WO2021239046A1 - rhFGF21融合蛋白、编码其的多核苷酸、包含其的组合物及其用途 - Google Patents

rhFGF21融合蛋白、编码其的多核苷酸、包含其的组合物及其用途 Download PDF

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WO2021239046A1
WO2021239046A1 PCT/CN2021/096292 CN2021096292W WO2021239046A1 WO 2021239046 A1 WO2021239046 A1 WO 2021239046A1 CN 2021096292 W CN2021096292 W CN 2021096292W WO 2021239046 A1 WO2021239046 A1 WO 2021239046A1
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fusion protein
rhfgf21
vpgxg
seq
recombinant
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PCT/CN2021/096292
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English (en)
French (fr)
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肖伟
李德山
于丹
刘芝航
王振中
尹成凯
孙旭
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江苏康缘瑞翱生物医药科技有限公司
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Priority to KR1020227044626A priority Critical patent/KR20230017804A/ko
Priority to EP21814253.7A priority patent/EP4159758A4/en
Priority to US17/999,903 priority patent/US20240261371A1/en
Priority to JP2022573594A priority patent/JP2023527093A/ja
Publication of WO2021239046A1 publication Critical patent/WO2021239046A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This application belongs to the field of biomedicine. Specifically, it relates to a fusion protein comprising recombinant human fibroblast growth factor 21 protein (rhFGF21) and elastin-like protein (VPGXG) n fused together, a polynucleotide encoding the same, Recombinant expression vectors and recombinant cells, compositions containing them, kits and uses thereof, and methods for producing the fusion protein.
  • rhFGF21 human fibroblast growth factor 21 protein
  • VPGXG elastin-like protein
  • Fibroblast growth factor 21 is a metabolic regulatory factor mainly produced by the liver, and it plays an effective role in reducing blood sugar and lipids in animal models of obesity and type 2 diabetes.
  • the main sites of FGF21 metabolism are adipose tissue, liver and pancreas. Experimental studies have shown that after administration of FGF21 to diabetic mice and primates, diabetes compensation and dyslipidemia are improved.
  • FGF21 has a very short circulating half-life in the body, and thus cannot achieve the desired efficacy.
  • Modifying FGF21 with polyethylene glycol (PEG) can effectively improve its pharmacokinetics, improve drug distribution, and improve its efficacy.
  • PEG polyethylene glycol
  • the current PEGylated FGF21 has disadvantages such as low reaction yield, difficulty in controlling the binding site and coupling stoichiometry, which increases the cost in production. Therefore, it is particularly important to develop a site-specific modification method with mild reaction conditions, simple steps, rapid, and high efficiency to ensure the efficacy of FGF21.
  • the pentapeptide repeat unit is a polypeptide with elastic function and temperature sensitivity composed of five amino acids VPGXG.
  • X ie, Xaa
  • X can be any amino acid except proline. It has good biocompatibility, but for different proteins, different cycle numbers (ie, the number of repetitions of the VPGXG unit) will have different effects on expression, half-life and activity. There is no report about the suitable cycle number of elastin that can ensure the activity of FGF21 and prolong its half-life, and it needs to be explored experimentally.
  • hFGF21 human fibroblast growth factor 21
  • the inventors provided a new recombinant human fibroblast growth factor 21 fusion protein through research. Furthermore, the inventors have also developed a prokaryotic expression system suitable for the fusion protein, so that the soluble expression of rhFGF21 fusion protein can be achieved with the desired efficiency.
  • the present application relates to a recombinant human fibroblast growth factor 21 fusion protein, wherein the fusion protein comprises rhFGF21 and (VPGXG) n fused together, and the rhFGF21 comprises the rhFGF21 shown in SEQ ID NO:1
  • X is A and/or V;
  • n is an integer selected from 20-80.
  • this application relates to polynucleotides encoding the aforementioned fusion protein.
  • this application relates to a recombinant expression vector containing a polynucleotide encoding the above-mentioned fusion protein.
  • the present application relates to a recombinant cell containing the above-mentioned recombinant expression vector or the above-mentioned polynucleotide integrated in its genome.
  • this application relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned fusion protein.
  • this application relates to a kit, wherein the kit comprises the above-mentioned fusion protein or the above-mentioned pharmaceutical composition.
  • the present application relates to the use of the above-mentioned fusion protein in the preparation of drugs for preventing or treating diseases related to abnormal blood glucose metabolism.
  • the present application relates to a method for preventing or treating diseases related to abnormal blood glucose metabolism, including administering the above-mentioned fusion protein to a subject in need.
  • the present application relates to a fusion protein for preventing or treating diseases related to abnormal blood glucose metabolism.
  • the present application relates to a method for producing the above-mentioned fusion protein of the present application, wherein the method comprises: culturing the above-mentioned recombinant cell, and then inducing the above-mentioned recombinant cell to express and purifying the expression product to obtain the Fusion protein.
  • diabetes model mice are used to evaluate the diabetes effect of the fusion protein described in this application.
  • the results of animal experiments show that the fusion protein of this application can effectively reduce the blood glucose level of diabetic model animals, and compared with wild-type hFGF21, this
  • the applied fusion protein also has the advantages of long duration of drug effect (ie, longer half-life) in reducing blood glucose levels.
  • the fusion protein described in the present application exhibits a good ability to promote sugar absorption, and moreover, the fusion protein can achieve the desired expression efficiency.
  • Figure 1 is an electrophoresis diagram of the expression of SUMO-rhFGF21-(VPGXG) n fusion protein.
  • the reference signs respectively indicate:
  • Fig. 2 is a graph showing changes in sugar absorption of ⁇ -Klotho-3T3-L1 adipocytes stimulated by different test samples.
  • rhFGF21 the sugar absorption level of ⁇ -Klotho-3T3-L1 adipocytes was about 35%.
  • rhFGF21-(VPGXG) 20 the sugar absorption level of Klotho-3T3-L1 adipocytes increased to about 39%.
  • Figure 3 is an HPLC chart for analyzing the purity of rhFGF21-(VPGXG) 40 protein. After HPLC analysis, the peak of rhFGF21-(VPGXG) 40 protein began to appear when the retention time was about 6 min, and its purity could reach more than 95%.
  • Figure 4 is a graph showing the hypoglycemic results of STZ model mice treated with different test samples for 12 hours, in which, compared with the model group, the hypoglycemic effect of the rhFGF21 treatment group gradually weakened after 12 hours of administration; rhFGF21- (VPGXG) 20 treatment group, rhFGF21-(VPGXG) 40 treatment group, rhFGF21-(VPGXG) 60 treatment group and rhFGF21-(VPGXG) 80 treatment group still show certain therapeutic effects, and rhFGF21-(VPGXG) 40 treatment group
  • the blood sugar lowering effect is the best.
  • Fig. 5 is a graph showing the results of an oral glucose tolerance test (OGTT). Thirty minutes after oral glucose, the blood glucose level of the model group increased significantly, and the blood glucose level of the rhFGF21-(VPGXG) 40 treatment group was significantly lower than that of the model group. Among them, * p ⁇ 0.05, ** p ⁇ 0.01, compared with the model group; # p ⁇ 0.05, ## p ⁇ 0.01, compared with the normal group.
  • Fig. 6 is a graph showing changes in blood glucose of mice in each group during 58 days.
  • the blood glucose of the mice in the model group always maintained a high level.
  • the blood glucose of the rhFGF21-(VPGXG) 40 treatment group was significantly lower than that of the model group. After treatment, the blood glucose remained at a low level and was close to the blood glucose level of the normal group. And the efficacy is longer than rhFGF21.
  • Fig. 7 is a graph showing the serum insulin content of mice in each group.
  • the serum insulin content of mice in the model group decreased significantly, while the serum insulin content of the rhFGF21-(VPGXG) 40 treatment group was significantly increased compared to the model group.
  • Fig. 8 is a graph showing the levels of glycosylated hemoglobin of mice in each group. After giving the corresponding test samples for 30 days, the glycosylated hemoglobin content of the model group mice was maintained at a higher level, while the glycosylated hemoglobin level of the rhFGF21-(VPGXG) 40 treatment group mice decreased significantly. Among them, * p ⁇ 0.05, ** p ⁇ 0.01, compared with the model group; # p ⁇ 0.05, ## p ⁇ 0.01, compared with the normal group.
  • Fig. 9 is a graph showing the relative expression level of G6Pase gene in the liver of each group of mice.
  • the expression of G6Pase in the liver of mice in the model group was significantly increased, while the expression of G6Pase in the liver of mice in the rhFGF21-(VPGXG) 40 treatment group was significantly decreased.
  • Fig. 10 is a graph showing the relative expression level of the PCK gene in the liver of each group of mice.
  • the PCK gene expression in the liver of mice in the model group was significantly increased, while the PCK gene expression in the liver of the rhFGF21-(VPGXG) 40 treatment group mice was significantly decreased.
  • FIG. 11 and 12 are graphs showing the expression levels of G6Pase and PCK protein in the liver of each group of mice.
  • the expression of G6Pase and PCK protein in the liver of mice in the model group increased significantly, while the expression of G6Pase and PCK protein in the liver of the rhFGF21-(VPGXG) 40 treatment group decreased significantly.
  • Figure 13 is a picture showing the results of HE staining and immunohistochemistry of the pancreas.
  • the pancreatic islets of the model group are more severely damaged, and the amount of insulin secreted is significantly reduced, while the pancreatic islets of the rhFGF21-(VPGXG) 40 treatment group are significantly improved compared with the model group.
  • Figure 14 shows the results of HPLC analysis of rhFGF21-(VPGXG) 40 protein stored in citrate buffer solutions of different pH values (pH 5.0 and 5.5) at 25°C for 14 days and the same protein stored at -80°C Spectrogram.
  • Figure 15 shows the HPLC of rhFGF21-(VPGXG) 40 protein stored at 25°C for 25 days in different concentrations of arginine aqueous solution (containing 50mM, 100mM and 150mM arginine) and the same protein stored at -80°C Spectra of the results of the analysis.
  • Figure 16 is a spectrum showing the results of HPLC analysis of rhFGF21-(VPGXG) 40 protein stored in arginine + citrate buffer solution (pH 5.5) at 25°C for 35 days and the same protein stored at -80°C picture.
  • Figure 17 shows the rhFGF21-(VPGXG) 40 protein stored in arginine + citrate buffer solutions of different pH values (pH 5.0 and 5.5) at 4°C for 60 days and the same protein stored at -80°C The spectrum of the result of HPLC analysis.
  • FIG. 18 is a graph showing the results of different doses (10 nmol, 100 nmol, and 1000 nmol) of rhFGF21-(VPGXG) 40 protein in promoting glucose uptake by HepG2 cells after storage at 25°C and -80°C for 35 days.
  • X is A and V
  • the ratio of A to V is 2:3.
  • model group and “model control group” can be used interchangeably, and the terms “normal group”, “blank group” and “normal control group” can be used interchangeably.
  • linker may be an amino acid sequence used to connect rhFGF21 and each repeating unit in (VPGXG) n and (VPGXG) n together (the length may be, for example, about 1 to About 5 amino acids), and can be used for identification.
  • it can include linker peptide sequences, restriction sites, etc., which are common in the art, but are not limited thereto.
  • treatment includes reversing, alleviating, alleviating, ameliorating, inhibiting, slowing or stopping the progress or severity of the related disorder.
  • the term “subject” can be used interchangeably with “individual” and “patient”, and includes vertebrates, such as birds, fish, and mammals, such as but not limited to mice, and large animals. Rats, guinea pigs, dogs, pigs, chickens, rabbits, monkeys (such as rhesus monkeys), humans, etc.
  • percent identity between sequences can be compared between two sequences by, for example, using freely available computer programs commonly used for this purpose on the World Wide Web (e.g. BLASTp or BLASTn with default settings) to make sure.
  • freely available computer programs commonly used for this purpose on the World Wide Web (e.g. BLASTp or BLASTn with default settings) to make sure.
  • the present application provides a recombinant human fibroblast growth factor 21 fusion protein, wherein the fusion protein includes rhFGF21 and (VPGXG) n fused together, and the rhFGF21 contains the following SEQ ID NO: The amino acid sequence shown in 1 or the amino acid sequence having at least 80% identity with SEQ ID NO:1; X is A and/or V; n is an integer selected from 20-80.
  • the rhFGF21 contains at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, or at least 88% of SEQ ID NO:1. , 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% identical amino acid sequence.
  • the rhFGF21 has an amino acid sequence shown in SEQ ID NO:1 (SEQ ID NO:1:
  • 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).
  • n is an integer selected from 30-80, for example, n is selected from 30-70, 30-60, 30-50, 40-80, 40-70, 40-60, 40- An integer in 50.
  • n is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75.
  • 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.
  • n is 40-60, such as 40.
  • the fusion protein consists of rhFGF21 and (VPGXG) n fused together and an optional linker.
  • the amino acid sequence of the linker is RS and/or GS.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO: 2 or has at least 80%, such as at least 81%, at least 82%, at least 83%, at least with SEQ ID NO: 2. 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% , Amino acid sequences with at least 97%, 98% or 99% identity.
  • the fusion protein has an amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO: 2:
  • a fusion protein comprising rhFGF21 having an amino acid sequence having at least 80% identity with SEQ ID NO: 1 or a fusion protein comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 2 has hypoglycemic activity.
  • the present application provides polynucleotides encoding the aforementioned fusion protein.
  • polynucleotide encoding the aforementioned fusion protein.
  • nucleic acid encoding the aforementioned fusion protein.
  • nucleic acid sequence encoding the aforementioned fusion protein.
  • gene encoding the aforementioned fusion protein.
  • the polynucleotide portion encoding rhFGF21 in the aforementioned fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 3 or has at least 80% of SEQ ID NO: 3, 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%, A nucleotide sequence that is at least 94%, at least 95%, at least 96%, at least 97%, 98%, or 99% identical.
  • polynucleotide portion encoding rhFGF21 in the aforementioned fusion protein has the nucleotide sequence shown in SEQ ID NO: 3 (SEQ ID NO: 3:
  • the polynucleotide portion encoding the linker in the above-mentioned fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 4 and/or SEQ ID NO: 5, or is combined with SEQ ID NO: 4 and/or SEQ ID NO: 5 has 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% identical nucleotide sequence.
  • the polynucleotide portion encoding the linker in the aforementioned fusion protein has the nucleotide sequence shown in SEQ ID NO: 4 and/or SEQ ID NO: 5 (SEQ ID NO: 4: AGATCT; SEQ ID NO: 5: GGATCC).
  • the polynucleotide encoding the above-mentioned fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 6 or has at least 80%, such as at least 81%, at least 82% of SEQ ID NO: 6. %, 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%, A nucleotide sequence that is at least 95%, at least 96%, at least 97%, 98%, or 99% identical.
  • polynucleotide encoding the aforementioned fusion protein has the nucleotide sequence shown in SEQ ID NO: 6 (SEQ ID NO: 6:
  • the present application provides a recombinant expression vector containing a polynucleotide encoding the aforementioned fusion protein.
  • the prokaryotic expression vector suitable for the prokaryotic expression system is considered in this application.
  • a prokaryotic expression vector carrying the molecular chaperone can be selected.
  • the molecular chaperone is preferably a small molecule ubiquitin-like modified protein (SUMO).
  • SUMO is a small molecule ubiquitin-like modified protein, which can promote the correct folding of the target protein and increase the expression and solubility of the target protein.
  • suitable expression vectors suitable for this application include, but are not limited to: pSUMO vectors, PET series vectors (such as PET30a vectors).
  • the present application provides a recombinant cell containing the aforementioned recombinant expression vector or the aforementioned polynucleotide integrated in its genome.
  • the host cell used to construct the recombinant cell is preferably a prokaryotic host cell.
  • the prokaryotic host cell include, but are not limited to: Escherichia coli (E. coli) (e.g. Escherichia coli Rossetta (DE3), Agrobacterium tumefaciens, Staphylococcus aureus, Staphylococcus albus, Lactobacillus acidophilus, Bacillus anthracis, Subtilis Bacillus (Bacillus subtilis) or Bacillus thuringiensis (Bacillus thuringiensis), etc.
  • Escherichia coli Escherichia coli
  • DE3 Escherichia coli Rossetta
  • Agrobacterium tumefaciens Staphylococcus aureus
  • Staphylococcus albus Lactobacillus acidophilus
  • Bacillus anthracis Subtilis Bacillus (Bacillus subtilis) or
  • the present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned fusion protein.
  • the pharmaceutical composition further comprises pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable pharmaceutical excipients can be selected from, for example, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regulators, stabilizers, glidants, flavors, preservatives, suspending agents, antioxidants, penetration enhancers, pH regulators, surfactants, diluents, etc.
  • solvents for example, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regulators, stabilizers, glidants, flavors, preservatives, suspending agents, antioxidants, penetration enhancers, pH regulators, surfactants, diluents, etc.
  • this application relates to a kit, wherein the kit comprises the above-mentioned fusion protein or the above-mentioned pharmaceutical composition.
  • the kit further comprises a citrate buffer solution with a pH of 5.0-5.5.
  • the kit further comprises an aqueous arginine solution, preferably 50 mmol/L-150 mmol/L arginine aqueous solution.
  • the kit further comprises an arginine + citrate buffer solution with a pH of 5.0-5.5.
  • the kit further comprises a citrate buffer solution of pH 5.0-5.5 and an aqueous arginine solution, and the fusion protein or the pharmaceutical composition, the pH 5.0-5.5
  • the citrate buffer solution and the arginine aqueous solution are packaged together or separately packaged separately.
  • the application relates to the use of the above-mentioned fusion protein in the preparation of a medicine for preventing or treating diseases related to abnormal blood glucose metabolism.
  • the present application relates to a method for preventing or treating diseases related to abnormal blood glucose metabolism, including administering the above-mentioned fusion protein to a subject in need.
  • the present application relates to a fusion protein for preventing or treating diseases related to abnormal blood glucose metabolism.
  • the disease related to abnormal blood glucose metabolism is selected from but not limited to: diabetes and diabetes-related metabolic diseases.
  • the diabetes-related metabolic disease is selected from diabetic nephropathy, dyslipidemia, obesity, cardiovascular disease, metabolic syndrome, lipid metabolism disorder, non-alcoholic fatty liver disease (NAFLD) or neurological disease (such as epilepsy, depression) Etc.), but not limited to this.
  • the present application provides a method for producing the aforementioned fusion protein of the present application, wherein the method comprises: culturing the aforementioned recombinant cell, and then inducing the aforementioned recombinant cell to express and purifying the expression product to obtain The fusion protein.
  • the recombinant cell may be a recombinant prokaryotic cell.
  • examples of the recombinant prokaryotic cell include but are not limited to: recombinant Escherichia coli (such as recombinant Escherichia coli Rosetta (DE3)), recombinant Agrobacterium , Recombinant Staphylococcus aureus, Recombinant Staphylococcus white, Recombinant Lactobacillus acidophilus, Recombinant Bacillus anthracis, Recombinant Bacillus subtilis or Recombinant Bacillus thuringiensis, etc.
  • the cultivation of recombinant cells can be carried out by those skilled in the art according to the types of recombinant cells to select conventional medium and culture conditions (for example, see the following record: "Manufacturing and Application of Microbial Culture Medium”, edited by Tianshou Chen, China Agriculture Press, 1995; “Practical Handbook of Microbial Culture Medium”, edited by Ma Lehao, etc., 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.).
  • inducers known to those skilled in the art can be used to induce expression of the aforementioned recombinant cells.
  • an inducer selected from the following is used to induce the expression of the above-mentioned recombinant cell: IPTG and/or lactose.
  • the expression product can be purified by conventional purification means known to those skilled in the art to obtain the fusion protein, such as ion exchange chromatography, adsorption chromatography, affinity chromatography, molecular sieve chromatography, hydrophobic chromatography or Any combination of them, etc., but not limited to this.
  • the purification of the expression product includes the following steps: (i) After the expression is induced, the recombinant cells are centrifuged to collect the bacterial cells, the bacterial cells are broken and the supernatant is collected by centrifugation (Ii) subjecting the supernatant to ion exchange chromatography to collect the eluent; (iii) subjecting the eluent to affinity chromatography to obtain the fusion protein.
  • the recombinant cells are centrifuged at 10000-12000 rpm/min to collect bacterial cells.
  • the bacteria are broken by adding lysozyme to the bacteria and performing ultrasound.
  • centrifugation is performed at 10000-12000 rpm/min to collect the supernatant.
  • an agarose gel such as DEAE agarose gel, is used for the ion exchange chromatography.
  • the supernatant is eluted with a buffer comprising the following composition: 25mmol/L Tris-HCl, 0.25mol/L NaCl, pH 8.0-8.2.
  • Ni ⁇ NTA resin is used for the affinity chromatography.
  • the affinity chromatography includes the following process: after equilibrating the affinity chromatography column with an equilibration buffer, loading the affinity chromatography column with the eluent and Wash with a washing buffer, and then use an elution buffer to elute the target protein.
  • the equilibration buffer includes the following composition: 50mmol/L Tris-HCl, 0.5mol/L NaCl, 10mmol/L imidazole, pH 8.0-8.2.
  • the washing buffer includes the following composition: 50mmol/L Tris-HCl, 500mmol/L NaCl, 50mmol/L imidazole, pH 8.0-8.2.
  • the elution buffer includes the following composition: 50mmol/L Tris-HCl, 500mmol/L NaCl, 500mmol/L imidazole, pH 8.0-8.2.
  • the above-mentioned method for producing a fusion protein further includes the following steps:
  • the polynucleotide encoding the fusion protein and the expression vector are subjected to restriction digestion and ligation to obtain a recombinant expression vector, the recombinant expression vector is introduced into a host cell, and the recombinant cell is obtained by screening.
  • rhFGF21 can be based on the protein sequence of rhFGF21 or its DNA or RNA sequence known in the art (for example, see 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), through codon optimization, using conventional gene synthesis methods to synthesize rhFGF21 Polynucleotide sequence.
  • the polynucleotide sequence encoding rhFGF21 includes the nucleotide sequence shown in SEQ ID NO: 3 or has at least 80% of SEQ ID NO: 3, 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% identical nucleotide sequence.
  • the polynucleotide encoding rhFGF21 has a nucleotide sequence shown in SEQ ID NO: 3.
  • the polynucleotide encoding (VPGXG) 20 comprises the nucleotide sequence shown in SEQ ID NO: 7 or has at least 80% of the nucleotide sequence shown in SEQ ID NO: 7 , 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%, A nucleotide sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, 98%, or 99% identical.
  • the polynucleotide encoding (VPGXG) 20 has a nucleotide sequence shown in SEQ ID NO: 7 (SEQ ID NO: 7:
  • a polynucleotide encoding a linker selected from the following is optionally attached to the 5'end and 3'end of the polynucleotide encoding (VPGXG) 20, respectively: RS and/or GS.
  • a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4 and SEQ ID NO: 5 is connected to the 5'end and 3'end of the polynucleotide encoding (VPGXG) 20, respectively .
  • step (2) BglII restriction endonuclease and BamHI restriction endonuclease are used to perform the restriction endonuclease.
  • Suitable routines can be selected by those skilled in the art based on general knowledge in the field (for example, see “Molecular Cloning Experiment Guide” (4th Edition), edited by J. Sambrook et al., translated by He Fuchu, Science Press, 2017) PCR experiment conditions and steps, or a commercially available PCR kit according to the instructions in the PCR reaction. Without limitation, other primer pairs and other gene synthesis methods known in the art can also be used to obtain the polynucleotide encoding the aforementioned fusion protein.
  • restriction digestion and ligation have the well-known meanings in the art.
  • Restriction restriction endonuclease is used to specifically cut two target DNA fragments or one target DNA fragment and the carrier DNA molecule. Then, the two target DNA fragments after digestion or the target DNA fragment after digestion are ligated with the ends of the vector DNA molecule by using a ligase to obtain a recombinant molecule or a recombinant vector.
  • step (3) BsaI restriction endonuclease and BamHI restriction endonuclease are used to perform the restriction endonuclease.
  • the recombinant expression vector is introduced into the host cell by transformation. Transformation refers to the use of some known methods in molecular biology and genetic engineering to treat host cells so that the treated host cells are in a competent state, and thereby contact with foreign DNA, so that the foreign DNA enters into a competent state In the host cell.
  • the host cell is a prokaryotic host cell.
  • the prokaryotic host cell include but are not limited to: Escherichia coli (such as Escherichia coli Rossetta (DE3)), Agrobacterium, Staphylococcus aureus, Staphylococcus albicans, Lactobacillus acidophilus, Bacillus anthracis, Bacillus subtilis or Bacillus thuringiensis, etc.
  • the positive recombinant cells can be screened by conventional methods such as resistance screening.
  • the cultivation of the recombinant cells can be carried out by those skilled in the art according to the types of recombinant cells to select a conventional medium and culture conditions (for example, see the following record: "Manufacturing and Application of Microbial Culture Medium”, edited by Chen Tianshou, China Agriculture Publishing House, 1995; "Practical Handbook of Microbial Culture Medium”, edited by Ma Lehao, etc., 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.).
  • the expression vector is selected from a pSUMO vector and a PET series vector (such as a PET30a vector), but it is not limited thereto.
  • a pSUMO vector carrying a small molecule ubiquitin-like modified protein (SUMO) as a molecular chaperone is used as the expression vector, and Escherichia coli Rosetta (DE3) is used as a host cell.
  • SUMO small molecule ubiquitin-like modified protein
  • DE3 Escherichia coli Rosetta
  • a recombinant pSUMO vector containing a polynucleotide encoding the aforementioned fusion protein is transformed into the host cell; the recombinant cells obtained by screening are cultured, and then the aforementioned recombinant cells are induced to express SUMO-fusion Protein, the expressed product is purified by ion exchange chromatography and affinity chromatography to obtain a preliminary purified SUMO-fusion protein.
  • the SUMO protease is used to excise the molecular chaperone SUMO connected to the fusion protein, and the fusion protein after SUMO excision is performed Purified by affinity chromatography to obtain the purified fusion protein.
  • a recombinant human fibroblast growth factor 21 fusion protein wherein the fusion protein comprises rhFGF21 and (VPGXG) n fused together, and the rhFGF21 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO: 1 has an amino acid sequence with at least 80% identity; X is A and/or V; n is an integer selected from 20-80.
  • n is an integer in the range of 40-60, preferably 40.
  • fusion protein according to any one of paragraphs 1-3, wherein the fusion protein consists of rhFGF21 and (VPGXG) n fused together and an optional linker.
  • fusion protein according to any one of paragraphs 1-5, wherein the fusion protein has an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with SEQ ID NO: 2 .
  • polynucleotide according to paragraph 7 or 8 wherein the polynucleotide portion encoding the linker in the fusion protein comprises the nucleotides shown in SEQ ID NO: 4 and/or SEQ ID NO: 5 Sequence, or a nucleotide sequence that has at least 80% identity with SEQ ID NO: 4 and/or SEQ ID NO: 5.
  • polynucleotide according to any of paragraphs 7-9, wherein the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 6 or has at least 80% identity with SEQ ID NO: 6 sexual nucleotide sequence.
  • prokaryotic host cell is selected from Escherichia coli, Agrobacterium, Staphylococcus aureus, Staphylococcus albicans, Lactobacillus acidophilus, Bacillus anthracis, Bacillus subtilis, or Bacillus thuringiensis Bacillus.
  • composition wherein the pharmaceutical composition comprises the fusion protein described in any of paragraphs 1-6.
  • kit wherein the kit comprises the fusion protein described in any of paragraphs 1-6 or the pharmaceutical composition described in paragraph 19 or 20.
  • kit according to paragraph 21 or 22, wherein the kit further comprises an aqueous arginine solution, preferably 50 mmol/L-150 mmol/L arginine aqueous solution.
  • kit according to any of paragraphs 21-23, wherein the kit further comprises an arginine+citrate buffer solution with a pH of 5.0-5.5.
  • kit further comprises an aqueous arginine solution and a citrate buffer solution of pH 5.0-5.5, and the fusion protein or the pharmaceutical composition, the The citrate buffer solution with pH 5.0-5.5 and the arginine aqueous solution are packaged together or separately packaged.
  • the diseases related to abnormal blood glucose metabolism are 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 disorder, non-alcoholic fatty liver disease or nervous system disease.
  • a method for producing the fusion protein of any of paragraphs 1-6 comprising: culturing the recombinant cell of any of paragraphs 16-18, and then inducing the recombinant cell to perform Express and purify the expression product to obtain the fusion protein.
  • the purification of the expression product comprises the following steps: (i) after inducing expression, centrifuging the recombinant cell to collect the bacterial cells, and subjecting the bacterial cells to After crushing, the supernatant is collected by centrifugation; (ii) the supernatant is subjected to ion exchange chromatography to collect the eluate; (iii) the eluate is subjected to affinity chromatography to obtain the fusion protein.
  • the polynucleotide encoding the fusion protein and the expression vector are subjected to restriction digestion and ligation to obtain a recombinant expression vector, the recombinant expression vector is introduced into a host cell, and the recombinant cell is obtained by screening.
  • the polynucleotide sequence encoding rhFGF21 comprises the nucleotide sequence shown in SEQ ID NO: 3 or has at least 80 percent with SEQ ID NO: 3. % Identical nucleotide sequence.
  • the polynucleotide encoding (VPGXG) 20 comprises the nucleotide sequence shown in SEQ ID NO: 7 or is the same as SEQ ID NO: 7 has a nucleotide sequence with at least 80% identity.
  • step (2) the 5'end and 3'end of the polynucleotide encoding (VPGXG) 20 are optionally linked with a code selected from The following linker polynucleotides: RS and/or GS.
  • step (2) the restriction endonuclease BglII and the restriction endonuclease BamHI are used to perform the digestion.
  • step (3) the restriction enzymes BsaI and BamHI are used to perform the restriction enzyme digestion.
  • step (3) the expression vector is selected from the group consisting of pSUMO vector and PET series vector.
  • step (3) the expression vector is a pSUMO vector, and the host cell is Escherichia coli Rosetta (DE3).
  • the gene synthesis company was commissioned to synthesize the linker-(VPGXG) 20 gene with the nucleotide sequence shown in SEQ ID NO: 9, which was encoded by the 5'end having the nucleotide sequence shown in SEQ ID NO: 4
  • the polynucleotide of the linker RS ie, the BglII restriction site
  • the polynucleotide encoding (VPGXG) 20 having the nucleotide sequence shown in SEQ ID NO: 7
  • the polynucleotide at the 3′ end having the sequence shown in SEQ ID NO
  • the composition of the polynucleotide encoding the linker GS (ie, the BamHI restriction site) of the nucleotide sequence shown in 5 (SEQ ID NO: 9:
  • primers P1 (SEQ ID NO: 11) and P2 (SEQ ID NO: 12) and rTaq enzyme were used to carry out PCR according to the 50 ⁇ L system described below, and to the obtained rhFGF21- (VPGXG)
  • the 5'end and 3'end of the nucleotide sequence of the 20 polypeptide introduce BsaI and BamHI restriction sites.
  • the nucleotide sequence introduced into the restriction site was ligated to the T vector by solution I ligase, and then digested with BamHI restriction endonuclease; at the same time, the linker-(VPGXG) 20 gene was passed through BglII Digest with BamHI restriction endonuclease, perform digestion according to the manufacturer's instructions, and use the DNA fragment gel recovery kit (purchased from Omega, catalog number: D6492) for the gel recovery of the above two digestion products, and use T4 DNA Ligase performs the ligation.
  • the linker-(VPGXG) 20 gene was passed through BglII Digest with BamHI restriction endonuclease, perform digestion according to the manufacturer's instructions, and use the DNA fragment gel recovery kit (purchased from Omega, catalog number: D6492) for the gel recovery of the above two digestion products, and use T4 DNA Ligase performs the ligation.
  • the rTaq enzyme was used to perform a PCR reaction (50 ⁇ L system), and a stop codon (TCA) was introduced:
  • primer P1 and primer P3 are as follows:
  • the obtained PCR product was recovered by using a DNA fragment gel recovery kit (purchased from Omega, article number: D6492) to obtain the rhFGF21-(VPGXG) 40 fusion protein gene.
  • the genes of fusion protein rhFGF21-(VPGXG) 20 , rhFGF21-(VPGXG) 60 and rhFGF21-(VPGXG) 80 were obtained.
  • the pSUMO and Escherichia coli Rosetta (DE3) involved in this example are commercially available.
  • the rhFGF21-(VPGXG) 40 target gene fragment obtained in Example 1 and the prokaryotic expression vector pSUMO were double digested with BsaI and BamHI enzymes according to the manufacturer’s instructions, and a DNA fragment gel recovery kit (purchased from Omega, catalog number: D6492) Purify the digested product, and then use T4 DNA ligase to ligate with a ligation reaction system of 10 ⁇ L according to the manufacturer's instructions, and ligate overnight at 16°C.
  • the recombinant plasmid pSUMO-rhFGF21-(VPGXG) 40 containing the correct sequence was transformed into competent cells of the expression strain Escherichia coli Rossetta (DE3). Spread the transformed competent cells on an LB plate containing 100 ⁇ g/mL ampicillin.
  • the soluble expressed target protein can account for more than 70% of the total target protein of the bacteria.
  • the supernatant was purified by DEAE Sepharose FF chromatography column, where it was eluted with buffer 1 (25mmol/L Tris-HCl, 0.25mol/L NaCl, pH 8.0), and collected to obtain ion exchange chromatography elution liquid.
  • the ion exchange chromatography eluate was subjected to Ni ⁇ NTA resin column affinity chromatography, in which equilibration buffer (50mmol/L Tris-HCl, pH 8.0, 0.5mol/L NaCl, 10mmol/L imidazole) was used first Equilibrate the Ni ⁇ NTA resin column, and then load the above eluate to the balanced resin column.
  • washing buffer 50mmol/L Tris-HCl, pH 8.0, 0.5mol/L NaCl, 50mmol
  • elution buffer 50mmol/L Tris-HCl, pH 8.0, 0.5mol/L NaCl, 500mmol/L imidazole. Collect each elution peak to obtain affinity chromatography eluate, and perform SDS-PAGE detection.
  • the affinity chromatography eluate was dialyzed overnight with PBS buffer (pH 7.4) at 4°C to obtain the imidazole-free fusion protein SUMO-rhFGF21-(VPGXG) 40 , and DTT was added to it to a final concentration of 2mmol/L and SUMO Protease-I (with His Tag) (cutting ratio is 1:50, that is, 1mg SUMO Protease-I: 50mg SUMO-rhFGF21-(VPGXG) 40 ), cut overnight at 4°C.
  • the resulting cleavage product was thoroughly mixed with Ni-NTA Agarose to collect the flow-through fluid, in which the non-specifically adsorbed rhFGF21-(VPGXG) 40 protein was eluted with PBS buffer (pH 7.4).
  • HPLC analysis mobile phase is PBS buffer, flow rate is 1mL/min, column: Agilent AdvanceBio SEC 300 2.7 ⁇ m, 7.8 ⁇ 300mm, article number: PL1180-5301) showed that the purity of the purified rhFGF21-(VPGXG) 40 protein (having the amino acid sequence shown in SEQ ID NO: 2) reached more than 95% (Figure 3) .
  • rhFGF21-(VPGXG) 20 The expression and purification of rhFGF21-(VPGXG) 20 , rhFGF21-(VPGXG) 60 and rhFGF21-(VPGXG) 80 protein were performed under the same expression and purification operations and conditions as the above-mentioned rhFGF21-(VPGXG) 40 protein.
  • the ⁇ -Klotho was cloned into the retroviral eukaryotic expression vector pBMN- ⁇ Klotho-IRES-EGFP, green fluorescent protein was used as the reporter gene, and the recombinant retroviral vector was transferred into 293 packaging cells by liposome transfection method
  • the virus supernatant was collected by centrifugation, and 3T3-L1 precursor cells (purchased from ATCC, catalog number: CL-173) were infected, and a stable ⁇ -Klotho-3T3-L1 cell line expressing ⁇ -Klotho was screened by flow cytometry. It is ⁇ -Klotho-3T3-L1 adipocyte.
  • Glucose concentration detection After the above-mentioned treatments, after incubating the cells for 24 hours, take 2 ⁇ L of the supernatant medium and put it into 200 ⁇ L of glucose detection solution to determine the glucose content. Repeat each concentration at least 3 times. After reacting at 37°C for 5-10 minutes, measure the OD value at a wavelength of 500nm.
  • the calculation method of the cell glucose consumption rate is as follows, and the experimental results are statistically analyzed.
  • Cellular glucose consumption rate (%) [(C blank glucose- C dosing glucose )/C blank glucose ] ⁇ 100%.
  • ⁇ -Klotho-3T3-L1 adipocytes were treated with rhFGF21 and the fusion proteins prepared above.
  • the results showed that the sugar absorption of untreated ⁇ -Klotho-3T3-L1 adipocytes was about 27%. It increased to about 35% after stimulation with 10nmol/L rhFGF21, increased to about 39% after stimulation with 10nmol/L rhFGF21-(VPGXG) 20 , and increased to about 46% after stimulation with 10nmol/L rhFGF21-(VPGXG) 40.
  • the established diabetes model mice were randomly divided into 6 groups with 8 mice in each group. At the same time, a normal mouse group was set as a blank control group, with 8 mice.
  • the model control group was injected with PBS buffer; the rhFGF21-(VPGXG) 40 treatment group was injected with the fusion protein at a dose of 1 mg/kg/d, rhFGF21-(VPGXG) 20 , rhFGF21-(VPGXG) 60 , rhFGF21-( The VPGXG) 80 treatment group was injected with the fusion protein at the same dose as the rhFGF21-(VPGXG) 40 treatment group.
  • Model control group Diabetic mice are injected subcutaneously with PBS buffer at around 8:00 am every day, with a single injection volume of 0.15 mL;
  • rhFGF21-(VPGXG) 40 treatment group diabetic rats were injected subcutaneously with rhFGF21-(VPGXG) 40 fusion protein (solvent is PBS buffer) at around 8:00 am every day, and the single injection dose was 1mg/kg/d rhFGF21-(VPGXG) 40 fusion protein (based on total protein), single injection volume is 0.15mL.
  • mice normal control group: healthy mice were injected subcutaneously with PBS buffer at around 8:00 am every day, with a single injection volume of 0.15 mL.
  • the blood glucose of each group of mice was tested every 2 days. The results showed that after treatment with rhFGF21-(VPGXG) 40 fusion protein for 3 days, the blood glucose level of this group of mice was significantly lower than that of the model group, and blood glucose remained at a lower level after 6 days of treatment And it was close to the blood glucose level of mice in the normal group. After 30 days of continuous administration, OGTT detected the glucose tolerance level of the mice in each group. The results showed that 30 minutes after oral glucose, the blood glucose of the model group mice increased significantly, and the decline after 30 minutes was relatively slow, the blood glucose of the rhFGF21-(VPGXG) 40 treatment group was significantly lower than that of the model group, and the blood glucose had dropped to close to that at 60 minutes.
  • mice in the normal group The results showed that after rhFGF21-(VPGXG) 40 fusion protein treatment, the glucose tolerance of mice was significantly improved.
  • the blood glucose of the model group mice was always maintained at a high level.
  • the blood glucose of the rhFGF21-(VPGXG) 40 treatment group was significantly lower than that of the model group.
  • the blood glucose remained at a low level and was close to normal.
  • the blood glucose level of mice in the group, and the drug effect was longer than rhFGF21 ( Figure 6).
  • the serum insulin level of mice in the model group was significantly lower than that of the normal group.
  • Example 4 Determination of stable storage conditions of human fibroblast growth factor 21 fusion protein
  • acetic acid buffer solution pH 4.0 pH 5.0 (prepared by dissolving acetic acid in water); citrate buffer solution pH 5.0, pH 5.5, pH 6.0 (prepared by dissolving citric acid and sodium citrate in water); Phosphate buffer solution has pH 6.5 and pH 7.0 (prepared by dissolving disodium hydrogen phosphate and sodium dihydrogen phosphate in water); Tris-HCl buffer solution has pH 7.5 and pH 8.0 (prepared by dissolving Tris-HCl in water).
  • the HiPrepTM 26/10 Desalting column Connect the HiPrepTM 26/10 Desalting column to the AKTA purifier100 system, first flush out the protective solution (20% ethanol) in the desalting column with 8-10 column volumes of distilled water, and then use 8-10 column volumes of the above-mentioned buffer
  • the solution equilibrates the desalting column, and uploads the sample (a solution of rhFGF21-(VPGXG) 40 protein at a concentration of 2 mg/mL in a phosphate buffer at pH 7.4).
  • the UV curve rises first, and when the UV curve drops to the baseline, the conductivity curve rises.
  • Use a collector to collect the protein at the corresponding UV absorption peak to obtain the rhFGF21-(VPGXG) 40 protein replaced by the buffer solution.
  • the 9 groups of rhFGF21-(VPGXG) 40 proteins after the replacement of the buffer solution were stored in a 25°C incubator and protected from light. The appearance of the protein was inspected at regular intervals and samples were taken for 15% SDS-PAGE electrophoresis and HPLC detection ( PBS buffer, 1mL/min, column: Agilent AdvanceBio SEC 300 , 2.7 ⁇ m, 7.8 ⁇ 300mm, article number: PL1180-5301); and the rhFGF21-(VPGXG) 40 protein aqueous solution stored at -80°C was used as a control.
  • sucrose solution 25mg/mL, 50mg/mL, 100mg/mL
  • trehalose solution 25mg/mL, 50mg/mL, 100mg/mL
  • glycine solution 50mmol/L, 100mmol) /L, 150mmol/L
  • arginine solution 50mmol/L, 100mmol/L, 150mmol/L
  • histidine solution 25mmol/L, 50mmol/L, 100mmol/L
  • mannitol solution (2.5% , 5%, 7.5%)
  • sorbitol solution 5%, 10%, 15%
  • NaCl solution 50mg/mL, 100mg/mL, 150mg/mL
  • dextran solution 2%, 3%, 5%
  • each protective agent solution is as described in the first part of this embodiment, using a collector to collect the protein at the corresponding ultraviolet absorption peak, that is, the rhFGF21-(VPGXG) 40 protein after the replacement of the protective agent solution is obtained.
  • the rhFGF21-(VPGXG) 40 protein after the replacement of the protective agent solution in each group was stored in a 25°C incubator and protected from light.
  • the fusion protein stored in the above three concentrations of arginine aqueous solution was analyzed by HPLC (PBS buffer, 1mL/min, column: Agilent AdvanceBio SEC 300 2.7 ⁇ m, 7.8 ⁇ 300mm, article number: PL1180-5301). It can be seen from the HPLC results that the arginine aqueous solution has a great protective effect on the stability of rhFGF21-VPGXG 40 protein at 25°C, and the protective effects of the three concentrations of arginine aqueous solutions are not significantly different (Figure 15 ).
  • Arginine, citric acid and sodium citrate were dissolved in water to prepare arginine (50mmol/L) + citrate buffer solutions at pH 5.5 and pH 5.0, respectively.
  • the storage solution replacement method of rhFGF21-(VPGXG) 40 protein is as described in the first part of this example.
  • the protein at the corresponding ultraviolet absorption peak is collected by a collector to obtain rhFGF21-(VPGXG) 40 protein after replacement of the storage solution.
  • the rhFGF21-VPGXG 40 protein after replacement of each histone storage solution was divided into 2 equal parts, one part was stored in a 25°C incubator and protected from light; the other part was stored in a 4°C display cabinet and protected from light.
  • rhFGF21-(VPGXG) 40 protein in the above-mentioned storage solution after storage at -80°C and 25°C for 35 days to HepG2 cells. After incubating at 37°C and 0.5% CO 2 for 24 hours, use GOD-POD glucose
  • the detection kit detects the remaining glucose content in the culture medium (DMEM, gibco, C11965500BT).

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Abstract

一种重组人成纤维细胞生长因子21(rhFGF21)融合蛋白、编码其的多核苷酸、重组表达载体、重组细胞、包含其的药物组合物和试剂盒、其用途及产生其的方法。通过基因工程方法高效可溶性地表达得到rhFGF21-(VPGXG) n融合蛋白，其中，通过rhFGF21与具有合适的循环数n的类弹性蛋白(VPGXG) n的融合，使得到的融合蛋白相较于野生型人成纤维细胞生长因子21具有更好的生物活性。而且，试验结果表明，所述rhFGF21-(VPGXG) n融合蛋白表达效率较高，并能有效地降低糖尿病动物体内的血糖水平。此外，相比于野生型hFGF21，所述融合蛋白在降低血糖水平方面还具有药效持续时间长的优点。

Description

rhFGF21融合蛋白、编码其的多核苷酸、包含其的组合物及其用途 技术领域
本申请属于生物医药领域，具体而言，涉及一种包含融合在一起的重组人成纤维细胞生长因子21蛋白(rhFGF21)与类弹性蛋白(VPGXG) n的融合蛋白、编码其的多核苷酸、重组表达载体和重组细胞、包含其的组合物、试剂盒及其用途和产生该融合蛋白的方法。
背景技术
成纤维细胞生长因子21(FGF21)是主要由肝脏产生的新陈代谢调控因子，其在肥胖症和2型糖尿病动物模型中发挥有效的降糖和降脂作用。FGF21代谢作用的主要位点是脂肪组织、肝脏和胰腺。实验研究已显示，在对糖尿病小鼠和灵长类施用FGF21后，糖尿病代偿和血脂异常有改善。
但是，FGF21在体内循环半衰期非常短，进而达不到理想的药效。用聚乙二醇(PEG)修饰FGF21能有效改善其药代动力学，改善药物分布，提高其疗效。然而，目前的PEG化FGF21存在如反应产率低、结合位点和偶联化学计量难以控制等弊端，在生产中增加了成本。因此，开发反应条件温和、步骤简单、快速、高效的位点特异性修饰方法对保证FGF21的药效尤为重要。
五肽重复序列单元是由五种氨基酸VPGXG组成的具有弹性功能和温度敏感性的多肽。X(即，Xaa)可以是除脯氨酸以外的任何氨基酸。它具有良好的生物相容性，但是针对不同蛋白，不同的循环数(即，VPGXG单元的重复次数)会对表达量、半衰期以及活性产生不同的影响。对于能保证FGF21活性并延长其半衰期的合适的弹性蛋白的循环数，目前尚未有报道，需要实验探究。
因此，需要开发出合适的融合蛋白以确保人成纤维细胞生长因子21(hFGF21)活性并延长其半衰期，同时，还需要进一步开发出适合该融合蛋白的体外表达体系，以高效得到具有活性的融合蛋白。
发明内容
本申请针对rhFGF21的半衰期短、活性无法保证等在药用过程中存在的问题，发明人通过研究而提供一种新的重组人成纤维细胞生长因子21融合蛋白。进一步地，发明人还开发了适合该融合蛋白的原核表达体系，从而能够以期望的效率实现rhFGF21融合蛋白的可溶性表达。
一个方面，本申请涉及一种重组人成纤维细胞生长因子21融合蛋白，其中，所述融合蛋白包括融合在一起的rhFGF21和(VPGXG) n，所述rhFGF21包含以SEQ ID NO:1示出的氨基酸序列或与SEQ ID NO:1具有至少80％一致性的氨基酸序列；X为A和/或V；n为选自20-80中的整数。
另一方面，本申请涉及编码上述的融合蛋白的多核苷酸。
另一方面，本申请涉及含有编码上述的融合蛋白的多核苷酸的重组表达载体。
另一方面，本申请涉及含有上述的重组表达载体或者在其基因组中整合有上述的多核苷酸的重组细胞。
另一方面，本申请涉及一种药物组合物，其中，所述药物组合物包含上述的融合蛋白。
另一方面，本申请涉及一种试剂盒，其中，所述试剂盒包含上述的融合蛋白或上述的药物组合物。
另一方面，本申请涉及上述的融合蛋白在制备用于预防或治疗与血糖代谢异常相关的疾病的药物中的用途。或者，本申请涉及一种预防或治疗与血糖代谢异常相关的疾病的方法，包括向有需要的受试者给予上述的融合蛋白。或者，本申请涉及一种用于预防或治疗与血糖代谢异常相关的疾病的融合蛋白。
又一方面，本申请涉及产生本申请上述的融合蛋白的方法，其中，所述方法包括：对上述的重组细胞进行培养，然后诱导上述的重组细胞进行表达并对表达产物进行纯化，得到所述融合蛋白。
本文中通过糖尿病模型鼠来评价本申请所述的融合蛋白治疗的糖尿病效果，动物学试验结果表明，本申请的融合蛋白能够有效降低糖尿病模型动物的血糖水平，而且与野生型hFGF21相比，本申请的融合蛋白在降低血糖水平方面还具有药效持续时间长(即，半衰期更长)等优点。同时，本申请所述的融合蛋白表现出良好的促进糖吸收的能力，而且，所述融合蛋白能够实现期望的表达效率。
附图说明
图1为SUMO-rhFGF21-(VPGXG) n融合蛋白表达量的电泳图。其中的各附图标记分别表示：
1：蛋白marker；2：IPTG诱导前的蛋白表达；3：SUMO-rhFGF21；4：SUMO-rhFGF21-(VPGXG) 20；5：SUMO-rhFGF21-(VPGXG) 40；6：SUMO-rhFGF21-(VPGXG) 60；7：SUMO-rhFGF21-(VPGXG) 80。对结果进行灰度分析可知，SUMO-rhFGF21-(VPGXG) 40表达量占总蛋白表达量的百分比相较于其它组更高，相同条件下，具有更高效的表达：SUMO-rhFGF21-(VPGXG) 40，11.2％；SUMO-rhFGF21-(VPGXG) 20，9.7％； SUMO-rhFGF21-(VPGXG) 60，10.1％；SUMO-rhFGF21-(VPGXG) 80，3.2％。
图2为示出经不同的测试样品刺激后的β-Klotho-3T3-L1脂肪细胞的糖吸收的变化的图表。经10nmol/L SUMO-rhFGF21刺激后(以“rhFGF21”示出)，β-Klotho-3T3-L1脂肪细胞的糖吸收水平为约35％，经10nmol/L rhFGF21-(VPGXG) 20刺激后，β-Klotho-3T3-L1脂肪细胞的糖吸收水平增加至约39％，经10nmol/L rhFGF21-(VPGXG) 40刺激后，β-Klotho-3T3-L1脂肪细胞的糖吸收水平增加至约46％，经10nmol/L rhFGF21-(VPGXG) 60刺激后，β-Klotho-3T3-L1脂肪细胞的糖吸收水平增加至约37％，经10nmol/L rhFGF21-(VPGXG) 80刺激后，β-Klotho-3T3-L1脂肪细胞的糖吸收水平增加至约32％。可见，与其它组相比，10nmol/L rhFGF21-(VPGXG) 40在细胞水平上具有更好的促进糖吸收的能力。
图3为对rhFGF21-(VPGXG) 40蛋白的纯度进行分析的HPLC谱图。经过HPLC分析，在保留时间为约6min时开始出现rhFGF21-(VPGXG) 40蛋白的峰，且其纯度可达95％以上。
图4为示出采用不同的测试样品对STZ模型鼠治疗12小时后的降糖结果的图表，其中，与模型组相比，rhFGF21治疗组在给予12小时后的降糖效果逐渐减弱；rhFGF21-(VPGXG) 20治疗组、rhFGF21-(VPGXG) 40治疗组、rhFGF21-(VPGXG) 60治疗组和rhFGF21-(VPGXG) 80治疗组仍然表现出一定的治疗效果，且rhFGF21-(VPGXG) 40治疗组的降糖效果最好。其中， *p<0.05， **p<0.01，与模型组相比； #p<0.05， ##p<0.01，与正常组相比。
图5为示出口服葡萄糖耐量试验(OGTT)的结果的图表。口服葡萄糖后30min，模型组的血糖水平显著上升，rhFGF21-(VPGXG) 40治疗组的血糖水平显著低于模型组。其中， *p<0.05， **p<0.01，与模型组相比； #p<0.05， ##p<0.01，与正常组相比。
图6为示出58天内各组小鼠的血糖的变化情况的图表。模型组小鼠的血糖始终维持较高的水平，rhFGF21-(VPGXG) 40治疗组小鼠的血糖较模型组显著下降，治疗后血糖维持在较低的水平并接近正常组小鼠的血糖水平，且药效比rhFGF21更持久。其中， *p<0.05， **p<0.01，与模型组相比； #p<0.05， ##p<0.01，与正常组相比。
图7为示出各组小鼠的血清胰岛素含量的图表。模型组小鼠的血清胰岛素含量显著下降，而rhFGF21-(VPGXG) 40治疗组小鼠的血清胰岛素含量相较于模型组得到显著的提高。其中， *p<0.05， **p<0.01，与模型组相比； #p<0.05， ##p<0.01，与正常组相比。
图8为示出各组小鼠的糖化血红蛋白水平的图表。给予相应的测试样品30天后，模型组小鼠的糖化血红蛋白含量维持在较高的水平，而rhFGF21-(VPGXG) 40治疗组小鼠的糖化血红蛋白水平显著下降。其中， *p<0.05， **p<0.01，与模型组相比； #p<0.05， ##p<0.01，与正常组相比。
图9为示出各组小鼠的肝脏内G6Pase基因的相对表达量的图表。模型组小鼠的肝脏内G6Pase表达量显著升高，而rhFGF21-(VPGXG) 40治疗组小鼠的肝脏内G6Pase表达量显著下降。其中， *p<0.05， **p<0.01，与模型组相比； #p<0.05， ##p<0.01，与正常组相比。
图10为示出各组小鼠的肝脏内PCK基因的相对表达量的图表。模型组小鼠的肝脏内PCK基因表达量显著升高，而rhFGF21-(VPGXG) 40治疗组小鼠的肝脏内PCK基因表达量显著下降。其中， *p<0.05， **p<0.01，与模型组相比； #p<0.05， ##p<0.01，与正常组相比。
图11和图12为示出各组小鼠的肝脏内的G6Pase和PCK蛋白的表达量的图。模型组小鼠的肝脏内G6Pase和PCK蛋白表达量显著升高，而rhFGF21-(VPGXG) 40治疗组小鼠的肝脏内G6Pase和PCK蛋白表达量显著下降。其中， *p<0.05， **p<0.01，与模型组相比； #p<0.05， ##p<0.01，与正常组相比。
图13为示出胰腺HE染色以及免疫组化结果的图片。模型组小鼠胰腺胰岛破坏比较严重，分泌胰岛素的量显著降低，而rhFGF21-(VPGXG) 40治疗组小鼠胰岛相较于模型组得到显著的改善。
图14为示出在不同pH值(pH 5.0和5.5)的柠檬酸盐缓冲溶液中于25℃下保存14天后的rhFGF21-(VPGXG) 40蛋白以及-80℃保存的相同蛋白的HPLC分析的结果的谱图。
图15为示出在不同浓度的精氨酸水溶液(含有50mM、100mM和150mM精氨酸)中于25℃下保存25天后的rhFGF21-(VPGXG) 40蛋白以及-80℃保存的相同蛋白的HPLC分析的结果的谱图。
图16为示出在精氨酸+柠檬酸盐缓冲溶液(pH 5.5)中于25℃下保存35天后的rhFGF21-(VPGXG) 40蛋白以及-80℃保存的相同蛋白的HPLC分析的结果的谱图。
图17为示出在不同pH值(pH 5.0和5.5)的精氨酸+柠檬酸盐缓冲溶液中于4℃下保存60天后的rhFGF21-(VPGXG) 40蛋白以及-80℃保存的相同蛋白的HPLC分析的结果的谱图。
图18为示出在25℃和-80℃下储存35天后的不同剂量(10nmol、100nmol和1000nmol)的rhFGF21-(VPGXG) 40蛋白促进HepG2细胞对葡萄糖的摄取的结果的图表。
在上述各图涉及的融合蛋白中，X为A和V，且A与V比例为2：3。
具体实施方式
下面结合具体的实施例来进一步描述本申请，本申请的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本申请的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本申请的精神和范围下可以对本申请技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本申请的保护范围内。
除非另外定义，否则本文使用的技术和科学术语具有与本公开所属领域的普通技术人员通常理解的含义相同的含义。参见如Singleton等，Dictionary of Microbiology and Molecular Biology 2nd ed.，J.Wiley&Sons(New York，NY 1994)；Sambrook等，Molecular Cloning，A Laboratory Manual，Cold Springs Harbor Press(Cold Springs Harbor，NY 1989)。
在本文中，除非另有说明，术语“模型组”和“模型对照组”可互换使用，术语“正常组”、“空白组”和“正常对照组”可互换使用。
在本文中，除非另有说明，术语“重组质粒”和“重组表达载体”可互换使用。
在本文中，除非另有说明，术语“接头”可为用于将rhFGF21和(VPGXG) n以及(VPGXG) n中的各重复单元连接在一起的氨基酸序列(其长度例如可为约1个至约5个氨基酸)，并可用于鉴定，例如可包括本领域常见的接头肽序列、酶切位点等，但不限于此。
在本文中，除非另有说明，单数术语涵盖复数的指代物，反之亦然。
在本文中，除非另有说明，术语“包含、包括和含有(comprise、comprises和comprising)”或其等同物(contain、containing、include、including)为开放式表述，意味着除所列出的要素、组分和步骤外，还可涵盖其它未指明的要素、组分和步骤。
在本文中，除非另有说明，术语“治疗”包括逆转、减轻、缓解、改善、抑制、减缓或停止有关的病症的进展或严重程度。
在本文中，除非另有说明，术语“受试者”可以和“个体”、“患者”互换使用，包括脊椎动物，例如鸟类、鱼类、哺乳动物，例如但不限于小鼠、大鼠、豚鼠、狗、猪、鸡、兔、猴(例如恒河猴)、人等。
在本文中，除非另有说明，本文所使用的表示成分的量、测量值或反应条件的所有数字应理解为在所有情况下均由术语“约”修饰。当与百分比相连时，术语“约”可以表示±1％。
在本文中，序列之间的一致性百分比(同源性程度)可以通过例如使用万维网上通常用于此目的的免费可用的计算机程序(例如具有默认设置的BLASTp或BLASTn)对两个序列进行比较来确定。
在一个实施方式中，本申请提供了一种重组人成纤维细胞生长因子21融合蛋白，其中，所述融合蛋白包括融合在一起的rhFGF21和(VPGXG) n，所述rhFGF21包含以SEQ ID NO:1示出的氨基酸序列或与SEQ ID NO:1具有至少80％一致性的氨基酸序列；X为A和/或V；n为选自20-80中的整数。
在一个优选的实施方式中，所述rhFGF21包含与SEQ ID NO:1具有至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、98％或99％一致性的氨基酸序列。
在一个优选的实施方式中，所述rhFGF21具有以SEQ ID NO:1示出的氨基酸序列(SEQ ID NO:1：
Figure PCTCN2021096292-appb-000001
在一个优选的实施方式中，X为A和V，且A与V比例为(1-3):(1-3)，例如A与V比例为(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)或3:(1-3)。在更优选的实施方式中，X为A和V，且A与V比例为2:3(即，此时(VPGXG) n为[(VPGAG) 2(VPGVG) 3] m，其中，m的值满足n＝m×(2+3))。
在一个优选的实施方式中，n为选自30-80中的整数，例如n为选自30-70、30-60、30-50、40-80、40-70、40-60、40-50中的整数。在一些具体的实施方式中，n为20、25、30、35、40、45、50、55、60、65、70或75。在一些具体的实施方式中，n为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或70。在一些更优选的实施方式中，n为40-60，例如40。
在一个优选的实施方式中，所述融合蛋白由融合在一起的rhFGF21和(VPGXG) n以及任选的接头组成。
在一个优选的实施方式中，所述接头的氨基酸序列为RS和/或GS。
在一个优选的实施方式中，所述融合蛋白具有以SEQ ID NO:2示出的氨基酸序列或与SEQ ID NO:2 具有至少80％、例如至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、98％或99％一致性的氨基酸序列。
在一个优选的实施方式中，所述融合蛋白具有以SEQ ID NO:2示出的氨基酸序列(SEQ ID NO:2：
Figure PCTCN2021096292-appb-000002
在本文中，包含与SEQ ID NO:1具有至少80％一致性的氨基酸序列的rhFGF21的融合蛋白或包含与SEQ ID NO:2具有至少80％一致性的氨基酸序列的融合蛋白具有降糖活性。
在一个实施方式中，本申请提供编码上述的融合蛋白的多核苷酸。在本文中，除非另有说明，术语“多核苷酸”、“核酸”、“核酸序列”和“基因”可互换使用。
在一些优选的实施方式中，编码上述的融合蛋白中的rhFGF21的多核苷酸部分包含以SEQ ID NO:3示出的核苷酸序列或与SEQ ID NO:3具有至少80％、例如至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、98％或99％一致性的核苷酸序列。
在进一步优选的实施方式中，编码上述的融合蛋白中的rhFGF21的多核苷酸部分具有以SEQ ID NO:3示出的核苷酸序列(SEQ ID NO:3：
Figure PCTCN2021096292-appb-000003
在一些优选的实施方式中，编码上述的融合蛋白中的接头的多核苷酸部分包含以SEQ ID NO:4和/或SEQ ID NO:5示出的核苷酸序列，或与SEQ ID NO:4和/或SEQ ID NO:5具有至少80％、例如至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、98％或99％一致性的核苷酸序列。
在进一步优选的实施方式中，编码上述的融合蛋白中的接头的多核苷酸部分具有以SEQ ID NO:4和/或SEQ ID NO:5示出的核苷酸序列(SEQ ID NO:4：AGATCT；SEQ ID NO:5：GGATCC)。
在一些优选的实施方式中，编码上述的融合蛋白的多核苷酸包含以SEQ ID NO:6示出的核苷酸序列或与SEQ ID NO:6具有至少80％、例如至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、98％或99％一致性的核苷酸序列。
在一个优选的实施方式中，编码上述的融合蛋白的多核苷酸具有以SEQ ID NO:6示出的核苷酸序列(SEQ ID NO:6：
Figure PCTCN2021096292-appb-000004
Figure PCTCN2021096292-appb-000005
在一个实施方式中，本申请提供含有编码上述的融合蛋白的多核苷酸的重组表达载体。
目前的表达载体的类型虽多，但对于特定的蛋白而言，选择合适的载体却并不容易，很多表达系统的蛋白的表达量虽高，但表达的蛋白可溶性较差。就本申请的融合蛋白而言，为了便于大量生产和后续的纯化，优选能够在原核表达体系中进行表达，因此，在本申请考虑之列的是适合于原核表达体系的原核表达载体。优选的，考虑到分子伴侣有助于协助蛋白质折叠，可以选择携带分子伴侣的原核表达载体。其中，所述的分子伴侣优选为小分子泛素样修饰蛋白(SUMO)。SUMO是一种小分子泛素样修饰蛋白，其能够促进目的蛋白的正确折叠进而增加目的蛋白的表达量和可溶性。
作为非限制性示例，适用于本申请的合适的表达载体例如但不限于：pSUMO载体、PET系列载体(如PET30a载体)。
在一个实施方式中，本申请提供含有上述的重组表达载体或者在其基因组中整合有上述的多核苷酸的重组细胞。
考虑到后续发酵和纯化的便利性，优选用于构建所述重组细胞的宿主细胞为原核宿主细胞，优选地，所述原核宿主细胞的实例包括但不限于：大肠杆菌(E.coli)(例如大肠杆菌Rossetta(DE3))、农杆菌(Agrobacterium tumefaciens)、金黄色葡萄球菌(Staphylococcus aureus)、白色葡萄球菌(Staphylococcus albus)、嗜酸乳杆菌(Lactobacillus acidophilus)、炭疽芽孢杆菌(Bacillus anthracis)、枯草芽孢杆菌(Bacillus subtilis)或苏云金芽孢杆菌(Bacillus thuringiensis)等。
在一个实施方式中，本申请提供一种药物组合物，其中，所述药物组合物包含上述的融合蛋白。
在优选的实施方式中，所述药物组合物进一步包含药学上可接受的辅料。所述药学上可接受的药用辅料可选自例如但不限于溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、抗氧剂、渗透促进剂、pH值调节剂、表面活性剂、稀释剂等。关于其它可用的药学上可接受的药用辅料，可参见例如《药用辅料手册》(第4版)，R.C.罗等著，郑泽民主译，2005年，化学工业出版社。
在一个实施方式中，本申请涉及一种试剂盒，其中，所述试剂盒包含上述的融合蛋白或上述的药物组合物。
在一些优选的实施方式中，所述试剂盒进一步包含pH 5.0-5.5的柠檬酸盐缓冲溶液。
在一些优选的实施方式中，所述试剂盒进一步包含精氨酸水溶液、优选50mmol/L-150mmol/L的精氨酸水溶液。
在一些优选的实施方式中，所述试剂盒进一步包含pH 5.0-5.5的精氨酸+柠檬酸盐缓冲溶液。
在一些优选的实施方式中，所述试剂盒进一步包含pH 5.0-5.5的柠檬酸盐缓冲溶液和精氨酸水溶液，并且，所述融合蛋白或所述药物组合物、所述pH 5.0-5.5的柠檬酸盐缓冲溶液、所述精氨酸水溶液包装在一起或者分别单独包装。
在一个实施方式中，本申请涉及上述的融合蛋白在制备用于预防或治疗与血糖代谢异常相关的疾病的药物中的用途。在一个替代的实施方式中，本申请涉及一种预防或治疗与血糖代谢异常相关的疾病的方法，包括向有需要的受试者给予上述的融合蛋白。在一个替代的实施方式中，本申请涉及一种用于预防或治疗与血糖代谢异常相关的疾病的融合蛋白。
在优选的实施方式中，所述与血糖代谢异常相关的疾病选自但不限于：糖尿病及糖尿病相关代谢疾病。优选地，所述糖尿病相关代谢疾病选自糖尿病肾病、血脂异常、肥胖症、心血管疾病、代谢综合征、脂代谢紊乱、非酒精性脂肪肝疾病(NAFLD)或神经系统疾病(如癫痫、抑郁等)，但不限于此。
在一个实施方式中，本申请提供产生本申请上述的融合蛋白的方法，其中，所述方法包括：对上述的重组细胞进行培养，然后诱导上述的重组细胞进行表达并对表达产物进行纯化，得到所述融合蛋白。
在一个优选的实施方式中，所述重组细胞可为重组原核细胞，优选地，所述重组原核细胞的实例包括但不限于：重组大肠杆菌(例如重组大肠杆菌Rossetta(DE3))、重组农杆菌、重组金黄色葡萄球菌、重组白色葡萄球菌、重组嗜酸乳杆菌、重组炭疽芽孢杆菌、重组枯草芽孢杆菌或重组苏云金芽孢杆菌等。
重组细胞的培养可由本领域技术人员根据重组细胞的种类选择常规的培养基和培养条件进行(例如可 参见如下的记载：《微生物培养基的制造与应用》，陈天寿主编，中国农业出版社，1995年；《微生物培养基实用手册》，马乐好等主编，吉林科学技术出版社，2005年；http://www.cgmcc.net/；以及https://www.sigmaaldrich.com/china-mainland/technical-documents/articles/biology/microbial-media.html等)。
可采用本领域技术人员已知的常规诱导剂，诱导上述的重组细胞进行表达。作为示例性的实施方式，例如采用选自如下的诱导剂，诱导上述的重组细胞进行表达：IPTG和/或乳糖。
可采用本领域技术人员已知的常规纯化手段对表达产物进行纯化以得到融合蛋白，所述常规纯化手段例如离子交换层析、吸附层析、亲和层析、分子筛层析、疏水层析或它们的任意组合等，但不限于此。在一个优选的实施方式中，所述表达产物的纯化包括如下步骤：(i)在诱导表达后，将所述重组细胞进行离心以收集菌体，将所述菌体进行破碎后离心收集上清；(ii)将所述上清进行离子交换层析，收集洗脱液；(iii)将所述洗脱液进行亲和层析，得到所述融合蛋白。
在一些优选的实施方式中，将所述重组细胞在10000-12000rpm/min下进行离心以收集菌体。
在一些优选的实施方式中，通过向所述菌体中加入溶菌酶并进行超声，将所述菌体进行破碎。
在一些优选的实施方式中，在10000-12000rpm/min下离心以收集所述上清。
在一些优选的实施方式中，采用琼脂糖凝胶、例如DEAE琼脂糖凝胶进行所述离子交换层析。
在一些优选的实施方式中，采用包括如下组成的缓冲液对所述上清进行洗脱：25mmol/L Tris-HCl，0.25mol/L NaCl，pH 8.0-8.2。
在一些优选的实施方式中，采用Ni·NTA树脂进行所述亲和层析。
在一些优选的实施方式中，所述亲和层析包括如下过程：在用平衡缓冲液对亲和层析柱进行平衡后，以所述洗脱液向所述亲和层析柱上样并采用洗涤缓冲液进行洗涤，然后，采用洗脱缓冲液对目的蛋白进行洗脱。
在一些优选的实施方式中，所述平衡缓冲液包括如下组成：50mmol/L Tris-HCl，0.5mol/L NaCl，10mmol/L咪唑，pH 8.0-8.2。
在一些优选的实施方式中，所述洗涤缓冲液包括如下组成：50mmol/L Tris-HCl，500mmol/L NaCl，50mmol/L咪唑，pH 8.0-8.2。
在一些优选的实施方式中，所述洗脱缓冲液包括如下组成：50mmol/L Tris-HCl，500mmol/L NaCl，500mmol/L咪唑，pH 8.0-8.2。
在一些优选的实施方式中，上述的产生融合蛋白的方法进一步包括如下步骤：
(1)合成编码rhFGF21的多核苷酸，并合成编码(VPGXG) 20的多核苷酸；
(2)将上述的编码rhFGF21的多核苷酸和编码(VPGXG) 20的多核苷酸进行酶切和连接，通过PCR反应，获得编码所述融合蛋白的多核苷酸；
(3)将编码所述融合蛋白的多核苷酸与表达载体进行酶切和连接，得到重组表达载体，将所述重组表达载体导入宿主细胞中，筛选得到所述重组细胞。
可通基于本领域已知的rhFGF21的蛋白序列或其DNA或RNA序列(例如，可参见https://www.ncbi.nlm.nih.gov/protein/NP_061986.1；https://www.ncbi.nlm.nih.gov/nuccore/NG_033945.1；或https://www.ncbi.nlm.nih.gov/nuccore/NM_019113.4)，通过密码子优化，利用常规的基因合成手段合成编码rhFGF21的多核苷酸序列。
在优选的实施方式中，在步骤(1)中，所述编码rhFGF21的多核苷酸序列包含以SEQ ID NO:3示出的核苷酸序列或与SEQ ID NO:3具有至少80％、例如至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、98％或99％一致性的核苷酸序列。
在优选的实施方式中，在步骤(1)中，所述编码rhFGF21的多核苷酸具有以SEQ ID NO:3示出的核苷酸序列。
在优选的实施方式中，在步骤(1)中，所述编码(VPGXG) 20的多核苷酸包含以SEQ ID NO:7示出的核苷酸序列或与SEQ ID NO:7具有至少80％、例如至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、98％或99％一致性的核苷酸序列。
在优选的实施方式中，在步骤(1)中，所述编码(VPGXG) 20的多核苷酸具有以SEQ ID NO:7示出的核苷酸序列(SEQ ID NO:7：
Figure PCTCN2021096292-appb-000006
在优选的实施方式中，在步骤(2)中，任选在所述编码(VPGXG) 20的多核苷酸的5′端和3′端分别连接有编码选自如下的接头的多核苷酸：RS和/或GS。优选地，在所述编码(VPGXG) 20的多核苷酸的5′端和3′端分别连接有具有以SEQ ID NO:4和SEQ ID NO:5示出的核苷酸序列的多核苷酸。
在优选的实施方式中，在步骤(2)中，利用BglII限制性内切酶与BamHI限制性内切酶进行所述酶切。
可由本领域技术人员基于本领域的普通知识(例如可参见《分子克隆实验指南》(第4版)，J.萨姆布鲁克等编著，贺福初主译，科学出版社，2017年)选择合适的常规的PCR实验条件和步骤、或者采用可商购的PCR试剂盒按照其中的说明书进行所述PCR反应。不受限制的，还可采用其它引物对和本领域已知的其它的基因合成手段来获得编码上述的融合蛋白的多核苷酸。
上述的酶切和连接，其具有本领域公知的含义，酶切和连接是指采用限制性核酸内切酶对两种目的DNA片段或者对一种目的DNA片段与载体DNA分子进行特异性切割，然后使用连接酶将进行酶切后的两种目的DNA片段或者酶切后的目的DNA片段与载体DNA分子的末端进行连接，从而得到重组分子或者重组载体。
在优选的实施方式中，在步骤(3)中，利用BsaI限制性内切酶与BamHI限制性内切酶进行所述酶切。
对于将重组表达载体导入宿主细胞中的具体手段，可采用本领域已知的任何常规手段，例如转化、转染等。优选地，通过转化将该重组表达载体导入宿主细胞中。转化是指通过采用分子生物学和基因工程中的一些已知方法处理宿主细胞，使经处理后的宿主细胞处于感受态，并由此与外源DNA接触，从而使外源DNA进入处于感受态的宿主细胞中。
在优选的实施方式中，在步骤(3)中，所述宿主细胞为原核宿主细胞，优选地，所述原核宿主细胞的实例包括但不限于：大肠杆菌(例如大肠杆菌Rossetta(DE3))、农杆菌、金黄色葡萄球菌、白色葡萄球菌、嗜酸乳杆菌、炭疽芽孢杆菌、枯草芽孢杆菌或苏云金芽孢杆菌等。
对于导入重组表达载体的宿主细胞，可通过抗性筛选等常规手段筛选得到阳性的重组细胞。所述重组细胞的培养可由本领域技术人员根据重组细胞的种类选择常规的培养基和培养条件进行(例如，可参见如下的记载：《微生物培养基的制造与应用》，陈天寿主编，中国农业出版社，1995年；《微生物培养基实用手册》，马乐好等主编，吉林科学技术出版社，2005年；http://www.cgmcc.net/；以及https://www.sigmaaldrich.com/china-mainland/technical-documents/articles/biology/microbial-media.html等)。
在优选的实施方式中，在步骤(3)中，所述表达载体选自pSUMO载体和PET系列载体(如PET30a载体)，但不限于此。在进一步优选的实施方式中，将携带小分子泛素样修饰蛋白(SUMO)作为分子伴侣的pSUMO载体用作所述表达载体，并将大肠杆菌Rossetta(DE3)用作宿主细胞。
在示例性的实施方式中，将含有编码上述的融合蛋白的多核苷酸的重组pSUMO载体转化至所述宿主细胞中；对筛选得到的重组细胞进行培养，然后诱导上述的重组细胞表达SUMO-融合蛋白，对表达产物进行离子交换层析和亲和层析纯化得到初步纯化的SUMO-融合蛋白，在透析后，利用SUMO蛋白酶切除与融合蛋白相连的分子伴侣SUMO，并对切除SUMO后的融合蛋白进行亲和层析纯化，得到经纯化的融合蛋白。
本申请的示例性的技术方案可通过如下编号段落的内容进行说明：
1.一种重组人成纤维细胞生长因子21融合蛋白，其中，所述融合蛋白包括融合在一起的rhFGF21和(VPGXG) n，所述rhFGF21包含以SEQ ID NO:1示出的氨基酸序列或与SEQ ID NO:1具有至少80％一致性的氨基酸序列；X为A和/或V；n为选自20-80中的整数。
2.如段落1所述的融合蛋白，其中，X为A和V，且A与V比例为(1-3):(1-3)、优选2:3。
3.如段落1或2所述的融合蛋白，其中，n为40-60中的整数、优选40。
4.如段落1-3中任一段所述的融合蛋白，其中，所述融合蛋白由融合在一起的rhFGF21和(VPGXG) n以及任选的接头组成。
5.如段落4所述的融合蛋白，其中，所述接头的氨基酸序列为RS和/或GS。
6.如段落1-5中任一段所述的融合蛋白，其中，所述融合蛋白具有以SEQ ID NO:2示出的氨基酸序列或与SEQ ID NO:2具有至少80％一致性的氨基酸序列。
7.一种编码段落1-6中任一段所述的融合蛋白的多核苷酸。
8.如段落7所述的多核苷酸，其中，编码所述融合蛋白中的rhFGF21的多核苷酸部分包含以SEQ ID NO:3示出的核苷酸序列或与SEQ ID NO:3具有至少80％一致性的核苷酸序列。
9.如段落7或8所述的多核苷酸，其中，编码所述融合蛋白中的接头的多核苷酸部分包含以SEQ ID NO:4和/或SEQ ID NO:5示出的核苷酸序列，或与SEQ ID NO:4和/或SEQ ID NO:5具有至少80％一致性的核苷酸序列。
10.如段落7-9中任一段所述的多核苷酸，其中，所述多核苷酸包含以SEQ ID NO:6示出的核苷酸序列或与SEQ ID NO:6具有至少80％一致性的核苷酸序列。
11.一种含有段落7-10中任一段所述的多核苷酸的重组表达载体。
12.如段落11所述的重组表达载体，其中，所述重组表达载体为原核重组表达载体。
13.如段落12所述的重组表达载体，其中，所述重组表达载体为携带分子伴侣的原核重组表达载体。
14.段落13所述的重组表达载体，其中，所述分子伴侣为小分子泛素样修饰蛋白。
15.如段落11或12所述的重组表达载体，其中，所述重组表达载体为重组的pSUMO载体或重组的PET系列载体。
16.一种含有段落11-15中任一段所述的重组表达载体或者在其基因组中整合有段落7-10中任一段所述的多核苷酸的重组细胞。
17.如段落16所述的重组细胞，其中，用于构建所述重组细胞的宿主细胞为原核宿主细胞。
18.如段落17所述的重组细胞，其中，所述原核宿主细胞选自大肠杆菌、农杆菌、金黄色葡萄球菌、白色葡萄球菌、嗜酸乳杆菌、炭疽芽孢杆菌、枯草芽孢杆菌或苏云金芽孢杆菌。
19.一种药物组合物，其中，所述药物组合物包含段落1-6中任一段所述的融合蛋白。
20.如段落19所述的药物组合物，其中，所述药物组合物进一步包含药学上可接受的辅料。
21.一种试剂盒，其中，所述试剂盒包含段落1-6中任一段所述的融合蛋白或者段落19或20所述的药物组合物。
22.如段落21所述的试剂盒，其中，所述试剂盒进一步包含pH 5.0-5.5的柠檬酸盐缓冲溶液。
23.如段落21或22所述的试剂盒，其中，所述试剂盒进一步包含精氨酸水溶液、优选50mmol/L-150mmol/L的精氨酸水溶液。
24.如段落21-23中任一段所述的试剂盒，其中，所述试剂盒进一步包含pH 5.0-5.5的精氨酸+柠檬酸盐缓冲溶液。
25.如段落21所述的试剂盒，其中，所述试剂盒进一步包含精氨酸水溶液和pH 5.0-5.5的柠檬酸盐缓冲溶液，并且，所述融合蛋白或所述药物组合物、所述pH 5.0-5.5的柠檬酸盐缓冲溶液、以及所述精氨酸水溶液包装在一起或者分别单独包装。
26.段落1-6中任一段所述的融合蛋白在制备用于预防或治疗与血糖代谢异常相关的疾病的药物中的用途。
27.如段落26所述的用途，其中，所述与血糖代谢异常相关的疾病选自糖尿病及糖尿病相关代谢疾病，优选地，所述糖尿病相关代谢疾病选自糖尿病肾病、血脂异常、肥胖症、心血管疾病、代谢综合征、脂代谢紊乱、非酒精性脂肪肝疾病或神经系统疾病。
28.一种产生段落1-6中任一段所述的融合蛋白的方法，其中，所述方法包括：对段落16-18中任一段所述的重组细胞进行培养，然后诱导所述重组细胞进行表达并对表达产物进行纯化，得到所述融合蛋白。
29.如段落28所述的方法，其中，采用选自如下的诱导剂，诱导所述重组细胞进行表达：IPTG和/或乳糖。
30.如段落28或29所述的方法，其中，所述表达产物的纯化包括如下步骤：(i)在诱导表达后，将所述重组细胞进行离心以收集菌体，将所述菌体进行破碎后离心收集上清；(ii)将所述上清进行离子交换层析，收集洗脱液；(iii)将所述洗脱液进行亲和层析，得到所述融合蛋白。
31.如段落28-30中任一段所述的方法，其中，所述方法还包括如下步骤：
(1)合成编码rhFGF21的多核苷酸，并合成编码(VPGXG) 20的多核苷酸；
(2)将上述的编码rhFGF21的多核苷酸和编码(VPGXG) 20的多核苷酸进行酶切和连接，通过PCR反应，获得编码所述融合蛋白的多核苷酸；
(3)将编码所述融合蛋白的多核苷酸与表达载体进行酶切和连接，得到重组表达载体，将所述重组表达载体导入宿主细胞中，筛选得到所述重组细胞。
32.如段落31所述的方法，在步骤(1)中，所述编码rhFGF21的多核苷酸序列包含以SEQ ID NO:3示出的核苷酸序列或与SEQ ID NO:3具有至少80％一致性的核苷酸序列。
33.如段落31或32所述的方法，在步骤(1)中，所述编码(VPGXG) 20的多核苷酸包含以SEQ ID NO:7示出的核苷酸序列或与SEQ ID NO:7具有至少80％一致性的核苷酸序列。
34.如段落31-33中任一段所述的方法，在步骤(2)中，任选在所述编码(VPGXG) 20的多核苷酸的5′端和3′端分别连接有编码选自如下的接头的多核苷酸：RS和/或GS。
35.如段落31-34中任一段所述的方法，在步骤(2)中，利用BglII限制性内切酶与BamHI限制性内切酶进行所述酶切。
36.如段落31-35中任一段所述的方法，在步骤(3)中，利用BsaI限制性内切酶与BamHI限制性内切酶进行所述酶切。
37.如段落31-36中任一段所述的方法，在步骤(3)中，所述表达载体选自pSUMO载体和PET系列载体。
38.如段落31-37中任一段所述的方法，在步骤(3)中，所述表达载体为pSUMO载体，且所述宿主细胞为大肠杆菌Rossetta(DE3)。
需要说明的是，本申请中涉及的基因的设计、合成和克隆，原核表达载体的构建，核酸提取及序列分析及鉴定，以及表达产物的分离和纯化等操作步骤，可按照本领域已知的技术进行(例如参见CURRENT PROTOCOLS IN MOLECULAR BIOLOGY的记载)。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。除非另外说明，下述实施例中使用的试剂、材料和设备均为可商购的试剂、材料和设备。
实施例
实施例1重组人成纤维细胞生长因子21融合蛋白的基因的制备
将rhFGF21的基因与(VPGXG) 20的基因之间通过BglII酶切位点相连，然后进行PCR在rhFGF21-(VPGXG) 20的5’端与3’端引入BsaI与BamHI酶切位点；再利用BamHI酶切位点连接至另一经BglII和BamHI酶切后的(VPGXG) 20的基因，得到rhFGF21-(VPGXG) 40，然后进行PCR在rhFGF21-(VPGXG) 40的5’端引入BsaI酶切位点，在3’端引入终止密码子和BamHI酶切位点。
1、带有酶切位点的rhFGF21的基因的合成
通过NCBI查询野生型hFGF21的氨基酸序列(SEQ ID NO:1)，并经密码子优化得到编码rhFGF21的核苷酸序列(SEQ ID NO:3)，委托基因合成公司合成带有BsaI酶切位点(SEQ ID NO:10：GGTCTCAGGT)和BglII酶切位点(SEQ ID NO:4：AGATCT)的基因(SEQ ID NO:8：
Figure PCTCN2021096292-appb-000007
2、接头-(VPGXG) 20的基因的制备
委托基因合成公司合成具有以SEQ ID NO:9示出的核苷酸序列的接头-(VPGXG) 20的基因，其由5′端的具有以SEQ ID NO:4示出的核苷酸序列的编码接头RS的多核苷酸(即，BglII酶切位点)、具有以SEQ ID NO:7示出的核苷酸序列的编码(VPGXG) 20的多核苷酸、以及3′端的具有以SEQ ID NO:5示出的核苷酸序列的编码接头GS的多核苷酸(即，BamHI酶切位点)组成(SEQ ID NO:9：
Figure PCTCN2021096292-appb-000008
3、rhFGF21-(VPGXG) 40融合蛋白的基因的制备
以上述得到的带有酶切位点的rhFGF21的基因和接头-(VPGXG) 20的基因作为模板，采用BglII限制性内切酶按制造商的说明书进行酶切并采用T4 DNA连接酶进行连接，得到rhFGF21-(VPGXG) 20多肽的核苷酸序列。
为了得到rhFGF21-(VPGXG) 40融合蛋白的基因，采用引物P1(SEQ ID NO:11)和P2(SEQ ID NO:12)以及rTaq酶按照下文所述的50μL体系进行PCR，向得到的rhFGF21-(VPGXG) 20多肽的核苷酸序列的5’端与3’端引入BsaI与BamHI酶切位点。
P1：5′- GGTCTCAGGTCATCCTATTCCTGACAGCT-3′(SEQ ID NO:11)
BsaI
P2：5′- GGATCCACCTACACCCGGAACA-3′(SEQ ID NO:12)
BamHI
将上述的引入酶切位点后的核苷酸序列，通过solution I连接酶连接至T载体后，利用BamHI限制性内切酶进行酶切；同时，将接头-(VPGXG) 20的基因通过BglII和BamHI限制性内切酶进行酶切，按制造商的说明书进行酶切，并将上述两种酶切产物采用DNA片段胶回收试剂盒(购自Omega，货号：D6492)进行胶回收后，采用T4 DNA连接酶进行连接。
然后，以连接产物作为模板，采用rTaq酶进行PCR反应(50μL体系)，引入终止密码子(TCA)：
Figure PCTCN2021096292-appb-000009
其中，引物P1和引物P3(SEQ ID NO:13)的序列如下：
P1：5′-GGTCTCAGGTCATCCTATTCCTGACAGCT-3′(SEQ ID NO:11)
BsaI
P3：5′- GGATCCTCAACCTACACCCGGAACA-3′(SEQ ID NO:13)
BamHI
将得到的PCR产物采用DNA片段胶回收试剂盒(购自Omega，货号：D6492)进行胶回收，得到rhFGF21-(VPGXG) 40融合蛋白的基因。
4、其它的融合蛋白的基因的制备
按照与上述制备rhFGF21-(VPGXG) 40融合蛋白的基因相同的操作和条件，得到融合蛋白rhFGF21-(VPGXG) 20、rhFGF21-(VPGXG) 60、rhFGF21-(VPGXG) 80的基因。
其中，为了制备融合蛋白rhFGF21-(VPGXG) 20的基因，直接在得到的rhFGF21-(VPGXG) 20多肽的核苷酸序列的3’端通过PCR引入终止密码子，而无需再引入酶切位点，其中，50μL的PCR体系同上文所述。实施例2人成纤维细胞生长因子21融合蛋白的制备
本实施例中涉及的pSUMO和大肠杆菌Rossetta(DE3)通过商购得到。
1、rhFGF21-(VPGXG) 40基因重组原核表达载体的构建
将实施例1得到的rhFGF21-(VPGXG) 40目的基因片段与原核表达载体pSUMO采用BsaI和BamHI酶按制造商的说明书进行双酶切，并采用DNA片段胶回收试剂盒(购自Omega，货号：D6492)对酶切产物进行纯化，随后利用T4 DNA连接酶以10μL的连接反应体系按制造商的说明书进行连接，16℃连接过夜。
经过BsaI和BamHI双酶切并将酶切产物进行琼脂糖凝胶电泳鉴定后，构建得到含有正确序列的重组质粒pSUMO-rhFGF21-(VPGXG) 40。
2、rhFGF21-(VPGXG) 40蛋白的表达和纯化
(1)诱导表达
将含有正确序列的重组质粒pSUMO-rhFGF21-(VPGXG) 40转化至表达菌株大肠杆菌Rossetta(DE3)感受态细胞中。将转化后的感受态细胞涂布至含有100μg/mL氨苄青霉素的LB平板上，37℃培养10-12h后，挑取转化后的阳性重组菌的单菌落接种至5mL含氨苄青霉素(100μg/mL)的LB培养基中，37℃培养10h，将培养液以1:100接种于500mL含氨苄青霉素(100μg/mL)的LB培养基中，37℃培养2h，A600＝0.3-0.6时，加入IPTG至终浓度为0.5mmol/L进行诱导(在诱导前，取培养物在12000rpm/min下离心30min得 到上清和沉淀，并将所述上清和沉淀进行15％SDS-PAGE电泳分析)，诱导3h后通过离心(12000rpm/min下离心30min)收集菌体。
(2)蛋白质纯化
通过上述的离心收集菌体后，向菌体中加入溶菌酶至终浓度1mg/mL，冰上静置1h，然后进行超声破碎并在破碎完成后离心(12000rpm/min，30min)收集上清，分别取得到的上清和沉淀进行15％SDS-PAGE电泳分析，其中，可溶性表达的目的蛋白可占菌体总目的蛋白的70％以上。将所述上清通过DEAE Sepharose FF层析柱进行纯化，其中，用缓冲液1(25mmol/L Tris-HCl，0.25mol/L NaCl，pH 8.0)进行洗脱，收集得到离子交换层析洗脱液。将所述离子交换层析洗脱液进行Ni·NTA树脂柱亲和层析，其中，先用平衡缓冲液(50mmol/L Tris-HCl，pH 8.0，0.5mol/L NaCl，10mmol/L咪唑)平衡Ni·NTA树脂柱，然后将上述的洗脱液上样到平衡好的树脂柱中，上样完毕后用洗涤缓冲液(50mmol/L Tris-HCl，pH 8.0，0.5mol/L NaCl，50mmol/L咪唑)进行洗涤。最后，以洗脱缓冲液(50mmol/L Tris-HCl，pH 8.0，0.5mol/L NaCl，500mmol/L咪唑)进行目的蛋白的洗脱。收集各洗脱峰得到亲和层析洗脱液，并进行SDS-PAGE检测。
将所述亲和层析洗脱液用PBS缓冲液(pH 7.4)4℃透析过夜，得到去除咪唑的融合蛋白SUMO-rhFGF21-(VPGXG) 40，向其中加入DTT至终浓度为2mmol/L和SUMO Protease-I(带有His Tag)(切割比例为1:50，即1mg SUMO Protease-I：50mg SUMO-rhFGF21-(VPGXG) 40)，4℃切割过夜。将得到的切割产物与Ni-NTA Agarose充分混合以收集流穿液，其中，用PBS缓冲液(pH 7.4)将非特异性吸附的rhFGF21-(VPGXG) 40蛋白洗脱。经过HPLC分析(流动相为PBS缓冲液，流速为1mL/min，色谱柱：Agilent AdvanceBio SEC 300
Figure PCTCN2021096292-appb-000010
2.7μm，7.8×300mm，货号：PL1180-5301)显示出，纯化后的rhFGF21-(VPGXG) 40蛋白(具有以SEQ ID NO:2示出的氨基酸序列)的纯度达到95％以上(图3)。
以与上述的rhFGF21-(VPGXG) 40蛋白相同的表达和纯化操作和条件，实施rhFGF21-(VPGXG) 20、rhFGF21-(VPGXG) 60、rhFGF21-(VPGXG) 80蛋白的表达和纯化。利用15％SDS-PAGE电泳对各蛋白的表达量进行分析(图1)。
实施例3人成纤维细胞生长因子21融合蛋白的活性试验
1、rhFGF21-(VPGXG) 40蛋白的细胞水平活性检测
将β-Klotho克隆至逆转录病毒真核表达载体pBMN-β Klotho-IRES-EGFP中，以绿色荧光蛋白作为报告基因，利用脂质体转染法将重组的逆转录病毒载体转入293包装细胞，离心收集病毒上清，感染3T3-L1前体细胞(购自ATCC，货号：CL-173)，利用流式细胞仪筛选到表达β-Klotho的β-Klotho-3T3-L1稳定细胞系，称为β-Klotho-3T3-L1脂肪细胞。将β-Klotho-3T3-L1脂肪细胞饥饿12h，用细胞培养基(DMEM，gibco，C11965500BT)稀释纯化后的rhFGF21-(VPGXG) 40蛋白，使其终浓度为10nmol/L、100nmol/L、1000nmol/L，以每孔1ml的量将不同浓度的稀释液加入到已分化成熟的脂肪细胞中。每个浓度至少设3个重复孔。
葡萄糖浓度的检测：在上述处理后分别孵育细胞24h后，取上清培养基2μL放入200μL的葡萄糖检测液中测定葡萄糖的含量。每个浓度至少重复3次，37℃反应5～10分钟后，在500nm波长下测OD值。细胞的葡萄糖消耗率计算方法如下，并对实验结果进行统计学分析。
计算培养基中残留的葡萄糖浓度(C)，公式为：
葡萄糖浓度(mmol/L)＝OD 样品/OD 标准×5.55mmol/L
计算细胞对葡萄糖的消耗率，公式为：
细胞葡萄糖消耗率(％)＝[(C 空白葡萄糖-C 给药葡萄糖)/C 空白葡萄糖]×100％。
用不同浓度的rhFGF21-(VPGXG) 40蛋白处理脂肪细胞24h后，经微量化的GOD-POD法葡萄糖检测试剂盒检测培养基中葡萄糖含量，统计学分析结果显示，用rhFGF21-(VPGXG) 40蛋白处理的细胞对葡萄糖的摄取利用显著增加，与未经任何处理的对照组相比，残存在培养基中的葡萄糖含量明显减少。
按照上述方法，采用rhFGF21和上述制备的各融合蛋白对β-Klotho-3T3-L1脂肪细胞进行处理，结果显示，未经处理的β-Klotho-3T3-L1脂肪细胞的糖吸收为约27％，经10nmol/L rhFGF21刺激后增加至约35％，经10nmol/L rhFGF21-(VPGXG) 20刺激后增加至约39％，经10nmol/L rhFGF21-(VPGXG) 40刺激后增加至约46％，经10nmol/L rhFGF21-(VPGXG) 60刺激后增加至约37％，经10nmol/L rhFGF21-(VPGXG) 80刺激后增加至约32％。结果表明，相较于同剂量的rhFGF21以及其它融合蛋白，10nmol/L rhFGF21-(VPGXG) 40融合蛋白具有最好的活性(图2)。
2、rhFGF21和不同的融合蛋白的动物体内降糖活性的比较
利用STZ(80mg/kg)诱导C57BL/6J小鼠(长春亿斯实验动物技术有限责任公司，动物质量合格证号SCXK(吉)-2011-0004)，获得糖尿病小鼠，血糖持续高于13.8mmol/L的小鼠被认为是糖尿病小鼠。
将建立好的糖尿病模型小鼠，随机分为6组，每组8只。同时设置正常小鼠组作为空白对照组，8只。通过皮下给药方式，模型对照组注射PBS缓冲液；rhFGF21-(VPGXG) 40治疗组以1mg/kg/d剂量注射融合蛋白，rhFGF21-(VPGXG) 20、rhFGF21-(VPGXG) 60、rhFGF21-(VPGXG) 80治疗组分别以与rhFGF21-(VPGXG) 40治疗组相同的剂量注射融合蛋白，注射完毕12小时后(在此期间，小鼠自由饮食)，检测各组小鼠的血糖水平。结果显示，在给药12小时后，与模型组相比，rhFGF21治疗组的降糖效果减弱；各融合蛋白治疗组表现出一定的治疗效果，其中，融合蛋白rhFGF21-(VPGXG) 40治疗组的小鼠的血糖水平较模型组显著下降(p<0.01)，且相较于其它组，rhFGF21-(VPGXG) 40治疗组的降糖效果最好(图4)。
3、rhFGF21-(VPGXG) 40蛋白的体内活性试验
(1)糖尿病小鼠模型检测rhFGF21-(VPGXG) 40蛋白活性
利用STZ(80mg/kg)诱导C57BL/6J小鼠(长春亿斯实验动物技术有限责任公司，动物质量合格证号SCXK(吉)-2011-0004)，获得糖尿病小鼠(血糖持续高于13.8mmol/L)，随机分为5组，每组8只。同时将正常小鼠作为正常对照组，8只。通过皮下给药方式，模型对照组注射PBS缓冲液；rhFGF21-(VPGXG) 40治疗组以1mg/kg/d剂量注射融合蛋白，连续注射58天。实验开始于上午8:00(此时模型动物血糖为一天内较高值)，实验过程中自由饮食。
模型对照组：糖尿病小鼠每天上午8:00左右皮下注射PBS缓冲液，单次注射体积为0.15mL；
rhFGF21-(VPGXG) 40治疗组：糖尿病鼠每天上午8:00左右皮下注射rhFGF21-(VPGXG) 40融合蛋白(溶剂为PBS缓冲液)，单次注射剂量为1mg/kg/d rhFGF21-(VPGXG) 40融合蛋白(以总蛋白计)，单次注射体积为0.15mL。
正常对照组：健康小鼠每天上午8:00左右皮下注射PBS缓冲液，单次注射体积为0.15mL。
每2天检测一次各组小鼠血糖，结果显示，经rhFGF21-(VPGXG) 40融合蛋白治疗3天，该组小鼠的血糖水平较模型组显著下降，治疗6天后血糖维持在较低的水平并接近正常组小鼠血糖水平。持续给药30天后，OGTT检测各组小鼠的糖耐受水平。结果显示，口服葡萄糖后30min，模型组小鼠的血糖显著上升，且30min后下降比较缓慢，rhFGF21-(VPGXG) 40治疗组小鼠的血糖显著低于模型组，并且血糖在60min已降至接近正常组小鼠血糖(图5)。结果表明，rhFGF21-(VPGXG) 40融合蛋白治疗后，小鼠的葡萄糖耐受得到显著的改善。在58天的注射期间，模型组小鼠的血糖始终维持较高的水平，rhFGF21-(VPGXG) 40治疗组小鼠的血糖较模型组显著下降，治疗后血糖维持在较低的水平并接近正常组小鼠的血糖水平，且药效比rhFGF21更持久(图6)。模型组小鼠的血清胰岛素水平较正常组显著下降，经rhFGF21-(VPGXG) 40融合蛋白治疗后，血清胰岛素含量较模型组得到显著的提高(图7)。模型组小鼠的糖化血红蛋白水平较正常组显著上升，rhFGF21-(VPGXG) 40融合蛋白治疗后，糖化血红蛋白较模型组得到显著的降低(图8)。通过实时荧光定量PCR和western印迹检测肝脏G6Pase和PCK表达量，结果显示，模型组小鼠的G6Pase和PCK表达量较正常组显著上升；rhFGF21-(VPGXG) 40治疗后，小鼠的G6Pase和PCK表达量较模型组显著下降(图9至图12)。此外，利用HE染色和免疫组化评价胰岛的损伤情况。结果显示，模型组小鼠的胰岛损伤严重，分泌胰岛素的量显著下降；rhFGF21-(VPGXG) 40治疗组小鼠的胰岛的损伤情况得到显著的改善，分泌胰岛素的量显著提高(图13)。
实施例4人成纤维细胞生长因子21融合蛋白的稳定储存条件的确定
1、rhFGF21-(VPGXG) 40蛋白的缓冲溶液的确定
分别配制如下缓冲液：醋酸缓冲溶液pH 4.0、pH 5.0(将醋酸溶于水配制)；柠檬酸盐缓冲溶液pH 5.0、pH 5.5、pH 6.0(将柠檬酸和柠檬酸钠溶于水配制)；磷酸盐缓冲溶液pH 6.5、pH 7.0(将磷酸氢二钠和磷酸二氢钠溶于水配制)；Tris-HCl缓冲溶液pH 7.5、pH 8.0(将Tris-HCl溶于水配制)。将HiPrepTM 26/10Desalting脱盐柱连接到AKTA purifier100系统中，先用8-10倍柱体积的蒸馏水冲掉脱盐柱中的保护液(20％乙醇)，再用8-10倍柱体积的上述目的缓冲溶液平衡脱盐柱，上载样品(处于pH 7.4的磷酸盐缓冲液中的浓度为2mg/mL的rhFGF21-(VPGXG) 40蛋白的溶液)。紫外曲线先上升，当紫外曲线下降至基线后电导曲线上升，利用收集器将相应紫外吸收峰处的蛋白收集，即获得缓冲溶液置换后的rhFGF21-(VPGXG) 40蛋白。将缓冲溶液置换后的9组rhFGF21-(VPGXG) 40蛋白置于25℃恒温箱中避光保存，每隔一段时间进行蛋白保存的外观检查并分别取样进行15％SDS-PAGE电泳及HPLC检测(PBS缓冲液，1mL/min，色谱柱：Agilent AdvanceBio SEC 300
Figure PCTCN2021096292-appb-000011
，2.7μm，7.8×300mm，货号：PL1180-5301)；并以于-80℃保存的rhFGF21-(VPGXG) 40蛋白水溶液作为对照。
结果显示，刚置换到pH 4.0的醋酸盐缓冲溶液中，rhFGF21-(VPGXG) 40蛋白就出现了肉眼可见的浑浊现象，在pH 4.5的醋酸盐缓冲溶液中在第7天出现了浑浊；在pH 8.0的Tris-HCl缓冲溶液中，在第三天出现了浑浊现象，在pH 7.5的Tris-HCl缓冲溶液中，在第7天出现了浑浊；任一pH的柠檬酸盐缓冲溶液和磷酸盐缓冲溶液在14天时，都没有发现肉眼可见的浑浊现象。分别取不同缓冲液保存的蛋白进行15％ SDS-PAGE分析、HPLC检测。由15％SDS-PAGE的结果可以看出：虽然两种pH的磷酸盐缓冲溶液保存的rhFGF21-(VPGXG) 40蛋白溶液都没有肉眼可见的浑浊现象，但都出现了大部分的蛋白降解；保存在pH 6.0柠檬酸盐缓冲溶液中的rhFGF21-(VPGXG) 40蛋白也有大部分的蛋白降解，相比之下，保存在pH 5.0和5.5的柠檬酸盐缓冲溶液中的rhFGF21-(VPGXG) 40蛋白有相对较少降解发生。由HPLC结果可知，以于-80℃保存的未加保护剂的rhFGF21-(VPGXG) 40蛋白的水溶液作为对照，只有pH 5.0和pH 5.5的柠檬酸盐缓冲溶液中保存的rhFGF21-(VPGXG) 40蛋白保持了较好的完整性(图14)。而且结果发现：pH 5.5的柠檬酸盐缓冲溶液对rhFGF21-(VPGXG) 40蛋白的保护作用略好于pH 5.0的柠檬酸盐缓冲溶液。
2、rhFGF21-(VPGXG) 40蛋白的保护剂的确定
分别用蒸馏水配制如下保护剂溶液：蔗糖溶液(25mg/mL、50mg/mL、100mg/mL)、海藻糖溶液(25mg/mL、50mg/mL、100mg/mL)、甘氨酸溶液(50mmol/L、100mmol/L、150mmol/L)、精氨酸溶液(50mmol/L、100mmol/L、150mmol/L)、组氨酸溶液(25mmol/L、50mmol/L、100mmol/L)、甘露醇溶液(2.5％、5％、7.5％)、山梨醇溶液(5％、10％、15％)、NaCl溶液(50mg/mL、100mg/mL、150mg/mL)、右旋糖酐溶液(2％、3％、5％)。各保护剂溶液置换方法如本实施例第1部分所述，利用收集器将相应紫外吸收峰处的蛋白收集，即获得保护剂溶液置换后的rhFGF21-(VPGXG) 40蛋白。将各组保护剂溶液置换后的rhFGF21-(VPGXG) 40蛋白置于25℃恒温箱中避光保存，每隔一段时间分别取样进行15％SDS-PAGE电泳及HPLC检测(PBS缓冲液，1mL/min，色谱柱：Agilent AdvanceBio SEC 300
Figure PCTCN2021096292-appb-000012
，2.7μm，7.8×300mm，货号：PL1180-5301)，并以于-80℃保存的未加保护剂的rhFGF21-(VPGXG) 40蛋白水溶液作为对照。
结果显示，由15％SDS-PAGE蛋白电泳图可以看出：在其它保护剂溶液中保存的蛋白在25℃保存25天后基本已经全部降解；而在精氨酸溶液中的rhFGF21-(VPGXG) 40蛋白在25℃保存25天后，发生相对较少的降解，仍能够具有很好的完整性，可见，精氨酸水溶液对融合蛋白的稳定性起到了很好的保护作用，并且三种浓度的精氨酸水溶液的保护效果的差别并不明显，为了更好的比较，对上述三种浓度的精氨酸水溶液中保存的融合蛋白进行HPLC分析(PBS缓冲液，1mL/min，色谱柱：Agilent AdvanceBio SEC 300
Figure PCTCN2021096292-appb-000013
2.7μm，7.8×300mm，货号：PL1180-5301)。由HPLC的结果可以看出，精氨酸水溶液对rhFGF21-VPGXG 40蛋白在25℃的稳定性起到了很大的保护作用，且三种浓度的精氨酸水溶液的保护效果差别不大(图15)。
3、rhFGF21-(VPGXG) 40蛋白的储存溶液的确定
将精氨酸、柠檬酸和柠檬酸钠溶于水分别配制pH 5.5和pH 5.0的精氨酸(50mmol/L)+柠檬酸盐缓冲溶液。rhFGF21-(VPGXG) 40蛋白的储存溶液置换方法如本实施例第1部分所述，利用收集器将相应紫外吸收峰处的蛋白收集，即获得储存溶液置换后的rhFGF21-(VPGXG) 40蛋白。将各组蛋白储存溶液置换后的rhFGF21-VPGXG 40蛋白分成2等份，一份置于25℃恒温箱中避光保存；另一份置于4℃展示柜中避光保存。每隔一段时间分别取样进行15％SDS-PAGE电泳及HPLC检测(PBS缓冲液，1mL/min，色谱柱：Agilent AdvanceBio SEC 300
Figure PCTCN2021096292-appb-000014
2.7μm，7.8×300mm，货号：PL1180-5301)，并以于-80℃保存的蛋白水溶液作为对照。
结果显示，将精氨酸和柠檬酸盐缓冲溶液混合后用于保存蛋白时，对蛋白的保存效果提高。在只有柠檬酸盐缓冲溶液保存的情况下，25℃放置14天时蛋白发生了降解。在只有精氨酸保存的情况下，在25℃放置25天后发生了降解。但是在精氨酸+柠檬酸盐缓冲溶液中，在25℃保存了35天后，保存在pH 5.0的精氨酸(50mmol/L)+柠檬酸盐缓冲溶液中的蛋白才有降解，而pH 5.5的精氨酸(50mmol/L)+柠檬酸盐缓冲溶液此时仍没有看到降解现象的发生。为了更精细地确定pH 5.5的精氨酸+柠檬酸盐缓冲溶液对蛋白的保护效果，将其与-80℃保存的蛋白进行HPLC分析(PBS缓冲液，1mL/min，色谱柱：Agilent AdvanceBio SEC 300
Figure PCTCN2021096292-appb-000015
2.7μm，7.8×300mm，货号：PL1180-5301)和比较，结果可以看出两者均几乎没有变化，这说明了将蛋白保存在pH 5.5的精氨酸+柠檬酸盐缓冲溶液中明显提高了蛋白的稳定性，在35天的保存期间保持了蛋白的物理及化学的稳定性(图16)。将蛋白在上述两种精氨酸+柠檬酸盐缓冲溶液中在4℃保存60天后，两者的SDS-PAGE电泳的结果看不出明显区别，均未显示出降解的带。但是经过HPLC检测(PBS缓冲液，1mL/min，色谱柱：Agilent AdvanceBio SEC 300
Figure PCTCN2021096292-appb-000016
2.7μm，7.8×300mm，货号：PL1180-5301)后可以看出，由pH 5.0的精氨酸+柠檬酸盐缓冲溶液保存的蛋白仅发生少量的降解(即，降解的带经SDS-PAGE电泳未分开)；而由pH 5.5的精氨酸+柠檬酸盐缓冲溶液保存的蛋白，经HPLC检测(PBS缓冲液，1mL/min，色谱柱：Agilent AdvanceBio SEC 300
Figure PCTCN2021096292-appb-000017
2.7μm，7.8×300mm，货号：PL1180-5301)未发现降解现象(图17)。可见，采用上述两种精氨酸+柠檬酸盐缓冲溶液在25℃保存35天或在4℃保存60天均能较好地保持蛋白的物理和化学稳定性；相对而言，pH 5.5的精氨酸+柠檬酸盐缓冲溶液能更好地保持蛋白的物理和化学稳定性。
4、蛋白储存溶液中的rhFGF21-(VPGXG) 40蛋白的活性检测
向HepG2细胞中加入在-80℃及25℃储存35天后的处于上述储存溶液中的rhFGF21-(VPGXG) 40蛋白， 在37℃、0.5％CO 2下孵育培养24h后，运用GOD-POD法葡萄糖检测试剂盒检测培养基(DMEM，gibco，C11965500BT)中剩余的葡萄糖含量。
结果显示，25℃储存的蛋白对细胞的葡萄糖吸收仍有很好的促进作用，且其促进作用呈现出剂量依赖关系；25℃储存的蛋白的3个剂量(10nmol、100nmol、1000nmol)的促进作用与相应剂量(10nmol、100nmol、1000nmol)的-80℃保存的蛋白相比，虽然都略微下降，但是它们之间的差异并不显著，说明在上述储存溶液中于25℃保存的蛋白保持很好的生物学活性(图18)。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请者可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。
本领域技术人员将认识到，本申请的范围并不限于上文描述的各种具体实施方式和实施例，而是能够在不脱离本申请的精神的情况下，进行各种修改、替换、或重新组合，这都落入了本申请的保护范围内。

Claims (15)

  1. 一种重组人成纤维细胞生长因子21融合蛋白，其中，所述融合蛋白包括融合在一起的rhFGF21和(VPGXG) n，所述rhFGF21包含以SEQ ID NO:1示出的氨基酸序列或与SEQ ID NO:1具有至少80％一致性的氨基酸序列；X为A和/或V；n为选自20-80中的整数。
  2. 如权利要求1所述的融合蛋白，其中，X为A和V，且A与V比例为(1-3):(1-3)、优选2:3；
    优选地，n为40-60中的整数、优选40。
  3. 如权利要求1或2所述的融合蛋白，其中，所述融合蛋白由融合在一起的rhFGF21和(VPGXG) n以及任选的接头组成；
    优选地，所述接头的氨基酸序列为RS和/或GS；
    优选地，所述融合蛋白具有以SEQ ID NO:2示出的氨基酸序列或与SEQ ID NO:2具有至少80％一致性的氨基酸序列。
  4. 一种编码权利要求1-3中任一项所述的融合蛋白的多核苷酸；
    优选地，编码所述融合蛋白中的rhFGF21的多核苷酸部分包含以SEQ ID NO:3示出的核苷酸序列或与SEQ ID NO:3具有至少80％一致性的核苷酸序列；
    优选地，编码所述融合蛋白中的接头的多核苷酸部分包含以SEQ ID NO:4和/或SEQ ID NO:5示出的核苷酸序列，或与SEQ ID NO:4和/或SEQ ID NO:5具有至少80％一致性的核苷酸序列。
  5. 如权利要求4所述的多核苷酸，其中，所述多核苷酸包含以SEQ ID NO:6示出的核苷酸序列或与SEQ ID NO:6具有至少80％一致性的核苷酸序列。
  6. 一种含有权利要求4或5所述的多核苷酸的重组表达载体；
    优选地，所述重组表达载体为原核重组表达载体；
    更优选地，所述重组表达载体为携带分子伴侣的原核重组表达载体；
    进一步优选地，所述分子伴侣为小分子泛素样修饰蛋白；
    或者进一步优选地，所述重组表达载体为重组的pSUMO载体或重组的PET系列载体。
  7. 一种含有权利要求6所述的重组表达载体或者在其基因组中整合有权利要求4或5所述的多核苷酸的重组细胞；
    优选地，用于构建所述重组细胞的宿主细胞为原核宿主细胞；
    更优选地，所述原核宿主细胞选自大肠杆菌、农杆菌、金黄色葡萄球菌、白色葡萄球菌、嗜酸乳杆菌、炭疽芽孢杆菌、枯草芽孢杆菌或苏云金芽孢杆菌。
  8. 一种药物组合物，其中，所述药物组合物包含权利要求1-3中任一项所述的融合蛋白；
    优选地，所述药物组合物进一步包含药学上可接受的辅料。
  9. 一种试剂盒，其中，所述试剂盒包含权利要求1-3中任一项所述的融合蛋白或者权利要求6所述的药物组合物；
    优选地，所述试剂盒进一步包含pH 5.0-5.5的柠檬酸盐缓冲溶液；
    优选地，所述试剂盒进一步包含精氨酸水溶液、优选50mmol/L-150mmol/L的精氨酸水溶液；
    优选地，所述试剂盒进一步包含pH 5.0-5.5的精氨酸+柠檬酸盐缓冲溶液；
    优选地，所述试剂盒进一步包含精氨酸水溶液和pH 5.0-5.5的柠檬酸盐缓冲溶液，并且，所述融合蛋白或所述药物组合物、所述pH 5.0-5.5的柠檬酸盐缓冲溶液、以及所述精氨酸水溶液包装在一起或者分别单独包装。
  10. 用于预防或治疗与血糖代谢异常相关的疾病的权利要求1-3中任一项所述的融合蛋白；
    优选地，所述与血糖代谢异常相关的疾病选自糖尿病及糖尿病相关代谢疾病，更优选地，所述糖尿病相关代谢疾病选自糖尿病肾病、血脂异常、肥胖症、心血管疾病、代谢综合征、脂代谢紊乱、非酒精性脂肪肝疾病或神经系统疾病。
  11. 一种产生权利要求1-3中任一项所述的融合蛋白的方法，其中，所述方法包括：对权利要求7所述的重组细胞进行培养，然后诱导所述重组细胞进行表达并对表达产物进行纯化，得到所述融合蛋白；
    优选地，采用选自如下的诱导剂，诱导所述重组细胞进行表达：IPTG和/或乳糖；
    优选地，所述表达产物的纯化包括如下步骤：(i)在诱导表达后，将所述重组细胞进行离心以收集菌体， 将所述菌体进行破碎后离心收集上清；(ii)将所述上清进行离子交换层析，收集洗脱液；(iii)将所述洗脱液进行亲和层析，得到所述融合蛋白。
  12. 如权利要求11所述的方法，其中，所述方法还包括如下步骤：
    (1)合成编码rhFGF21的多核苷酸，并合成编码(VPGXG) 20的多核苷酸；
    (2)将上述的编码rhFGF21的多核苷酸和编码(VPGXG) 20的多核苷酸进行酶切和连接，通过PCR反应，获得编码所述融合蛋白的多核苷酸；
    (3)将编码所述融合蛋白的多核苷酸与表达载体进行酶切和连接，得到重组表达载体，将所述重组表达载体导入宿主细胞中，筛选得到所述重组细胞。
  13. 如权利要求12所述的方法，在步骤(1)中，所述编码rhFGF21的多核苷酸序列包含以SEQ ID NO:3示出的核苷酸序列或与SEQ ID NO:3具有至少80％一致性的核苷酸序列；
    优选地，在步骤(1)中，所述编码(VPGXG) 20的多核苷酸包含以SEQ ID NO:7示出的核苷酸序列或与SEQ ID NO:7具有至少80％一致性的核苷酸序列。
  14. 如权利要求12或13所述的方法，在步骤(2)中，任选在所述编码(VPGXG) 20的多核苷酸的5′端和3′端分别连接有编码选自如下的接头的多核苷酸：RS和/或GS；
    优选地，在步骤(2)中，利用BglII限制性内切酶与BamHI限制性内切酶进行所述酶切。
  15. 如权利要求12-14中任一项所述的方法，在步骤(3)中，利用BsaI限制性内切酶与BamHI限制性内切酶进行所述酶切；
    优选地，在步骤(3)中，所述表达载体选自pSUMO载体和PET系列载体；
    优选地，在步骤(3)中，所述表达载体为pSUMO载体，且所述宿主细胞为大肠杆菌Rossetta(DE3)。
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