WO2012062078A1 - N-terminal deletion variant of human fibroblast growth factor 21 and conjugate thereof - Google Patents

N-terminal deletion variant of human fibroblast growth factor 21 and conjugate thereof Download PDF

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WO2012062078A1
WO2012062078A1 PCT/CN2011/071948 CN2011071948W WO2012062078A1 WO 2012062078 A1 WO2012062078 A1 WO 2012062078A1 CN 2011071948 W CN2011071948 W CN 2011071948W WO 2012062078 A1 WO2012062078 A1 WO 2012062078A1
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growth factor
fibroblast growth
human fibroblast
conjugate
terminal deletion
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French (fr)
Chinese (zh)
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范开
陈勇
陈海容
张淳
梅翔
张益�
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重庆富进生物医药有限公司
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    • 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 factors [FGF]
    • C07K14/501—Fibroblast growth factors [FGF] acidic FGF [aFGF]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of DNA recombination technology and medicine. More specifically, the present invention relates to a variant of N-terminally truncated human fibroblast growth factor 21 (FGF-21), a corresponding DNA molecule, an expression vector, and covalent attachment of the variant to polyethylene glycol And use.
  • FGF-21 N-terminally truncated human fibroblast growth factor 21
  • Fibroblast Growth Factor is a structurally related protein encoded by FGF gene family members. It has many mesodermal and neuroectodermal-derived cells such as endothelial cells, fibroblasts, and smooth muscle cells. It promotes DNA synthesis and cell division, and is mainly secreted by fibroblasts, endothelial cells, bone cells, and polymorphic cells. The relative molecular weight is generally 17 ⁇ 34kD. Amino acids in the central region of FGF family members have 30%-70% homology. FGF-21 is the latest member of a FGFs found to belong to the FGF-19 subfamily. The FGF-19 subfamily includes FGF-15, FGF-19, FGF-21, and FGF-23.
  • FGF-21 is mainly expressed in mature liver and thymus. It was first isolated from mouse embryos by Nishimura et al. (Nishimura T et al., Biochim Biophys Acta, 2000, 1492: 203-206). Its amino acid sequence and FGF were first obtained. The homology between -19 and FGF-23 was 35% and 24%, respectively. Subsequently, FGF-21 mRNA was also found in pancreas, adipose tissue and muscle tissue (Inagaki T et al, Cell Metab, 2007, 5(6): 415-425).
  • the human FGF-21 gene is located on chromosome 19, and the full-length FGF-21 protein consists of a 181 amino acid mature FGF-21 protein and a N-terminal 28 amino acid signal peptide.
  • the amino acid sequence is similar to the mouse FGF-21. 75% are the same.
  • FGF-21 plays a very important role in blood sugar, blood lipid metabolism and body weight control.
  • the N-terminal region and the C-terminal region play different biological functions, that is, the N-terminal amino acid participates in the interaction with the FGF receptor, and the C-terminal amino acid participates in a mutual interaction with a protein called ⁇ -Klotho.
  • Role Micanovic R et al, J Cell Phys, 2009, 219(2): 227-234). Kharitonenkov A et al.
  • FGF-21 glucose uptake by FGF-21 is non-insulin dependent and can increase glucose uptake when co-existing with insulin (Alexei Kharitonenkov et al, Biodrugs, 2008, 22(1): 37-44) .
  • FGF-21 can improve the function of islet beta cells. After 8 weeks of continuous treatment of FGF-21, db/db mice had normal blood glucose after meal, glucose clearance rate was significantly improved, insulin transcription, expression and secretion increased, but the number of islet cells did not change.
  • FGF-21 blocks glycolipid toxicity and cytokine-mediated apoptosis, protects the number and function of islet beta cells, and inhibits glucose-mediated glucagon release (Wente W et al, Diabetes, 2006) , 55: 2470-2478).
  • injection of recombinant human FGF-21 can lower LDL cholesterol, increase HDL cholesterol, and reduce the risk of cardiovascular disease (K aritonenkov A et al, Endocrinology, 2007, 148: 774- 781).
  • no side effects such as hypoglycemia, edema and obesity, which are often found in other diabetes, have shown good application prospects and safety.
  • Lilly Company discloses the amino acid sequence of human FGF-21 and its recombinant preparation, use (US7259248, WO/2009/020802), fusion protein of human FGF-21 protein with IgG4 or HSA (US20070237768), and a human FGF21
  • the biological effects are reduced to varying degrees, in which the simple N-terminal deletion of the 4 amino acid variants remains 90% (US 7622445) o
  • the researchers of the present invention found that the recombinant human wild-type FGF-21 has poor stability and exhibits an N-terminal break.
  • the N-terminal 9 amino acid sequence of the human FGF21 protein molecule was deleted (codenamed AN9FGF-21), which has higher stability and biological activity.
  • the above-mentioned characteristics of recombinant deletion human FGF21 were confirmed by using genetic engineering recombinant protein technology to prepare the deletion type AN9FGF21, including three major expression systems of E. coli, Pichia Pastoris and mammalian cells (CHO). .
  • the inventors of the present invention found that the conjugate obtained by modifying the polyethylene glycol of the deletion variant maintains the activity of the deletion variant in vivo and improves the deletion variant in vivo. Pharmacokinetic properties.
  • the deletion variant and its conjugate are more suitable as a pharmaceutical combination for the treatment or prevention of obesity, diabetes, high glucose or hyperlipidemia. Summary of the invention
  • One of the objects of the present invention is to provide a deletion type human fibroblast growth factor 21 ( ⁇ 9 FGF-21) variant. It has the amino acid sequence signature of SEQ ID No: 1, which is compared with the sequence of wild-type human fibroblast growth factor 21 (FGF-21), and has a total of 9 amino acids, 172 amino acids, truncated at the N-terminus of HPIPDSSPL. composition.
  • FGF-21 wild-type human fibroblast growth factor 21
  • the N-terminus may carry a methionine (Met) residue, which is expressed as Met-AN9FGF-21, and its amino acid sequence is SEQ ID NO: 3.
  • the N-terminal deletion of the N-terminal amino acid human fibroblast 21 (AN9FGF-21) of the present invention has Met at the N-terminus, and does not affect its biological properties.
  • the amino acid sequence of human AN9FGF-21 (SEQ ID NO: 1): Leu Gin Phe Gly Gly Gin 3 ⁇ 41 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp 1 5 1 0 15
  • Another object of the present invention is to provide a DNA molecule encoding the sequence of the deletion human human fibroblast growth factor 21 variant (SEQ ID NO: 1), and a recombinant expression vector containing the DNA molecule.
  • the expression vector may be derived from a prokaryotic or eukaryotic organism system, and in addition to the gene sequence encoding AN9FGF-21, further comprises a regulatory sequence for expression of the protein of interest such as a promoter and a screening marker such as ampicillin, tetracycline, neomycin, G418, Zeocin, DHFR, etc.
  • Another object of the present invention is to provide a host cell for use in the transformation of the above expression vector.
  • the host cell can be an E. coli, yeast or mammalian cell.
  • Another object of the present invention is to provide a method for culturing the above-described transformed host cells and isolating and purifying the recombinant deletion type human FGF-21 variant from the culture solution.
  • Another object of the present invention is to provide an analog of human fibroblast growth factor 21 which is improved in both stability and biological activity, i.e., human AN9FGF-21.
  • the polyethylene glycol derivative is covalently linked to the N-terminal amino group or the Lys amino group of the N-terminal deletion variant of human fibroblast growth factor 21.
  • the polyethylene glycol derivative is an aldehyde-activated monomethoxypolyethylene glycol molecule, and the aldehyde group is covalently linked to the Lys amino group.
  • the polyethylene glycol derivative is characterized by a linear or branched chain or a star shape and has a molecular weight of 5 KD to 40 KD.
  • the polyethylene glycol derivative has a molecular weight of 20 KD of linear monomethoxy polyethylene glycol propionaldehyde.
  • Another object of the present invention is to provide a conjugate of an N-terminal deletion variant of human fibroblast growth factor 21.
  • the conjugate is formed by covalent attachment of an activated polyethylene glycol molecule to the N-terminal amino group or Lys amino group of human AN9FGF-21, wherein the polyethylene glycol is mainly at the 47th position in the sequence of SEQ ID NO: 1.
  • the 50th, 60th and 113th amino groups of Lys are covalently linked, wherein the polyethylene glycol molecule may be linear or branched or star-shaped, and has a molecular weight of 5KD-40KD.
  • the N-terminal deletion variant of the human fibroblast growth factor 21 and its conjugate protein are more useful for the treatment or prevention of obesity, diabetes, high fat and hyperglycemia.
  • a therapeutic or prophylactic drug or combination of drugs can be prepared by techniques known in the art at an effective dosage range.
  • injections, lyophilizates, sustained release agents, tablets or capsules can be prepared by known public formulation methods.
  • Figure 1 Recombinant human AN9FGF-21 protein expression and purification SDS-PAGE electropherogram. Wherein, M is a protein standard, 1 is uninduced, 2 is induced, 3 is purified by Phenyl, and 4 is purified by reverse phase.
  • Figure 2 PEG modification of recombinant human AN9FGF-21 and its purification SDS-PAGE electropherogram.
  • M is a protein standard
  • 1 is a pre-modification sample
  • 2 is a modified sample
  • 3 is a purified PEG-AN9FGF21.
  • Figure 3 Effect of recombinant human AN9FGF21 and PEG-AN9FGF21 on glucose uptake rate of 3T3-L1 cells (gross content is glucose content (mMol/L), abscissa is time (hour), where ⁇ is the control group, ⁇ ⁇ N9FGF21 , ⁇ is PEG-Z N9FGF21.
  • the present inventors conducted extensive and intensive research, using the preferred expression vector pET-3c and host cell BL21 (DE3) PlysS for expression and isolation of recombinant deletion-type human fibroblast growth factor 21 variant, using a molecular weight of 20KD.
  • the linear carboxy group-activated monomethoxypolyethylene glycol molecule modifies the deleted human fibroblast growth factor 21 variant.
  • the examples are for illustrative purposes only and are not to be construed as limiting the invention.
  • Example 1 Construction of recombinant human AN9FGF-21 protein expression engineering bacteria
  • the N-terminal 9-position His Pro lie Pro Asp Ser Ser Pro Leu sequence was deleted as the amino acid sequence of AN9FGF-21 (SEQ ID No: 1), and follows The codons preferred by E. coli were optimized to encode the cDNA encoding human AN9FGF-21 (SEQ ID NO: 2).
  • Entrusted Biotechnology (Dalian) Co., Ltd. for whole gene synthesis in which the Nde l restriction site (CAT ATG) was introduced at the 5' end of the cDNA, and the stop codon (TCA) and BamH I restriction site (GGA) were introduced at the 3' end. TCC), after inserting pMD-19-T simple vector, transferred to host DH5cx, the positive clone was confirmed by cDNA sequencing and the design sequence was identical, and named pMD-AN9FGF21.
  • the recombinant plasmid pMD-AN9FGF21 was extracted and digested with Nde I and BamH I.
  • the 540 bp fragment was recovered and ligated with the expression plasmid pET-3c (Invitrogen) which was also digested with Nde I and BamH I by T4 DNA ligase. .
  • CaThe CaC12 method was used to transform the E. coli host strain DH5a, and positive clones were selected.
  • the correct recombinant plasmid was identified by enzyme digestion and PCR as pET-3C- AN9FGF21 o
  • the plasmid was transformed into E. coli expression host strain by CaC12 method.
  • Example 2 Recombinant expression and preparation of recombinant human AN9FGF-21 in E. coli.
  • LA (Vg/ml Amp) agar plates were added to LB medium, and cultured overnight at 37 ° C. The lawns were picked from the overnight cultured LA plates and inoculated in LB-containing liquid culture.
  • Base tryptone 10g, yeast extract 5g, NaCl lOg, add water to lOOOmL, 121 °C, autoclave for 30min
  • lOOOmL 121 °C
  • autoclave for 30min a test tube, activate at 37 °C for 12 hours, then transfer to 1% In a 1000 mL flask containing 200 mL of LB medium, it was cultured overnight at 37 ° C to become the upper tank seed solution.
  • the upper tank seed solution was inoculated in a 5% ratio in a 30 L fermentor containing 20 L of M9+YT medium, 37 °. C culture, during the whole fermentation process, by adjusting the rotation speed, the amount of air, and the amount of pure oxygen to maintain dissolved oxygen>30%, adjust the pH with 28% ammonia water and keep at 7.0.
  • the fermentation broth was centrifuged at 8000 rpm for 10 min at 4 ° C in a frozen high speed centrifuge, and the cells were collected and stored at -20 ° C for 24 hours.
  • 10 ml of sterilizing buffer 50 mmol/LTris, 5 mmol/L EDTA, pH 10.3 per lg of bacteria, stir for 1 hour in a water bath at 30 ° C, and use ATS pressure homogenizer (Model: AH-BASIC ) 500Pa Homogenize twice and observe with a microscope. The cells are completely broken and flocculated. Then, it was centrifuged at 1000 °C for 10 min at 4 ° C, and the supernatant was collected.
  • solution B was 10 mmol/L JL (pH 8.0) and 0.5 M NaCl.
  • the target protein peak containing AN9FGF-21 (about 50% gradient) was collected.
  • C18 reverse phase chromatography The separation medium was WelchXB-C18, solution A was 5% acetonitrile and 0.1% TFA, and solution B was 75% acetonitrile (containing 0.1% TFA).
  • the elution peak of the Q column was adjusted to pH 2.0 to 3.0 with TFA, and eluted with a gradient of solution A to solution B.
  • AN9FGF-21 elution peak (approximately 35% gradient) was collected. After lyophilization, a sample of recombinant human ⁇ N9FGF-21 was formed.
  • the sample is purified by the above steps, that is, salting out, hydrophobic chromatography, ion exchange chromatography, reverse phase chromatography, and the purity is over 95%.
  • recombinant human AN9FGF-21 is soluble, and each 100 g of bacteria can be purified to prepare recombinant human ⁇ N9FGF-21 protein about 100 mg.
  • Example 3 Modification and purification of AN9FGF-21 by mPEG-ButylALD-20KD
  • the ⁇ N9FGF-21 solution was prepared from Example 2, mPEG-ButylALD-20KD (relative molecular mass 20x10 3 ), and sodium cyanoborohydride (CH3BNNa) solution as the activation of ⁇ - ⁇ 2 .
  • the AN9FGF-21 solution was dialyzed against 100 mM PB pH 5.0 and added to a final concentration of CH3BNNa of 20 mM. Take AN9FGF-21 and mPEG-ButylALD-20KD at a mass ratio of 1:2, stir at 100 r/min at 4 °C for 24 hr, and sample for SDS-PAGE electrophoresis (Fig. 3) to determine the modification ratio of PEG-AN9FGF21.
  • the modification rate reaches 75%.
  • the modified sample PEG-20-Z ⁇ N9FGF21 was diluted 5-10 times with DDW and then adjusted to pH 8.0. Further separation and purification using Source 15-Q. After equilibration with an equilibration solution (50 mM Tris-HCl pH 8.0), the sample was re-equilibrated. The elution peak of PEG-AN9FGF21 containing no unmodified AN9FGF-21 was collected by linear elution with 0-500 mM NaCl.
  • the recombinantly prepared AN9FGF-21 protein was automatically analyzed by N-terminal amino acid sequence (Edman degradation method), and the N-terminal 15 amino acid sequence was: Met-Leu-Gln-Phe-Gly-Gly-Gln-Val-Arg-Gln-Arg- Tyr-Leu-Tyr-Thr, for
  • the 15% SDS-PAGE reduction electrophoresis showed that the prepared recombinant human AN9FGF-21 was a single-electrophoresis band with a molecular weight of 19 000 Daltons.
  • the HPLC (CHPLC) C18 reverse phase column showed that the purity was greater than 98.0%.
  • 3T3-L1 preadipocytes (Shanghai Cell Biology Cell Bank) were cultured in high glucose DMEM containing 10% FBS at 37 ° C, 5% CO 2 , and added 0.5 after 2 days.
  • Glucose transport assay 3T3-L1 adipocytes in 24-well plates were simultaneously added with recombinant human AN9FGF-21, PEG-AN9FGF21 and recombinant human FGF-21 (rhFGF-21) (Beijing Ai Dibo Biotechnology Co., Ltd.) The medium (0.2% BSA high glucose DMEM) was incubated at 37 ° C for 24 hours at final concentrations of 0, 0.032, 0.16, 0.8, 4, 20 and ⁇ , respectively.
  • Glucose uptake assay using heated (37 ° C) KRP buffer (NaCl 131.2 mmol / Lol / L, KC1 4.7 mmol / Lol / L, MgS0 4 1.2 mmol / Lol / L, CaCl 2 2.5 mmol / Lol / L , NaH 2 P0 4 2.5 mmoVLol/L, pH 7.4) After washing twice; using KRP containing 1% BSA, 0.2 ⁇ ( ⁇ /hole 2-deoxy-[14C]-glucose (Beijing Institute of Atomic Energy) ( ⁇ ) The buffer was incubated for 1 hr, and lOumol/1 cytochalasin B was added to stop the glucose uptake.
  • KRP buffer NaCl 131.2 mmol / Lol / L, KC1 4.7 mmol / Lol / L, MgS0 4 1.2 mmol / Lol / L, CaCl 2 2.5 m
  • ⁇ /L cytochalasin B Another set of ⁇ /L cytochalasin B was used as the non-specific uptake rate of 2-deoxy-(14C)-glucose. All values were subtracted from this value as the glucose uptake rate (CPM, liquid scintillation counter count per minute) for each group of cells. Results showed that recombinant human AN9FGF-21 was more than human The activity of FGF-21 for extracting glucose from fat cells in vitro was about 45% higher under the same experimental conditions. The activity of PEG-AN9FGF21 to promote glucose extraction from fat cells in vitro under the same experimental conditions was about 70%.
  • Glucose utilization analysis After 3T3-L1 was induced into adipocytes (the method is the same as above), PEG-AN9FGF21 and human ⁇ N9FGF-21 samples (using low-sugar DMEM + 5% FCS) were added to prepare a final concentration of 4nM at 37 °C. The culture supernatant was taken at 12 hr, 24 hr and 48 hr in 5% C0 2 , and the glucose content in the supernatant was determined by a glucose assay kit and plotted (Fig. 3). As shown in the figure, recombinant human AN9FGF-21 and PEG-AN9FGF21 significantly promoted the utilization of glucose by 3T3-L1 cells.
  • Example 6 Pharmacokinetics of recombinant human AN9FGF-21 and PEG-AN9FGF21 in CD-I mice
  • mice Twenty-four CD-I mice were randomly divided into two groups (AN9FGF-21 group and PEG-AN9FGF21 group). Each group was injected subcutaneously at 0.4 mg/kg, and blood was taken at intervals of 0 to 144 hrs after the injection. The blood concentration of FGF-21 in the blood was measured using a FGF-21 ELISA Kit (Kang Peptide Bios) to calculate the half-life.
  • Example 7 Stability of recombinant human AN9FGF-21
  • the recombinant human AN9FGF-21 protein solution was replaced by 10 mmol/L Mol/L phosphate buffer (pH 7.0) and B 0.15 Mol/L sodium chloride by dialysis treatment to adjust the protein concentration (measured by Lowry method) to 1.0. Mg/ml. Sterile filtration was carried out in a 4 °C refrigerator and a 25 °C constant temperature chamber for stability testing. Samples were taken at different time points for in vitro activity, SDS-PAGE electrophoresis and reverse HPLC analysis.
  • Example 8 Recombinant human AN9FGF-21 expressed in Pichia pastoris
  • the cDNA sequence encoded by AN9FGF-21 was inserted into the pIC9K (AOX promoter) or pPICZ ci (GAP promoter) plasmid according to Invitrogen's instructions to construct a corresponding secretion-inducible (methanol-induced) or constitutive recombinant expression vector.
  • pIC9K AOX promoter
  • pPICZ ci GAP promoter
  • the Pichia pastoris containing the foreign gene of AN9FGF-21 is cultured to express the target protein, and recombinant AN9FGF-21 can be obtained by a common separation technique such as centrifugation, ultrafiltration, concentration, precipitation, column chromatography and the like.
  • the AN9FGF-21 protein (N-terminal no Met) can also be recombinantly expressed in the S. cerevisiae expression plasmid containing the GAL-1 gene promoter.
  • the obtained recombinant human AN9FGF-21 protein was analyzed by the method of Example 3 to prove that it also had the same biological characteristics.
  • Example 9 Hypoglycemia and hypolipidemic effects of AN9FGF-21 in a db/db mouse model
  • mice of 8 weeks old were randomly divided into three groups (AN9FGF-21 group, Met-AN9FGF-21 group and model control group), subcutaneously injected at 2 mg/kg daily for 10 days. .
  • the model control group was injected with normal saline in the same manner and volume, and the animals were routinely fed in SPF.
  • the animals were treated for blood and blood lipids (triglycerides and cholesterol).
  • the results showed that Met-AN9FGF-21 and AN9FGF-21 had the same hypoglycemic and blood lipid effects in the diabetic model animals as compared with the model group.

Abstract

Provided is a variant of human fibroblast growth factor 21 which has a deletion of 9 amino acids at N-terminal of natural wild type human fibroblast growth factor 21. Also provided is a conjugate of the variant, which is obtained by chemical modification of the variant with polyethylene glycol derivatives. Also provided is the use of the variant and conjugate in the manufacture of a medicament for the treatment or prevention of obesity, diabetes, hyperglycemia or hyperlipidemia.

Description

人成纤维细胞生长因子 21的 N末端缺失型变异体及其偶联物 技术领域  N-terminal deletion variant of human fibroblast growth factor 21 and its conjugate
本发明涉及 DNA重组技术和药物领域。 更具体地， 本发明涉及一种 N末端截短的人成 纤维细胞生长因子 21 (FGF-21 ) 的变异体， 相应的 DNA分子、 表达载体、 该变异体与聚乙 二醇的共价连接及用途。  The present invention relates to the field of DNA recombination technology and medicine. More specifically, the present invention relates to a variant of N-terminally truncated human fibroblast growth factor 21 (FGF-21), a corresponding DNA molecule, an expression vector, and covalent attachment of the variant to polyethylene glycol And use.
发明背景 Background of the invention
成纤维细胞生长因子 （Fibroblast Growth Factor, FGF) 是一类由 FGF基因家族成员编码 的结构相关的蛋白质， 对内皮细胞、成纤维细胞、平滑肌细胞等多种中胚层和神经外胚层来 源的细胞有促进 DNA合成和细胞***作用， 主要由成纤维细胞、 内皮细胞、骨细胞、 多形细 胞分泌， 相对分子量一般在 17~34kD。 FGF家族成员的中心区域氨基酸具有 30%-70%的同源 性。 FGF-21是最新被发现的一个 FGFs成员， 属于 FGF-19亚科。 FGF-19亚科包括 FGF-15、 FGF-19、 FGF-21和 FGF-23, 与其他 FGF成员不同的是， 他们以内分泌的形式对代谢起调控 作用,且不具有与肝素结合的功能。 FGF-21主要在成熟肝脏和胸腺中表达， 最早由 Nishimura 等 （Nishimura T等， Biochim Biophys Acta, 2000， 1492： 203-206) 于 2000年首次在小鼠胚 胎中分离得到， 其氨基酸序列与 FGF-19和 FGF-23的同源性分别为 35%和 24%。 随后在胰脏、 脂肪组织和肌肉组织同样发现了 FGF-21的 mRNA (Inagaki T等， Cell Metab, 2007, 5 ( 6) ： 415-425 )。人源 FGF-21基因位于 19号染色体，全长 FGF-21蛋白由 181个氨基酸的成熟 FGF-21 蛋白和位于 N端的 28个氨基酸的信号肽组成， 其氨基酸序列与鼠源 FGF-21约有 75%相同。  Fibroblast Growth Factor (FGF) is a structurally related protein encoded by FGF gene family members. It has many mesodermal and neuroectodermal-derived cells such as endothelial cells, fibroblasts, and smooth muscle cells. It promotes DNA synthesis and cell division, and is mainly secreted by fibroblasts, endothelial cells, bone cells, and polymorphic cells. The relative molecular weight is generally 17~34kD. Amino acids in the central region of FGF family members have 30%-70% homology. FGF-21 is the latest member of a FGFs found to belong to the FGF-19 subfamily. The FGF-19 subfamily includes FGF-15, FGF-19, FGF-21, and FGF-23. Unlike other FGF members, they regulate metabolism in an endocrine form and do not function in combination with heparin. FGF-21 is mainly expressed in mature liver and thymus. It was first isolated from mouse embryos by Nishimura et al. (Nishimura T et al., Biochim Biophys Acta, 2000, 1492: 203-206). Its amino acid sequence and FGF were first obtained. The homology between -19 and FGF-23 was 35% and 24%, respectively. Subsequently, FGF-21 mRNA was also found in pancreas, adipose tissue and muscle tissue (Inagaki T et al, Cell Metab, 2007, 5(6): 415-425). The human FGF-21 gene is located on chromosome 19, and the full-length FGF-21 protein consists of a 181 amino acid mature FGF-21 protein and a N-terminal 28 amino acid signal peptide. The amino acid sequence is similar to the mouse FGF-21. 75% are the same.
近年来研究发现， FGF-21对血糖、血脂的代谢和体重控制起非常重要的作用。在这些作 用过程中， 其 N端区域和 C端区域发挥着不同的生物学功能， 即 N端氨基酸参与与 FGF受体的 相互作用， C端氨基酸参与与一种叫做 β-Klotho的蛋白的相互作用 （Micanovic R等， J Cell Phys, 2009, 219(2)： 227-234) 。 Kharitonenkov A等在 2005年首次报道了重组人 FGF-21蛋白 可以刺激分化的小鼠 3T3-L1细胞和人前脂肪细胞的葡萄糖摄取 （Kharitonenkov A等， J Clin Invest, 2005, 115 (6) ： 1627-1635) 。 随后的研究进一步表明， FGF-21是通过增力口 3T3-L1 细胞和人前脂肪细胞中葡萄糖载体 -1 (GLUT-1 ) 的表达来增加非胰岛素依赖的葡萄糖摄取 的 （Amer P等， FEBS Lett, 2008, 582 ( 12) ： 1725-1730 ) 。 与胰岛素介导的快速利用葡 萄糖不同， FGF-21在葡萄糖摄取方面的效应可持续几个小时。 更重要的是， FGF-21作用下 的葡萄糖摄取是非胰岛素依赖性的，而且在与胰岛素共同存在时可以增加摄取葡萄糖的效应 (Alexei Kharitonenkov等， Biodrugs, 2008, 22 ( 1 ) ： 37-44) 。 FGF-21可以改善胰岛 β细胞 的功能。 FGF-21持续处理 8周的 db/db小鼠餐后血糖正常， 葡萄糖清除率得到明显改善， 胰岛 素的转录、表达和分泌增加， 但胰岛细胞数量没有变化。 另外， 在分离的小鼠胰岛和 INS-1E 细胞中， FGF-21可以阻止糖脂毒性和细胞因子介导的编程性细胞死亡， 保护胰岛 β细胞的数 量和功能，抑制葡萄糖介导的胰高血糖素的释放（ Wente W等， Diabetes， 2006, 55： 2470-2478)。 在非人类灵长类糖尿病动物模型方面， 注射重组人 FGF-21可以降低 LDL胆固醇含量， 增加 HDL胆固醇含量，降低患心血管方面疾病的风险（K aritonenkov A等， Endocrinology, 2007， 148： 774-781 ) 。 在目前进行的重组人 FGF-21动物体内药效学实验中， 未发现有低血糖、 水 肿和肥胖增多等其他糖尿病经常出现的副反应出现， 显示出了良好的应用前景和安全性。 In recent years, studies have found that FGF-21 plays a very important role in blood sugar, blood lipid metabolism and body weight control. During these processes, the N-terminal region and the C-terminal region play different biological functions, that is, the N-terminal amino acid participates in the interaction with the FGF receptor, and the C-terminal amino acid participates in a mutual interaction with a protein called β-Klotho. Role (Micanovic R et al, J Cell Phys, 2009, 219(2): 227-234). Kharitonenkov A et al. first reported in 2005 that recombinant human FGF-21 protein can stimulate glucose uptake in differentiated mouse 3T3-L1 cells and human preadipocytes (Kharitonenkov A et al, J Clin Invest, 2005, 115 (6): 1627- 1635). Subsequent studies further showed that FGF-21 increases non-insulin-dependent glucose uptake by increasing the expression of glucose carrier-1 (GLUT-1) in 3T3-L1 cells and human preadipocytes (Amer P et al, FEBS Lett) , 2008, 582 ( 12) : 1725-1730 ). Unlike insulin-mediated rapid glucose utilization, the effect of FGF-21 on glucose uptake can last for several hours. More importantly, glucose uptake by FGF-21 is non-insulin dependent and can increase glucose uptake when co-existing with insulin (Alexei Kharitonenkov et al, Biodrugs, 2008, 22(1): 37-44) . FGF-21 can improve the function of islet beta cells. After 8 weeks of continuous treatment of FGF-21, db/db mice had normal blood glucose after meal, glucose clearance rate was significantly improved, insulin transcription, expression and secretion increased, but the number of islet cells did not change. In addition, in isolated mouse islets and INS-1E In cells, FGF-21 blocks glycolipid toxicity and cytokine-mediated apoptosis, protects the number and function of islet beta cells, and inhibits glucose-mediated glucagon release (Wente W et al, Diabetes, 2006) , 55: 2470-2478). In the non-human primate diabetes animal model, injection of recombinant human FGF-21 can lower LDL cholesterol, increase HDL cholesterol, and reduce the risk of cardiovascular disease (K aritonenkov A et al, Endocrinology, 2007, 148: 774- 781). In the current pharmacodynamic experiments of recombinant human FGF-21 animals, no side effects such as hypoglycemia, edema and obesity, which are often found in other diabetes, have shown good application prospects and safety.
Lilly 公司公开了人 FGF-21 的氨基酸序列和重组制备、 用途 （ US7259248， WO/2009/020802), 人 FGF-21蛋白与 IgG4或 HSA的融合蛋白专利 （US20070237768), 同 时公布了一种人 FGF21的 N端的缺失 4个氨基酸的突变体以及 153位 Leu, 167位 Ala和 118位及 134位 Cys的突变体。但生物效应不同程度降低， 其中单纯 N端缺失 4位氨基酸变 异体活性保留 90% (US 7622445 ) o  Lilly Company discloses the amino acid sequence of human FGF-21 and its recombinant preparation, use (US7259248, WO/2009/020802), fusion protein of human FGF-21 protein with IgG4 or HSA (US20070237768), and a human FGF21 The N-terminal deletion of the 4 amino acid mutant and the 153 Leu, 167 Ala and 118 and 134 Cys mutants. However, the biological effects are reduced to varying degrees, in which the simple N-terminal deletion of the 4 amino acid variants remains 90% (US 7622445) o
本发明研究人员发现， 重组人野生型 FGF-21 的稳定性差， 表现为 N末端断裂。 将人 FGF21蛋白分子的 N末端 9个氨基酸序列缺失后（代号为 AN9FGF-21 )，具有更高的稳定性 和生物活性。 通过采用基因工程重组蛋白技术制备缺失型 AN9FGF21， 包括大肠杆菌 （E. coli)、 酵母 （Pichia Pastoris)和哺乳动物细胞（CHO) 的三大表达体系， 均证明了重组缺失 型人 FGF21的以上特性。  The researchers of the present invention found that the recombinant human wild-type FGF-21 has poor stability and exhibits an N-terminal break. The N-terminal 9 amino acid sequence of the human FGF21 protein molecule was deleted (codenamed AN9FGF-21), which has higher stability and biological activity. The above-mentioned characteristics of recombinant deletion human FGF21 were confirmed by using genetic engineering recombinant protein technology to prepare the deletion type AN9FGF21, including three major expression systems of E. coli, Pichia Pastoris and mammalian cells (CHO). .
同时， 本发明研究人员发现， 通过对该缺失型变异体的聚乙二醇修饰后所得的偶联物， 保持了该缺失型变异体在体内的活性，改善了该缺失型变异体在体内的药物代谢动力学性质。 该缺失型变异体及其偶联物更适合作为一种药物组合用于治疗或预防肥胖、 糖尿病、 高糖或 高脂血症。 发明内容  At the same time, the inventors of the present invention found that the conjugate obtained by modifying the polyethylene glycol of the deletion variant maintains the activity of the deletion variant in vivo and improves the deletion variant in vivo. Pharmacokinetic properties. The deletion variant and its conjugate are more suitable as a pharmaceutical combination for the treatment or prevention of obesity, diabetes, high glucose or hyperlipidemia. Summary of the invention
本发明的目的之一是提供一种缺失型人成纤维细胞生长因子 21 ( ΔΝ9 FGF-21 )变异体。 它具有 SEQ ID No: 1的氨基酸序列特征， 该特征序列与野生型人成纤维细胞生长因子 21 (FGF-21 )的序列比较，在 N末端截短了 HPIPDSSPL共 9个氨基酸， 由 172个氨基酸组成。 在原核生物如大肠杆菌中重组表达时， 其 N末端可带有甲硫氨酸 （Met) 残基， 表述为 Met -AN9FGF-21 , 其氨基酸序列为 SEQ ID NO: 3。 本发明所述涉及的 N末端缺失 9位氨基酸 人成纤维细胞 21 ( AN9FGF-21 ) 其 N端带有 Met与否， 不影响其生物特性。  One of the objects of the present invention is to provide a deletion type human fibroblast growth factor 21 (ΔΝ9 FGF-21) variant. It has the amino acid sequence signature of SEQ ID No: 1, which is compared with the sequence of wild-type human fibroblast growth factor 21 (FGF-21), and has a total of 9 amino acids, 172 amino acids, truncated at the N-terminus of HPIPDSSPL. composition. When recombinantly expressed in a prokaryote such as E. coli, the N-terminus may carry a methionine (Met) residue, which is expressed as Met-AN9FGF-21, and its amino acid sequence is SEQ ID NO: 3. The N-terminal deletion of the N-terminal amino acid human fibroblast 21 (AN9FGF-21) of the present invention has Met at the N-terminus, and does not affect its biological properties.
人 AN9FGF-21的氨基酸序列 （SEQ ID NO: 1 ) ： Leu Gin Phe Gly Gly Gin ¾1 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp 1 5 1 0 15 The amino acid sequence of human AN9FGF-21 (SEQ ID NO: 1): Leu Gin Phe Gly Gly Gin 3⁄41 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp 1 5 1 0 15
Ala Gin Gin Thr Glu Ala His Leu Glu He Arg Glu Asp Gly Thr \¾1  Ala Gin Gin Thr Glu Ala His Leu Glu He Arg Glu Asp Gly Thr \3⁄41
20 25 30  20 25 30
Gly Gly Ala Ala Asp Gin Ser Pro Glu Ser Leu Leu Gin Leu Lys Ala  Gly Gly Ala Ala Asp Gin Ser Pro Glu Ser Leu Leu Gin Leu Lys Ala
35 40 45  35 40 45
Leu Lys Pro Gly \¾1 lie Gin lie Leu Gly \¾1 Lys Thr Ser Arg Phe  Leu Lys Pro Gly \3⁄41 lie Gin lie Leu Gly \3⁄41 Lys Thr Ser Arg Phe
50 55 60  50 55 60
Leu Cys Gin Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 65 70 75 SO Leu Cys Gin Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 65 70 75 SO
Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn
S5 90 95  S5 90 95
Val Tyr Gin Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn  Val Tyr Gin Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn
1 00 1 05 1 1 0  1 00 1 05 1 1 0
Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu  Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu
11 5 1 20 1 25  11 5 1 20 1 25
Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly lie Leu  Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly lie Leu
1 30 135 140  1 30 135 140
Ala Pro Gin Pro Pro Asp \¾1 Gly Ser Ser Asp Pro Leu Ser Met \¾1 145 1 50 1 55 1 60 Ala Pro Gin Pro Pro Asp \3⁄41 Gly Ser Ser Asp Pro Leu Ser Met \3⁄41 145 1 50 1 55 1 60
Gly Pro Ser Gin Gly Arg Ser Pro Ser Tjr Ala SerGly Pro Ser Gin Gly Arg Ser Pro Ser Tjr Ala Ser
Figure imgf000004_0001
Figure imgf000004_0001
本发明的另一目的是提供编码缺失型人成纤维细胞生长因子 21变异体的序列 （SEQ ID NO: 1 ) 的 DNA分子， 以及含有此 DNA分子的重组表达载体。 其表达载体可以是源于原核 生物或真核生物体系， 除含有编码 AN9FGF-21的基因序列外， 还包含目的蛋白表达的调控 序列如启动子和筛选标记如氨苄青霉素、 四环素、 新霉素、 G418、 Zeocin, DHFR等。  Another object of the present invention is to provide a DNA molecule encoding the sequence of the deletion human human fibroblast growth factor 21 variant (SEQ ID NO: 1), and a recombinant expression vector containing the DNA molecule. The expression vector may be derived from a prokaryotic or eukaryotic organism system, and in addition to the gene sequence encoding AN9FGF-21, further comprises a regulatory sequence for expression of the protein of interest such as a promoter and a screening marker such as ampicillin, tetracycline, neomycin, G418, Zeocin, DHFR, etc.
本发明的另一目的是提供一种用于上述表达载体转化的宿主细胞。该宿主细胞可以是大 肠杆菌， 酵母或哺乳动物细胞。  Another object of the present invention is to provide a host cell for use in the transformation of the above expression vector. The host cell can be an E. coli, yeast or mammalian cell.
本发明的另一目的是提供一种通过培养上述的转化宿主细胞和从培养液中分离和纯化 重组缺失型人 FGF-21变异体的方法。  Another object of the present invention is to provide a method for culturing the above-described transformed host cells and isolating and purifying the recombinant deletion type human FGF-21 variant from the culture solution.
本发明的另一目的是提供了一种稳定性和生物活性均获得提高的人成纤维细胞生长因 子 21的类似物， 即人 AN9FGF-21。  Another object of the present invention is to provide an analog of human fibroblast growth factor 21 which is improved in both stability and biological activity, i.e., human AN9FGF-21.
一种人成纤维细胞生长因子 21的 N末端缺失型变异体的偶联物， 以聚乙二醇衍生物化 学修饰人成纤维细胞生长因子 21的 N末端缺失型变异体而形成的 AN9FGF-21偶联物。  A conjugate of an N-terminal deletion variant of human fibroblast growth factor 21, an AN9FGF-21 formed by chemically modifying an N-terminal deletion variant of human fibroblast growth factor 21 with a polyethylene glycol derivative Conjugate.
聚乙二醇衍生物与人成纤维细胞生长因子 21的 N末端缺失型变异体的 N末端氨基或 Lys 氨基的共价连接。 所述的聚乙二醇衍生物为醛基活化的单甲氧基聚乙二醇分子， 醛基与 Lys氨基的共价连 接。 The polyethylene glycol derivative is covalently linked to the N-terminal amino group or the Lys amino group of the N-terminal deletion variant of human fibroblast growth factor 21. The polyethylene glycol derivative is an aldehyde-activated monomethoxypolyethylene glycol molecule, and the aldehyde group is covalently linked to the Lys amino group.
所述的聚乙二醇衍生物， 其特征为直链或分支链或星型状， 分子量为 5KD-40KD。  The polyethylene glycol derivative is characterized by a linear or branched chain or a star shape and has a molecular weight of 5 KD to 40 KD.
所述的聚乙二醇衍生物的分子量为 20KD的直链单甲氧基聚乙二醇丙醛。 本发明的另一个目的是提供人成纤维细胞生长因子 21 的 N末端缺失型变异体的偶联 物。 该偶联物是通过活化的聚乙二醇分子与人 AN9FGF-21的 N末端氨基或 Lys氨基的共价 连接而成， 其中聚乙二醇主要与 SEQ ID NO: 1序列中第 47位， 第 50位， 第 60位和第 113 位 Lys 的氨基共价连接， 其中聚乙二醇分子可以为直链或分支链或星型状， 其分子量为 5KD-40KD。  The polyethylene glycol derivative has a molecular weight of 20 KD of linear monomethoxy polyethylene glycol propionaldehyde. Another object of the present invention is to provide a conjugate of an N-terminal deletion variant of human fibroblast growth factor 21. The conjugate is formed by covalent attachment of an activated polyethylene glycol molecule to the N-terminal amino group or Lys amino group of human AN9FGF-21, wherein the polyethylene glycol is mainly at the 47th position in the sequence of SEQ ID NO: 1. The 50th, 60th and 113th amino groups of Lys are covalently linked, wherein the polyethylene glycol molecule may be linear or branched or star-shaped, and has a molecular weight of 5KD-40KD.
该人成纤维细胞生长因子 21的 N末端缺失型变异体及其偶联物蛋白更具有用于治疗或 预防肥胖、糖尿病、 高脂和高糖血症的应用价值。 为实现其药物应用价值， 在有效剂量范围 下， 通过本领域已知的技术可制备成相应治疗或预防用的药物或药物组合。根据药物的给药 方式或途径的不同， 用已知的公开制剂方法可制成注射液、 冻干剂、 缓释剂、 片剂或胶囊。 附图说明  The N-terminal deletion variant of the human fibroblast growth factor 21 and its conjugate protein are more useful for the treatment or prevention of obesity, diabetes, high fat and hyperglycemia. To achieve their pharmaceutical utility value, a therapeutic or prophylactic drug or combination of drugs can be prepared by techniques known in the art at an effective dosage range. Depending on the mode or route of administration of the drug, injections, lyophilizates, sustained release agents, tablets or capsules can be prepared by known public formulation methods. DRAWINGS
附图 1 重组人 AN9FGF-21蛋白表达和纯化 SDS-PAGE电泳图谱。 其中， M为蛋白质 标准， 1为未诱导， 2为诱导表达后， 3为经 Phenyl纯化后， 4为反相纯化后。  Figure 1 Recombinant human AN9FGF-21 protein expression and purification SDS-PAGE electropherogram. Wherein, M is a protein standard, 1 is uninduced, 2 is induced, 3 is purified by Phenyl, and 4 is purified by reverse phase.
附图 2重组人 AN9FGF-21的 PEG修饰及其纯化 SDS-PAGE电泳图谱。 其中， M为蛋 白质标准， 1为修饰前样品， 2为修饰后样品， 3为纯化后 PEG-AN9FGF21。  Figure 2 PEG modification of recombinant human AN9FGF-21 and its purification SDS-PAGE electropherogram. Wherein, M is a protein standard, 1 is a pre-modification sample, 2 is a modified sample, and 3 is a purified PEG-AN9FGF21.
附图 3重组人 AN9FGF21 和 PEG-AN9FGF21对 3T3-L1细胞葡萄糖摄取率的作用 （纵 坐标为葡萄糖含量（mMol/L)， 横坐标为时间 （小时）， 其中▲为对照组， ·为△N9FGF21 , ■ 为 PEG-Z N9FGF21。  Figure 3 Effect of recombinant human AN9FGF21 and PEG-AN9FGF21 on glucose uptake rate of 3T3-L1 cells (gross content is glucose content (mMol/L), abscissa is time (hour), where ▲ is the control group, · △N9FGF21 , ■ is PEG-Z N9FGF21.
具体实施方式  detailed description
本发明人经过广泛而深入的研究， 用优选的表达载体 pET-3c和宿主细胞 BL21 (DE3) PlysS进行重组缺失型人成纤维细胞生长因子 21 变异体的表达和分离制备， 采用分子量为 20KD 的直链由醛基活化的单甲氧基聚乙二醇分子 (mPEG-ButylALD-20KD)修饰缺失型人成 纤维细胞生长因子 21变异体。 所述实例仅为说明目的， 不应理解为对本发明的限制。  The present inventors conducted extensive and intensive research, using the preferred expression vector pET-3c and host cell BL21 (DE3) PlysS for expression and isolation of recombinant deletion-type human fibroblast growth factor 21 variant, using a molecular weight of 20KD. The linear carboxy group-activated monomethoxypolyethylene glycol molecule (mPEG-ButylALD-20KD) modifies the deleted human fibroblast growth factor 21 variant. The examples are for illustrative purposes only and are not to be construed as limiting the invention.
实施例 1 : 重组人 AN9FGF-21蛋白表达工程菌的构建  Example 1 : Construction of recombinant human AN9FGF-21 protein expression engineering bacteria
根据野生型人 FGF-21的氨基酸序列（来源于 Genebank), 将 N末端 9位 His Pro lie Pro Asp Ser Ser Pro Leu序列缺失后， 作为 AN9FGF-21的氨基酸序列 （SEQ ID No: 1 ) ，并按照 大肠杆菌偏爱的密码子， 优化设计编码相对应的人 AN9FGF-21的 cDNA ( SEQ ID NO: 2)。 委托宝生物（大连）有限公司进行全基因合成，其中在 CDNA 5'端引入 Nde l酶切位点（CAT ATG), 3'端引入终止密码子（TCA)和 BamH I酶切位点（GGA TCC) ,*** pMD-19-T simple vector后， 转入宿主菌 DH5cx， 阳性克隆经 cDNA测序验证与设计序列完全一致后， 命名为 pMD-AN9FGF21。 According to the amino acid sequence of wild type human FGF-21 (derived from Genebank), the N-terminal 9-position His Pro lie Pro Asp Ser Ser Pro Leu sequence was deleted as the amino acid sequence of AN9FGF-21 (SEQ ID No: 1), and Follow The codons preferred by E. coli were optimized to encode the cDNA encoding human AN9FGF-21 (SEQ ID NO: 2). Entrusted Biotechnology (Dalian) Co., Ltd. for whole gene synthesis, in which the Nde l restriction site (CAT ATG) was introduced at the 5' end of the cDNA, and the stop codon (TCA) and BamH I restriction site (GGA) were introduced at the 3' end. TCC), after inserting pMD-19-T simple vector, transferred to host DH5cx, the positive clone was confirmed by cDNA sequencing and the design sequence was identical, and named pMD-AN9FGF21.
人 Z\N9FGF- 21的 cDNA序列 （SEQ ID NO: 2) ： The cDNA sequence of human Z\N9FGF-21 (SEQ ID NO: 2):
GGC CCG AGC CAG GGC CGT AGC CCG AGC TAT GCG AGC -3 ' GGC CCG AGC CAG GGC CGT AGC CCG AGC TAT GCG AGC -3 '
提取 pMD-AN9FGF21重组质粒， 用 Nde I和 BamH I双酶切后， 回收 540bp左右目的 片段，用 T4 DNA连接酶与同样经 Nde I和 BamH I酶切回收的表达质粒 pET-3c ( Invitrogen) 连接。釆用 CaC12方法转化大肠杆菌宿主菌 DH5a，挑选阳性克隆， 用酶切和 PCR验证正确 的重组质粒命名为 pET-3C- AN9FGF21 o 该质粒经 CaC12方法转化大肠杆菌表达宿主菌 BL21 (DE3 ) PlysS (Novagen ) 感受态， 筛选并克隆高效表达重组人 AN9FGF-21蛋白的菌 株， 并于 -80 °C保存。 实施例 2 ： 重组人 AN9FGF-21在大肠杆菌中的重组表达及制备。 The recombinant plasmid pMD-AN9FGF21 was extracted and digested with Nde I and BamH I. The 540 bp fragment was recovered and ligated with the expression plasmid pET-3c (Invitrogen) which was also digested with Nde I and BamH I by T4 DNA ligase. . CaThe CaC12 method was used to transform the E. coli host strain DH5a, and positive clones were selected. The correct recombinant plasmid was identified by enzyme digestion and PCR as pET-3C- AN9FGF21 o The plasmid was transformed into E. coli expression host strain by CaC12 method. BL21 (DE3) PlysS (Novagen) competent, screened and cloned strains that efficiently expressed recombinant human AN9FGF-21 protein, and stored at -80 °C. Example 2: Recombinant expression and preparation of recombinant human AN9FGF-21 in E. coli.
将表达最佳工程菌株划线接种于 LA (LB培养基中加入 10(Vg/ml Amp)琼脂平板， 37°C 培养过夜。 从过夜培养的 LA平板上挑取菌苔接种于含 LB液体培养基（胰蛋白胨 10g， 酵母 提取物 5g， NaCl lOg, 加水定溶至 lOOOmL, 121 °C， 高压灭菌 30min即可）试管中， 37 °C活 化 12小时， 然后按 1%的比例转接到含 200mL LB培养液的 lOOOmL三角瓶中， 37 °C培养过 夜即成为上罐种子液。将上罐种子液按 5%的比例接种于含 20L M9+YT培养液的 30L发酵罐 中， 37 °C培养， 整过发酵过程中， 通过调节转速、 通空气量、 通纯氧量来保持溶氧 >30%， 用 28%的氨水调节 pH并保持在 7.0。 培养菌液 OD600=4.0〜6.0时， 将培养温度降至 28°C， 至 菌液 OD600=8.0〜10.0时， 加入终浓度为 0.5mmol/L IPTG， 继续培养 6小时后停止发酵， 收 集菌液， 8000rpm离心 10分钟， 弃上清， 收集菌体放入 -20 °C冰箱保存备用。 The best engineered strains were streaked into LA (10 (Vg/ml Amp) agar plates were added to LB medium, and cultured overnight at 37 ° C. The lawns were picked from the overnight cultured LA plates and inoculated in LB-containing liquid culture. Base (tryptone 10g, yeast extract 5g, NaCl lOg, add water to lOOOmL, 121 °C, autoclave for 30min) in a test tube, activate at 37 °C for 12 hours, then transfer to 1% In a 1000 mL flask containing 200 mL of LB medium, it was cultured overnight at 37 ° C to become the upper tank seed solution. The upper tank seed solution was inoculated in a 5% ratio in a 30 L fermentor containing 20 L of M9+YT medium, 37 °. C culture, during the whole fermentation process, by adjusting the rotation speed, the amount of air, and the amount of pure oxygen to maintain dissolved oxygen>30%, adjust the pH with 28% ammonia water and keep at 7.0. When the culture liquid OD600=4.0~6.0 , the culture temperature was lowered to 28 ° C, until the bacterial solution OD 600 = 8.0 ~ 10.0, the final concentration was added 0.5 mmol / L IPTG, continue to culture for 6 hours, then stop the fermentation, collect the bacterial solution, centrifuge at 8000 rpm for 10 minutes, discard Clear, collect the cells and store in a refrigerator at -20 °C for later use.
发酵液用冷冻高速离心机在 4°C下 8000rpm离心 lOmin,收集菌体，置 -20°C冻存 24小时。 每 lg菌体加入 10ml破菌缓冲液（50mmol/LTris， 5mmol/L EDTA, pH10.3 ), 30°C水浴搅拌 1 小时后， 用 ATS公司的压力匀浆机 （型号： AH-BASIC ) 500Pa匀浆两次， 用显微镜观察， 菌体完全破碎， 呈絮状。 然后 4°C下 lOOOOrpm离心 10min， 收集上清。  The fermentation broth was centrifuged at 8000 rpm for 10 min at 4 ° C in a frozen high speed centrifuge, and the cells were collected and stored at -20 ° C for 24 hours. Add 10 ml of sterilizing buffer (50 mmol/LTris, 5 mmol/L EDTA, pH 10.3) per lg of bacteria, stir for 1 hour in a water bath at 30 ° C, and use ATS pressure homogenizer (Model: AH-BASIC ) 500Pa Homogenize twice and observe with a microscope. The cells are completely broken and flocculated. Then, it was centrifuged at 1000 °C for 10 min at 4 ° C, and the supernatant was collected.
上清中补入 10%的饱和硫酸铵溶液， 4°C放置 30min后， 4°C下 8000rpm离心 10min， 除 去沉淀物。 随后 Phenyl Sepharose(FF)柱层析方法； 溶液 A为 10mmol/L PB， 15%饱和硫酸铵 溶液（pH8.0 ) : 溶液 B为 10mmol/L PB (pH8.0)。 上清上样后， 用 A液复平衡， 再用 B液洗 脱收集含 AN9FGF-21 洗脱峰。 Q-Srpharose ( FF)柱层析方法： 溶液 A 为 10mmol/LJL PB The supernatant was supplemented with 10% saturated ammonium sulfate solution, placed at 4 ° C for 30 min, and centrifuged at 8000 rpm for 10 min at 4 ° C to remove the precipitate. Subsequent Phenyl Sepharose (FF) column chromatography; solution A was 10 mmol/L PB, 15% saturated ammonium sulfate solution (pH 8.0): Solution B was 10 mmol/L PB (pH 8.0). After the supernatant was applied, the solution was re-equilibrated with solution A, and then eluted with solution B to collect the elution peak containing AN9FGF-21. Q-Srpharose (FF) column chromatography method: solution A is 10mmol/L JL PB
(PH8.0 ), 溶液 B为 lOmmol/LJL ( PH8.0 ) 和 0.5M NaCl。 将巯水层析洗脱峰上样后， 用溶 液 A至溶液 B的离子梯度洗脱， 收集含 AN9FGF-21的目的蛋白峰（约 50%梯度）。 C18反相 层析方法：分离介质为 WelchXB-C18，溶液 A为 5%乙腈和 0.1%TFA，溶液 B为 75%乙腈（含 0.1%TFA)。 Q柱洗脱峰用 TFA调 PH至 2.0~3.0后上样， 用溶液 A至溶液 B的梯度进行洗脱(pH 8.0), solution B was 10 mmol/L JL (pH 8.0) and 0.5 M NaCl. After loading the hydrophobic chromatography elution peak, elution with the ion gradient of Solution A to Solution B, the target protein peak containing AN9FGF-21 (about 50% gradient) was collected. C18 reverse phase chromatography: The separation medium was WelchXB-C18, solution A was 5% acetonitrile and 0.1% TFA, and solution B was 75% acetonitrile (containing 0.1% TFA). The elution peak of the Q column was adjusted to pH 2.0 to 3.0 with TFA, and eluted with a gradient of solution A to solution B.
( 10 个柱体积）， 收集 AN9FGF-21 洗脱峰 （约 35%梯度）。 再经冷冻干燥后成重组人 △N9FGF-21的样品。 (10 column volumes), AN9FGF-21 elution peak (approximately 35% gradient) was collected. After lyophilization, a sample of recombinant human ΔN9FGF-21 was formed.
样品经过上述步骤纯化， 即盐析、疏水层析、离子交换层析、反相层析后，纯度达到 95% 以上。 经工艺研究， 重组人 AN9FGF-21 为可溶性表达， 每 100g 菌体可纯化制备重组人 △N9FGF-21蛋白 lOOmg左右 实施例 3: mPEG-ButylALD-20KD对 AN9FGF-21的修饰和纯化 The sample is purified by the above steps, that is, salting out, hydrophobic chromatography, ion exchange chromatography, reverse phase chromatography, and the purity is over 95%. Through process research, recombinant human AN9FGF-21 is soluble, and each 100 g of bacteria can be purified to prepare recombinant human △N9FGF-21 protein about 100 mg. Example 3: Modification and purification of AN9FGF-21 by mPEG-ButylALD-20KD
△N9FGF-21溶液由实例 2制备， mPEG-ButylALD-20KD (相对分子质量为 20xl03)， 氰基硼氢化钠（CH3BNNa)溶液作为 α-ΝΗ2的活化齐 ^。 AN9FGF-21溶液经对 100 mM PB pH5.0 透析后， 加入 CH3BNNa终浓度为 20 mM。 取 AN9FGF-21与 mPEG-ButylALD-20KD质量比 1:2, 4°C条件下， 100 r/min搅拌， 反应 24 hr, 取样进行 SDS-PAGE 电泳 （图 3 )， 确定 PEG-AN9FGF21的修饰比例，其修饰率达到 75%。修饰后样品 PEG- 20- Z\N9FGF21 ,用 DDW 稀释 5-10倍后， 调 pH至 8.0。采用 Source 15-Q进一步分离纯化。用平衡液（50mM Tris-HCl pH8.0)平衡后，上样再平衡。用 0-500mM的 NaCl线性洗脱， 收集不含未修饰的 AN9FGF-21 的 PEG-AN9FGF21洗脱峰。 The ΔN9FGF-21 solution was prepared from Example 2, mPEG-ButylALD-20KD (relative molecular mass 20x10 3 ), and sodium cyanoborohydride (CH3BNNa) solution as the activation of α-ΝΗ 2 . The AN9FGF-21 solution was dialyzed against 100 mM PB pH 5.0 and added to a final concentration of CH3BNNa of 20 mM. Take AN9FGF-21 and mPEG-ButylALD-20KD at a mass ratio of 1:2, stir at 100 r/min at 4 °C for 24 hr, and sample for SDS-PAGE electrophoresis (Fig. 3) to determine the modification ratio of PEG-AN9FGF21. The modification rate reaches 75%. The modified sample PEG-20-Z\N9FGF21 was diluted 5-10 times with DDW and then adjusted to pH 8.0. Further separation and purification using Source 15-Q. After equilibration with an equilibration solution (50 mM Tris-HCl pH 8.0), the sample was re-equilibrated. The elution peak of PEG-AN9FGF21 containing no unmodified AN9FGF-21 was collected by linear elution with 0-500 mM NaCl.
实施例 4: 重组人 AN9FGF-21生物学特性检测  Example 4: Recombinant human AN9FGF-21 biological characteristics test
1、 N末端氨基酸序列检测  1, N-terminal amino acid sequence detection
重组制备的 AN9FGF-21蛋白经 N端氨基酸序列自动分析 （Edman降解法） ， N末端 15个 氨基酸序列为： Met-Leu-Gln-Phe-Gly-Gly-Gln-Val-Arg-Gln-Arg-Tyr-Leu-Tyr-Thr， 为  The recombinantly prepared AN9FGF-21 protein was automatically analyzed by N-terminal amino acid sequence (Edman degradation method), and the N-terminal 15 amino acid sequence was: Met-Leu-Gln-Phe-Gly-Gly-Gln-Val-Arg-Gln-Arg- Tyr-Leu-Tyr-Thr, for
Met-AN9FGF-21 Met-AN9FGF-21
2、 分子量和纯度  2, molecular weight and purity
15% SDS-PAGE还原电泳显示制备的重组人 AN9FGF-21为分子量 19， 000道尔顿的单 一电泳带， 高效液相色谱 （HPLC) C18反相柱检测结果显示， 其纯度大于 98.0%。  The 15% SDS-PAGE reduction electrophoresis showed that the prepared recombinant human AN9FGF-21 was a single-electrophoresis band with a molecular weight of 19 000 Daltons. The HPLC (CHPLC) C18 reverse phase column showed that the purity was greater than 98.0%.
实施例 5: 体外活性测定  Example 5: In vitro activity assay
细胞培养及诱导分化： 在 37°C、 5%C02的条件下， 3T3-L1前脂肪细胞（上海细胞生物所 细胞库）在含 10%FBS 的高糖 DMEM 中培养， 2天后加入含 0.5mmol/Lol/L IBMX、 Ιμπιοΐ/L ***、 10mg/L人胰岛素的高糖 DMEM培养 48 hr,再换含 10mg/L人胰岛素的高糖 DMEM 培养 48hr， 诱导分化 8~12天的 3T3-L1细胞 90%左右呈脂肪细胞表型， 用于以下实验。 Cell culture and induced differentiation: 3T3-L1 preadipocytes (Shanghai Cell Biology Cell Bank) were cultured in high glucose DMEM containing 10% FBS at 37 ° C, 5% CO 2 , and added 0.5 after 2 days. Methyl alcohol/Lol/L IBMX, Ιμπιοΐ/L dexamethasone, 10mg/L human insulin in high glucose DMEM for 48 hr, and then changed to 10mg/L human insulin in high glucose DMEM for 48hr, induced differentiation for 8-12 days of 3T3 -L1 cells are about 90% fat cell phenotype and are used in the following experiments.
1) 葡萄糖转运分析： 24孔板中 3T3-L1脂肪细胞， 同时分别加入含重组人 AN9FGF-21、 PEG-AN9FGF21和重组人 FGF-21 (rhFGF-21 ) (北京爱迪博生物科技公司）的培养基（ 0.2%BSA 的高糖 DMEM) ， 使终浓度分别为 0、 0.032、 0.16、 0.8、 4、 20和 ΙΟΟηΜ, 37°C下培养 24小时。 葡萄糖被摄取检测：用加热（37°C )的 KRP缓冲液（NaCl 131.2 mmol/Lol/L, KC1 4.7 mmol/Lol/L, MgS04 1.2 mmol/Lol/L, CaCl2 2.5 mmol/Lol/L, NaH2P042.5 mmoVLol/L, pH 7.4)洗涤两次后； 用含有 1%BSA、 0.2μ(ή/孔 2-脱氧- 〔14C〕 -葡萄糖 （北京原子能研究所） （ΙΟΟμΙ) 的 KRP缓 冲液孵育 lhr，加入 lOumol/1细胞松弛素 B终止葡萄糖的摄取。另设一组加 ΙΟμπιοΙ/L细胞松弛素 B (cytochalasin B ) 作为 2-脱氧- ( 14C) -葡萄糖的非特异摄取率， 所有数据减去此值， 作为 各组细胞的葡萄糖摄取率（CPM， 每分钟液闪仪计数值）。 结果显示重组人 AN9FGF-21比人 FGF-21在相同实验条件下测定的体外促进脂肪细胞提取葡萄糖的活性高 45%左右， PEG-AN9FGF21在相同实验条件下测定的体外促进脂肪细胞提取葡萄糖的活性保留 70%左 右。 1) Glucose transport assay: 3T3-L1 adipocytes in 24-well plates were simultaneously added with recombinant human AN9FGF-21, PEG-AN9FGF21 and recombinant human FGF-21 (rhFGF-21) (Beijing Ai Dibo Biotechnology Co., Ltd.) The medium (0.2% BSA high glucose DMEM) was incubated at 37 ° C for 24 hours at final concentrations of 0, 0.032, 0.16, 0.8, 4, 20 and ΙΟΟηΜ, respectively. Glucose uptake assay: using heated (37 ° C) KRP buffer (NaCl 131.2 mmol / Lol / L, KC1 4.7 mmol / Lol / L, MgS0 4 1.2 mmol / Lol / L, CaCl 2 2.5 mmol / Lol / L , NaH 2 P0 4 2.5 mmoVLol/L, pH 7.4) After washing twice; using KRP containing 1% BSA, 0.2μ (ή/hole 2-deoxy-[14C]-glucose (Beijing Institute of Atomic Energy) (ΙΟΟμΙ) The buffer was incubated for 1 hr, and lOumol/1 cytochalasin B was added to stop the glucose uptake. Another set of ΙΟμπιοΙ/L cytochalasin B was used as the non-specific uptake rate of 2-deoxy-(14C)-glucose. All values were subtracted from this value as the glucose uptake rate (CPM, liquid scintillation counter count per minute) for each group of cells. Results showed that recombinant human AN9FGF-21 was more than human The activity of FGF-21 for extracting glucose from fat cells in vitro was about 45% higher under the same experimental conditions. The activity of PEG-AN9FGF21 to promote glucose extraction from fat cells in vitro under the same experimental conditions was about 70%.
葡萄糖利用分析： 取 3T3-L1诱导成脂肪细胞后 （方法同上） ， 分别加入 PEG-AN9FGF21和人 △N9FGF-21样品（用低糖的 DMEM+5%FCS )配制成终浓度 4nM，于 37°C， 5%C02 中培养 12hr、 24hr和 48hr时间点分别取培养上清，用葡萄糖测定试剂盒测定上清中葡萄糖的含量并作图（图 3 ) ， 由图可知， 重组人 AN9FGF- 21和 PEG- AN9FGF21可显著促进 3T3- L1细胞利用葡萄糖。 实例 6: 重组人 AN9FGF-21和 PEG-AN9FGF21在 CD-I小鼠体内的药代动力学 Glucose utilization analysis: After 3T3-L1 was induced into adipocytes (the method is the same as above), PEG-AN9FGF21 and human △N9FGF-21 samples (using low-sugar DMEM + 5% FCS) were added to prepare a final concentration of 4nM at 37 °C. The culture supernatant was taken at 12 hr, 24 hr and 48 hr in 5% C0 2 , and the glucose content in the supernatant was determined by a glucose assay kit and plotted (Fig. 3). As shown in the figure, recombinant human AN9FGF-21 and PEG-AN9FGF21 significantly promoted the utilization of glucose by 3T3-L1 cells. Example 6: Pharmacokinetics of recombinant human AN9FGF-21 and PEG-AN9FGF21 in CD-I mice
取 CD-I小鼠 24只， 随机分成两组 （AN9FGF-21组和 PEG-AN9FGF21组）。 每组按 0.4mg/kg皮下注射， 于注射后 0到 144hr间， 间隔多次取血。 采用 FGF-21 ELISA Kit (康肽 生物公司）测定血中 FGF-21的血药浓度， 计算半衰期。  Twenty-four CD-I mice were randomly divided into two groups (AN9FGF-21 group and PEG-AN9FGF21 group). Each group was injected subcutaneously at 0.4 mg/kg, and blood was taken at intervals of 0 to 144 hrs after the injection. The blood concentration of FGF-21 in the blood was measured using a FGF-21 ELISA Kit (Kang Peptide Bios) to calculate the half-life.
Figure imgf000009_0001
Figure imgf000009_0001
实验结果表明， 重组人 AN9FGF-21的半衰期为 4.1hr; 而 PEG-AN9FGF21的半衰期达 到 39.5hr， PEG化 AN9FGF-21的体内半衰期显著延长。  The experimental results showed that the half-life of recombinant human AN9FGF-21 was 4.1 hr; while the half-life of PEG-AN9FGF21 reached 39.5 hr, the half-life of PEGylated AN9FGF-21 was significantly prolonged.
实例 7: 重组人 AN9FGF-21的稳定性  Example 7: Stability of recombinant human AN9FGF-21
重组制备的人 AN9FGF-21蛋白溶液， 经透析处理替换为 10mmol/L Mol/L磷酸盐缓冲 (PH7.0)禾 B 0.15Mol/L氯化钠中， 调整蛋白浓度 （Lowry法测定） 为 1.0mg/ml。 无菌过滤 后分别放置 4°C冰箱和 25 °C恒温考察箱中进行稳定性考査， 于不同时间点取样做体外活性， SDS-PAGE电泳和反向 HPLC分析。  The recombinant human AN9FGF-21 protein solution was replaced by 10 mmol/L Mol/L phosphate buffer (pH 7.0) and B 0.15 Mol/L sodium chloride by dialysis treatment to adjust the protein concentration (measured by Lowry method) to 1.0. Mg/ml. Sterile filtration was carried out in a 4 °C refrigerator and a 25 °C constant temperature chamber for stability testing. Samples were taken at different time points for in vitro activity, SDS-PAGE electrophoresis and reverse HPLC analysis.
ic 25 C  Ic 25 C
1个月 3个月 6个月 12 个 1个月 2个月 3个月  1 month 3 months 6 months 12 months 1 month 2 months 3 months
月  Month
生物活性保留 98.7 954 963 942 ~ 94.5 80.2 73.1 52.4Biological activity retention 98.7 954 963 942 ~ 94.5 80.2 73.1 52.4
(%) (%)
纯度 （HPLC禾 B 98.4% 98.1% 96.3% 95.1% 93.7% 74.2% ND ND 电泳） 以上结果证明重组人 AN9FGF-21在接近生理条件下具有很强稳定性。 Purity (HPLC Wo B 98.4% 98.1% 96.3% 95.1% 93.7% 74.2% ND ND electrophoresis) The above results demonstrate that recombinant human AN9FGF-21 has strong stability under physiological conditions.
实例 8: 重组人 AN9FGF-21在毕赤酵母中表达 Example 8: Recombinant human AN9FGF-21 expressed in Pichia pastoris
将 AN9FGF-21编码的 cDNA序列按 Invitrogen公司说明书*** pIC9K (AOX启动子） 或 pPICZ ci (GAP启动子）质粒中， 构建相应的分泌诱导型 （甲醇诱导） 或组成型的重组表 达载体。 经电转化 GS115 或 X33 宿主菌后， 用 G418或 Zencin抗性筛选重组表达子。 含 AN9FGF-21外源基因的毕赤酵母菌经发酵培养表达目的蛋白， 用离心、 超滤、 浓縮、 沉淀、 柱层析等常见分离技术可获得重组 AN9FGF-21。 同样可用含 GAL-1基因启动子的酿酒酵母 表达质粒中进行重组表达 AN9FGF-21蛋白（N末端无 Met)。获得的重组人 AN9FGF-21蛋白 用实例 3的方法进行分析， 证明同样具有相同的生物特性。 The cDNA sequence encoded by AN9FGF-21 was inserted into the pIC9K (AOX promoter) or pPICZ ci (GAP promoter) plasmid according to Invitrogen's instructions to construct a corresponding secretion-inducible (methanol-induced) or constitutive recombinant expression vector. After electroporation host strain GS115 or X33, 418, or by recombinant expressor G Zencin resistance selection. The Pichia pastoris containing the foreign gene of AN9FGF-21 is cultured to express the target protein, and recombinant AN9FGF-21 can be obtained by a common separation technique such as centrifugation, ultrafiltration, concentration, precipitation, column chromatography and the like. The AN9FGF-21 protein (N-terminal no Met) can also be recombinantly expressed in the S. cerevisiae expression plasmid containing the GAL-1 gene promoter. The obtained recombinant human AN9FGF-21 protein was analyzed by the method of Example 3 to prove that it also had the same biological characteristics.
实例 9: AN9FGF-21在 db/db小鼠模型中降血糖和降血脂作用 Example 9: Hypoglycemia and hypolipidemic effects of AN9FGF-21 in a db/db mouse model
取 8周龄的 db/db小鼠共 30只， 随机分成三组（AN9FGF-21组、 Met-AN9FGF-21组和 模型对照组）， 按 2mg/kg每日皮下注射， 连续给药 10天。模型对照组按相同方法和体积注射 生理盐水， 动物在 SPF中常规喂养， 于实验结束时处理动物取血测定血糖和血脂 （甘油三脂 和胆固醇）。 结果显示， 与模型组相比 Met-AN9FGF-21和 AN9FGF-21具有相同的降低糖尿 病模型动物的血糖和血脂作用。  A total of 30 db/db mice of 8 weeks old were randomly divided into three groups (AN9FGF-21 group, Met-AN9FGF-21 group and model control group), subcutaneously injected at 2 mg/kg daily for 10 days. . The model control group was injected with normal saline in the same manner and volume, and the animals were routinely fed in SPF. At the end of the experiment, the animals were treated for blood and blood lipids (triglycerides and cholesterol). The results showed that Met-AN9FGF-21 and AN9FGF-21 had the same hypoglycemic and blood lipid effects in the diabetic model animals as compared with the model group.

Claims

权 利 要 求 Rights request
1. 一种人成纤维细胞生长因子 21的 N末端缺失型变异体，其氨基酸序列如 SEQ ID N01或 SEQ ID N03所示。 An N-terminal deletion variant of human fibroblast growth factor 21, the amino acid sequence of which is shown in SEQ ID NO: 01 or SEQ ID NO.
2. 缺失型人成纤维细胞因子 21变异体的 DNA分子，该 DNA分子含有偏码权利要求 1所示 的人成纤维细胞生长因子 21的 N末端缺失型变异体的核苷酸序列。  2. A DNA molecule of a deletion-type human fibroblast factor 21 variant comprising a nucleotide sequence of the N-terminal deletion variant of human fibroblast growth factor 21 according to claim 1.
3. 根据权利要求 3所述的 DNA分子， 其核苷酸序列如 SEQ ID N02所示。  The DNA molecule according to claim 3, which has a nucleotide sequence as shown in SEQ ID NO: 2.
4. 一种重组表达权利要求 1 的人成纤维细胞生长因子 21 的 N末端缺失型变异体的表达载 体， 该表达载体含有权利要求 2的 DNA分子和用于该 DNA分子表达的调控序列。 An expression vector for recombinantly expressing the N-terminal deletion variant of human fibroblast growth factor 21 of claim 1, which comprises the DNA molecule of claim 2 and a regulatory sequence for expression of the DNA molecule.
5. 权利要求 4所述的表达载体转化的宿主细胞， 所述宿主细胞为大肠杆菌、 酵母或哺乳动 物。 The host cell transformed with the expression vector of claim 4, which is an Escherichia coli, a yeast or a mammal.
6. 一种人成纤维细胞生长因子 21的 N末端缺失型变异体的偶联物，其特征为它是以聚乙二 醇衍生物化学修饰权利要求 1所示的人成纤维细胞生长因子 21的 N末端缺失型变异体而 形成的 AN9FGF-21偶联物。  A conjugate of an N-terminal deletion variant of human fibroblast growth factor 21, characterized in that it chemically modifies human fibroblast growth factor 21 according to claim 1 with a polyethylene glycol derivative. An AN9FGF-21 conjugate formed by an N-terminal deletion variant.
7. 根据权利要求 6所述的偶联物， 其特征是聚乙二醇衍生物与权利要求 1所示的人成纤维 细胞生长因子 21的 N末端缺失型变异体的 N末端氨基或 Lys氨基的共价连接。  The conjugate according to claim 6, characterized by comprising a polyethylene glycol derivative and an N-terminal amino group or a Lys amino group of the N-terminal deletion variant of human fibroblast growth factor 21 according to claim 1. Covalent connection.
8. 根据权利要求 7所述的偶联物， 所述的聚乙二醇衍生物为醛基活化的单甲氧基聚乙二醇 分子， 醛基与 Lys氨基的共价连接。  8. The conjugate according to claim 7, wherein the polyethylene glycol derivative is an aldehyde-activated monomethoxypolyethylene glycol molecule, and the aldehyde group is covalently linked to the Lys amino group.
9. 根据权利要求 8所述的偶联物， 所述的聚乙二醇衍生物为直链或分支链或星型状， 分子 量为 5KD-40KD。  The conjugate according to claim 8, wherein the polyethylene glycol derivative is linear or branched or star-shaped, and has a molecular weight of 5 KD to 40 KD.
10. 根据权利要求 9所述的偶联物， 所述的聚乙二醇衍生物的分子量为 20KD的直链单甲氧 基聚乙二醇丙醛。  The conjugate according to claim 9, wherein the polyethylene glycol derivative has a molecular weight of 20 KD of linear monomethoxy polyethylene glycol propionaldehyde.
11. 权利要求 1所述的一种人成纤维细胞生长因子 21 的 N末端缺失型变异体， 或权利要求 6-10 所述的偶联系在制备用于用于治疗或预防肥胖、 糖尿病、 高糖或高脂血症的药物中 的应用。  11. An N-terminal deletion variant of human fibroblast growth factor 21 according to claim 1, or an even association as claimed in claims 6-10 for use in the treatment or prevention of obesity, diabetes, high Application in drugs for sugar or hyperlipidemia.
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