffer at pH 7.5. 40 The protein did not appear cloudy to the naked eye in any of the citrate and phosphate buffer solutions at any pH value on day 14. The proteins stored in various buffers were sampled for 15% SDS-PAGE analysis and HPLC assay. The results of 15% SDS-PAGE showed that the rhFGF21-(VPGXG) stored in the phosphate buffer solutions at two different pH values ​​was cloudy. 40 Although the protein solutions were free of visible turbidity, most of the protein degradation occurred at both pH levels. Most of the protein degradation occurred in rhFGF21-(VPGXG) stored in citrate buffer at pH 6.0. 40 In contrast, rhFGF21-(VPGXG) stored in citrate buffer solutions at pH 5.0 and 5.5 40 The protein showed relatively little degradation. HPLC results show that the control, rhFGF21-(VPGXG) without protectant stored at -80°C, 40Using aqueous solutions of proteins, rhFGF21-(VPGXG) stored in citrate buffer solutions at pH 5.0 and pH 5.5 40 Only the protein maintained relatively good integrity (Figure 14). The results showed that the citrate buffer solution at pH 5.5 was superior to the citrate buffer solution at pH 5.0 in the rhFGF21-(VPGXG) protein synthesis. 40 The protection against proteins was slightly better.

[0160] 2. rhFGF21-(VPGXG) 40 Determining Protective Agents for Proteins The following protectant solutions were prepared in distilled water: sucrose solution (25 mg / mL, 50 mg / mL, 100 mg / mL), trehalose solution (25 mg / mL, 50 mg / mL, 100 mg / mL), glycine solution (50 mmol / L, 100 mmol / L, 150 mmol / L), arginine solution (50 mmol / L, 100 mmol / L, 150 mmol / L), histidine solution (25 mmol / L, 50 mmol / L, 100 mmol / L), mannitol solution (2.5%, 5%, 7.5%), sorbitol solution (5%, 10%, 15%), NaCl solution (50 mg / mL, 100 mg / mL, 150 mg / mL), and dextran solution (2%, 3%, 5%). The exchange method for each solution of protectant was as described in Section 1 of this Example, and the protein at the corresponding UV absorption peak was collected using a collector, i.e., rhFGF21-(VPGXG) exchanged into multiple protectants. 40 Protein was obtained. rhFGF21-(VPGXG) exchanged with the protective agent in each group 40 The protein was stored in a 25°C incubator protected from light and sampled at regular intervals for 15% SDS-PAGE electrophoresis and HPLC assay (PBS buffer, 1 mL / min, chromatography column: Agilent AdvanceBio SEC 300 Å, 2.7 μm, 7.8 × 300 mm, Cat. No.: PL1180-5301). The protein was stored at -80°C. 40 An aqueous solution of the protein was used as a control.

[0161] As shown by the results of 15% SDS-PAGE protein electrophoresis, the proteins stored in the solutions of other protectants were essentially completely degraded after 25 days of storage at 25°C. On the other hand, the proteins stored in the arginine solution of rhFGF21-(VPGXG) were completely degraded. 40 The protein showed relatively little degradation and still had good integrity after being stored at 25°C for 25 days. It can be seen that the arginine aqueous solution had a very good protective effect on the stability of the fusion protein, and the differences in the protective effects of the arginine aqueous solutions at three different concentrations were not significant. For better comparison, the fusion protein stored in the arginine aqueous solutions at the above three concentrations was analyzed by HPLC (PBS buffer, 1 mL / min, chromatography column: Agilent AdvanceBio SEC 300Å, 2.7 μm, 7.8 × 300 mm, Cat. No.: PL1180-5301). The HPLC results showed that the arginine aqueous solution had a significant effect on the stability of rhFGF21-VPGXG at 25°C. 40 It was found that arginine played an important role in protecting protein stability, and that there was no significant difference in the protective effect of the three concentrations of arginine aqueous solutions (Figure 15).

[0162] 3. rhFGF21-(VPGXG) 40 Determining a storage solution for proteins Arginine (50 mmol / L) + citrate buffer solutions of pH 5.5 and pH 5.0 were prepared by dissolving arginine, citric acid, and sodium citrate in water. 40 The exchange method for the protein storage solution was as described in Section 1 of this Example, and a collector was used to collect the protein at the corresponding UV absorption peak, i.e., rhFGF21-(VPGXG) being exchanged into the storage solution. 40 Protein was obtained. rhFGF21-(VPGXG) was exchanged into protein stock solution. 40Each group of proteins was divided into two aliquots, one of which was stored in an incubator at 25°C and the other in a display cabinet at 4°C. Samples were taken at regular intervals for 15% SDS-PAGE electrophoresis and HPLC assay (PBS buffer, 1 mL / min, chromatography column: Agilent AdvanceBio SEC 300 Å, 2.7 μm, 7.8 × 300 mm, Cat. No.: PL1180-5301), and an aqueous solution of the protein stored at -80°C was used as a control.

[0163] The results showed that mixing arginine with citrate buffer solution for protein storage improved the protein storage effect. When stored in citrate buffer solution alone, protein degradation occurred after 14 days at 25°C. When stored in arginine alone, degradation occurred after 25 days at 25°C. However, in the case of arginine + citrate buffer solution, protein degradation only occurred after 35 days of storage at 25°C in arginine (50 mmol / L) + citrate buffer solution at pH 5.0, while no degradation was observed at this time in arginine (50 mmol / L) + citrate buffer solution at pH 5.5. To more accurately determine the protective effect of arginine citrate buffer solution at pH 5.5 on proteins, proteins stored at -80°C were analyzed and compared by HPLC (PBS buffer, 1 mL / min, chromatography column: Agilent AdvanceBio SEC 300Å, 2.7 μm, 7.8 × 300 mm, Cat. No.: PL1180-5301). The results showed that both solutions were largely unchanged, indicating that storing proteins in arginine citrate buffer solution at pH 5.5 significantly improved protein stability and maintained the physical and chemical stability of the protein over 35 days of storage (Figure 16). After storing proteins in the two arginine citrate buffer solutions at 4°C for 60 days, SDS-PAGE electrophoresis showed no significant difference, and neither solution showed any degraded bands. However, after HPLC assay (PBS buffer, 1 mL / min, chromatography column: Agilent AdvanceBio SEC 300 Å, 2.7 μm, 7.8 × 300 mm, Cat. No.: PL1180-5301), it was found that only a small amount of degradation occurred in the protein stored in arginine + citrate buffer solution at pH 5.0 (i.e., degraded bands were not resolved by SDS-PAGE electrophoresis).On the other hand, the protein stored in the arginine + citrate buffer solution at pH 5.5 showed no degradation as detected by HPLC (PBS buffer, 1 mL / min, chromatography column: Agilent AdvanceBio SEC 300Å, 2.7 μm, 7.8 × 300 mm, Cat. No.: PL1180-5301) (Figure 17). It can be seen that the physical and chemical stability of the protein was maintained relatively well using the above two arginine + citrate buffer solutions after storage at 25°C for 35 days or at 4°C for 60 days. Relatively speaking, the physical and chemical stability of the protein was maintained better with the arginine + citrate buffer solution at pH 5.5.

[0164] 4. rhFGF21-(VPGXG) in Protein Stock Solution 40 Protein activity assay rhFGF21-(VPGXG) in the above storage solution after 35 days of storage at -80°C and 25°C 40 The protein was added to HepG2 cells, and the cells were incubated at 37°C, 0.5% CO2 for 24 hours, and then the residual glucose content in the medium (DMEM, Gibco, C11965500BT) was measured using a glucose assay kit (GOD-POD method).

[0165] The results showed that the protein stored at 25°C still had a good promoting effect on cellular glucose uptake, and the promoting effect showed a dose-dependent relationship. Compared with the corresponding doses (10 nmol, 100 nmol, 1000 nmol) of protein stored at -80°C, the promoting effects of all three doses (10 nmol, 100 nmol, 1000 nmol) of protein stored at 25°C were slightly reduced, but the differences between them were not significant. This indicates that the protein stored at 25°C in the above storage solution maintained good biological activity (Figure 18).

[0166] For purposes of illustration and disclosure, all patents, patent applications, and other publications are hereby expressly incorporated by reference. These publications are provided solely for their disclosure prior to the filing date of the present application. Any statement as to the date of these documents or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any reference herein to these publications, in any country, does not constitute an admission that the publications are part of the common knowledge in the art.

[0167] Those skilled in the art will understand that the scope of the present application is not limited to the various specific embodiments and examples described above, and that various modifications, substitutions or combinations can be made without departing from the spirit of the present application, all of which fall within the scope of protection of the present application.

Claims

1. 1. A recombinant human fibroblast growth factor 21 fusion protein comprising: the fusion protein has the amino acid sequence set forth in SEQ ID NO:2; or the portion of rhFGF21 in the fusion protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and the fusion protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 2; Recombinant human fibroblast growth factor 21 fusion protein.

2. A polynucleotide encoding the fusion protein of claim 1.

3. The portion of the polynucleotide encoding rhFGF21 in the fusion protein comprises the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence having at least 90% identity to SEQ ID NO:3; and / or the portion of the polynucleotide encoding the linker in the fusion protein comprises a nucleotide sequence set forth in SEQ ID NO: 4 and / or SEQ ID NO: 5, or a nucleotide sequence having at least 90% identity to SEQ ID NO: 4 and / or SEQ ID NO: 5; The polynucleotide of claim 2.

4. 4. The polynucleotide of claim 2 or 3, comprising the nucleotide sequence set forth in SEQ ID NO:6 or a nucleotide sequence having at least 90% identity to SEQ ID NO:

6.

5. A recombinant expression vector comprising the polynucleotide of any one of claims 2 to 4.

6. The recombinant expression vector of claim 5, wherein the recombinant expression vector is a prokaryotic recombinant expression vector, or the recombinant expression vector is a prokaryotic recombinant expression vector carrying a molecular chaperone.

7. The recombinant expression vector described in claim 6, wherein the molecular chaperone is a small ubiquitin-like modified protein.

8. A recombinant expression vector described in any one of claims 5 to 7, wherein the recombinant expression vector is a recombinant pSUMO vector or a recombinant PET-based vector.

9. A recombinant cell comprising a recombinant expression vector according to any one of claims 5 to 8 or having a polynucleotide according to any one of claims 2 to 4 integrated into its genome.

10. The recombinant cell of claim 9, wherein the host cell for constructing the recombinant cell is a prokaryotic host cell.

11. The recombinant cell of claim 10, wherein the prokaryotic host cell is selected from E. coli, Agrobacterium tumefaciens, Staphylococcus aureus, Staphylococcus albus, Lactobacillus acidophilus, Bacillus anthracis, Bacillus subtilis, or Bacillus thuringiensis.

12. A pharmaceutical composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable excipient.

13. 13. A kit comprising the fusion protein of claim 1 or the pharmaceutical composition of claim 12.

14. The kit further comprises a citrate buffer solution of pH 5.0 to 5.5, or the kit further comprises an aqueous solution of arginine; or the kit further comprises an arginine and citrate buffer solution at pH 5.0 to 5.5; or The kit further comprises an arginine aqueous solution and a citrate buffer solution of pH 5.0-5.5, and the fusion protein or pharmaceutical composition, the citrate buffer solution of pH 5.0-5.5, and the arginine aqueous solution are packaged together or separately.

14. The kit of claim 13.

15. The kit described in claim 14, further comprising an aqueous solution of arginine at 50 mmol / L to 150 mmol / L.

16. A composition comprising the fusion protein of claim 1 for use in the prevention or treatment of a disease associated with abnormal glucose metabolism.

17. The composition described in claim 16, wherein the disease associated with abnormal glucose metabolism is selected from diabetes and diabetes-related metabolic diseases.

18. The composition described in claim 17, wherein the diabetes-related metabolic disease is selected from diabetic nephropathy, dyslipidemia, obesity, cardiovascular disease, metabolic syndrome, lipid metabolism disorder, non-alcoholic fatty liver disease, or nervous system disease.

19. 12. A method for producing the fusion protein of claim 1, comprising culturing the recombinant cell of any one of claims 9 to 11, then inducing the recombinant cell to express an expression product, and purifying the expression product to obtain the fusion protein.

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