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WO2018032929A1 - 一种高活性长效降糖融合蛋白及其制备方法与医药用途 - Google Patents

一种高活性长效降糖融合蛋白及其制备方法与医药用途 Download PDF

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WO2018032929A1
WO2018032929A1 PCT/CN2017/093699 CN2017093699W WO2018032929A1 WO 2018032929 A1 WO2018032929 A1 WO 2018032929A1 CN 2017093699 W CN2017093699 W CN 2017093699W WO 2018032929 A1 WO2018032929 A1 WO 2018032929A1
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fusion protein
long
mhigg1fc
exenatide
seq
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French (fr)
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谭树华
古丽丽
付剑
张永波
田清华
王玥
龚晓健
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China Pharmaceutical University
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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
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • 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
    • A61K38/22—Hormones
    • 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
    • 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
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C12N15/63—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
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention belongs to the field of biotechnology pharmacy, and particularly relates to a high activity long-acting hypoglycemic fusion protein and application thereof in preparing medicine for treating diabetes.
  • Diabetes is an endocrine and metabolic disorder characterized by hyperglycemia due to defects in insulin secretion and/or insufficient action of insulin. It is mainly classified into type 1 diabetes and type 2 diabetes. 90% to 95% of patients with diabetes are type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM).
  • NIDDM non-insulin-dependent diabetes mellitus
  • the main pathological manifestations are tissue-insensitive to insulin (insulin resistance) and due to islet function. Significantly reduced insulin secretion is relatively insufficient, resulting in persistent hyperglycemia, and may produce a variety of fatal complications.
  • the main drugs for the treatment of type 2 diabetes are sulfonylureas, biguanides, glinides, thiazolidinediones, ⁇ -glucosidase inhibitors, dipeptidyl peptidase IV (DPP-4) inhibitors, and pancreas Glucagon-like peptide-1 (GLP-1) receptor agonist and sodium-glucose cotransporter 2 (SGLT2) inhibitor and the like.
  • DPP-4 inhibitors and GLP-1 receptor agonists have the advantages of lowering blood sugar, rarely causing hypoglycemia, safety and tolerance, and also multi-systems such as digestion, central nervous system and cardiovascular system. It has a protective effect.
  • Glucagon-like peptide-1 (GLP-1) is an important endogenous insulin, which is synthesized and secreted by Langerhans cells in the small intestine. When combined with GLP-1 receptor, it stimulates islet ⁇ cells to secrete insulin and inhibit pancreatic high. Glucagon secretion, enhance tissue sensitivity to insulin, thereby reducing blood glucose levels.
  • GLP-1 is easily degraded by dipeptidylpeptidase IV (DPP-4) after its secretion into the blood, and its half-life is less than 2 min (Vilsboll T. J Clin Endocrinol Metab. 2003; 88: 220-4).
  • DPP-4 is a serine protease that specifically cleaves the GLP-1 N-terminal dipeptide to inactivate it.
  • Exendin-4 is an exogenous GLP-1 receptor polypeptide agonist found in the saliva of the scorpion venom of northwestern North America. It consists of 39 amino acids and has a GLP-1 amino acid sequence. About 53% homology. Its physiological function in mammals is similar to that of GLP-1, and it can stimulate glucose-dependent insulin secretion, that is, it only works when the body's blood sugar level is high, and it does not work when blood sugar is normal or low.
  • Exenatide was developed by Amylin and Eli Lilly in the United States in April 2005 and was launched in China in August 2009. It is the first GLP-1 receptor agonist to be marketed. Exenatide is insensitive to dipeptidylpeptidase IV (DPP-4), so its in vivo half-life is significantly increased compared to GLP-1, reaching 3.3 to 4 hours (Barnett AH. Drugs Today (Barc) .2005;41:563-78;Kolterman OG.Am J Health Syst Pharm. 2005; 62: 173-81).
  • DPP-4 dipeptidylpeptidase IV
  • Exenatide is clinically combined with sulfonylureas, metformin or thiazolidinediones to effectively control blood glucose levels in patients with type 2 diabetes, and has good safety and tolerability in the body, rarely Leading to hypoglycemia, has become the first-line treatment for type 2 diabetes, but the disadvantage is that the frequency of medication is higher, the patient needs to inject no less than 2 times a day.
  • Exenatide is an exogenous GLP-1 receptor agonist peptide, and its molecular structure and interaction with GLP-1 receptor have been studied clearly (Doyle ME. Regul Pept. 2003; 114: 153 -8; Al-Sabah S. Br J Pharmacol. 2003; 140: 339-46; Donnelly D. Br J Pharmacol. 2012; 166: 27-41.).
  • the N-terminus of Exenatide is an irregular coil, the 7-28 amino acid residue forms an ⁇ -helix, the C-terminus is an irregular hydrophilic fragment, and its N-terminus is a key region that activates the receptor signaling pathway, the middle part and C. The end is the receptor binding region. Therefore, based on the molecular structure of exenatide and its interaction with the GLP-1 receptor, it is possible to obtain an exenatide mutant with better pharmacological activity by further optimizing the structure of exenatide.
  • Immunoglobulin IgG is one of the most abundant proteins in the blood and has an in vivo half-life of up to 21 days. Therefore, human IgGFc fragments (ie, the hinge region and constant region CH2-CH3 of human IgG) in the biopharmaceutical field have been used to fuse with other active proteins or polypeptides to prolong their half-life in vivo, thereby reducing the frequency of administration and improving patient resistance. Compliance and tolerance of medications.
  • Romiplostim which was developed by Amgen, USA, is a fusion protein composed of a thrombopoietin receptor (TPO) binding peptide and human IgG1 Fc, which maintains the original binding and activates platelet-promoting.
  • TPO hormone-forming receptor
  • ITP chronic immune thrombocytopenic purpura
  • PTH-Fc PTH-Fc
  • Trebananib Amg386
  • AMG819 AMG819
  • IgG Fc fragment of human IgG
  • the present invention provides an Fc fragment of a human immunoglobulin IgG1 using an optimized mutation.
  • the modified high activity long-acting hypoglycemic fusion protein is obtained by linking exenatide or a mutant thereof to a peptide or directly to an Fc fragment of a human immunoglobulin IgG1 which is optimized for mutation.
  • the fusion protein of the invention not only has a long-term hypoglycemic effect, but also can be solublely expressed in E. coli of Escherichia coli, and the expression product can be directly isolated and purified from the bacterial supernatant liquid supernatant, which greatly simplifies. The production process improves the product yield and reduces the production cost.
  • a highly active long-acting hypoglycemic fusion protein formed by linking a highly active exenatide mutant to a peptide or directly to an Fc fragment of a human immunoglobulin IgG1 which is optimized for mutation, the Fc fragment including a person who optimizes the mutation
  • the hinge region of IgG1 and the CH2 and CH3 of the human IgG1 constant region, the amino acid sequence of the mutated human IgG1 hinge (mhIgG1 hinge region) of the optimized mutation is -SGGGGSDKTHTCPPCP-(SEQ ID NO: 6),
  • the native human IgG1 hinge region prototype sequence - VEPKSCDKTHTCPPCP- (SEQ ID NO: 5) was mutated.
  • the CH2 and CH3 sequences of the human IgG1 constant region are set forth in SEQ ID NO: 7.
  • linker peptide means a flexible peptide rich in Gly and/or Ala and/or Ser, and is between 1 and 50 amino acid residues in length, and preferably the amino acid sequence of the linker peptide is shown in SEQ ID NO: 8.
  • the high activity exenatide mutant of the present invention is mutated from the 21st Leu of wild type exenatide (SEQ ID NO: 1) to Lys (EX-L21K, SEQ. ID NO: 2), Arg (SEQ .ID NO: 3) or His (SEQ. ID NO: 4).
  • the fusion protein comprises: an exenatide mutant EX-L21K with enhanced pharmacological activity, a Gly-rich linker peptide, and a hinge region of a human IgG1 optimized for mutation (mutated human IgG1hinge, abbreviated as mhIgG1 hinge region) And human IgG1 constant region CH2-CH3.
  • the fusion protein is abbreviated as EX-L21K-mhIgG1Fc, and its amino acid sequence is shown in SEQ ID NO:11.
  • the fusion protein comprises: wild type exenatide, a Gly-rich linker peptide, a mutated human IgG1 hinge (mhIgG1 hinge region) and a human IgG1 constant region. CH2-CH3.
  • EX-mhIgG1Fc the amino acid sequence of the protein is shown in SEQ ID NO: 9.
  • the hinge region of the optimized mutant human IgG1 provided by the present invention - SGGGGSDKTHTCPPCP- (SEQ ID NO: 6) is mutated from the native human IgG1 hinge region prototype sequence - VEPKSCDKTHTCPPCP- (SEQ ID NO: 5), and the mutated human hIgG1
  • the N-terminal region of the hinge region is rich in Gly and Ser, which enhances the flexibility and hydrophilicity of the N-terminus of the hinge region, and helps maintain the biological activity and solubility of the fusion protein while eliminating The possibility that the first Cys at the N-terminus of the original hinge region forms a mismatched disulfide bond during protein expression.
  • Another object of the present invention is to provide a method for preparing the above high activity long-acting hypoglycemic fusion protein, which comprises the following steps:
  • the long-acting fusion proteins EX-mhIgG1Fc and EX-L21K-mhIgG1Fc of the present invention have a significantly lower hypoglycemic activity and hypoglycemic duration in vivo than wild-type exenatide Exendin-4, wherein EX-L21K-mh IgG1Fc The role is even better.
  • Another object of the present invention is to provide a use of the high activity long-acting hypoglycemic fusion protein of the present invention for the preparation of a blood glucose lowering drug.
  • the long-acting fusion proteins EX-mhIgG1Fc and EX-L21K-mhIgG1Fc of the present invention can be stably expressed in E. coli, and the expression product exists in the form of soluble dimer, which can be directly from E. coli.
  • the separation and purification of the supernatant of the broken wall liquid avoids the complicated downstream denaturation and renaturation process due to the formation of inclusion bodies.
  • the fusion protein consisting of wild-type exenatide and native human IgG1 Fc (hIgG1Fc) is expressed as inclusion bodies in E. coli in E. coli, and complicated downstream denaturation and renaturation processes cannot be avoided.
  • the long-acting fusion proteins EX-mhIgG1Fc and EX-L21K-mhIgG1Fc of the present invention have the remarkable advantages of high hypoglycemic activity and long duration of hypoglycemic action, which are advantageous for reducing the dosage for obtaining therapeutic effects and reducing administration.
  • Frequency, improved adherence to medication can be used to treat diabetes and other diseases that benefit from lowering blood sugar.
  • Figure 1 is a schematic view showing the structure of the high activity long-acting hypoglycemic fusion protein EX-mhIgG1Fc of the present invention.
  • A Schematic diagram of the long-acting fusion protein EX-mhIgG1 Fc of wild type exenatide (Exendin-4), which mainly comprises: starting methionine Met, wild type exenatide (Exendin-4), ligation Peptide (-GGGG-), optimized mutated human IgG1 hinge region (mutated human IgG1hinge, abbreviated as mhIgG1 hinge region), constant region CH2-CH3.
  • B Schematic diagram of the long-acting fusion protein EX-mhIgG1 Fc of wild type exenatide (Exendin-4), which mainly comprises: starting methionine Met, wild type exenatide (Exendin-4), ligation Peptide (-GGGG-), optimized mutated human IgG1 hinge region (mutated human IgG
  • FIG. 2 is a schematic view showing the structure of the high activity long-acting hypoglycemic fusion protein EX-L21K-mhIgG1Fc of the present invention.
  • A Schematic diagram of the long-acting fusion protein EX-L21K-mhIgG1Fc structure of the high-activity exenatide mutant (EX-L21K), which mainly comprises: an initiating methionine Met, a highly active exenatide mutant ( EX-L21K), ligation peptide (-GGGG-), (mutated human IgG1hinge, abbreviated as mhIgG1 hinge region), constant region (CH2-CH3).
  • EX-L21K highly active exenatide mutant
  • -GGGG- ligation peptide
  • mhIgG1 hinge region constant region
  • CH2-CH3 constant region
  • EX-L21K-mhIgG1Fc Complete amino acid sequence of EX-L21K-mhIgG1Fc and its synthetic gene sequence
  • the shade shows the portion of the highly active exenatide mutant (EX-L21K), which is underlined to show the mutated human IgG1hinge of the mutated human IgG1.
  • the asterisk is the stop codon).
  • FIG. 3 is a schematic diagram showing the structure of the long-acting fusion protein EX-hIgG1Fc of wild type exenatide (Exendin-4), which mainly comprises: starting methionine Met, wild type exenatide (Exendin-4) ), a linker peptide (-GGGG-), a hinge region of the native human IgG1 (hIgG1 hinge region), and a constant region CH2-CH3.
  • B The complete amino acid sequence of EX-hIgG1Fc and its synthetic gene sequence, shaded as the wild type exenatide (Exendin-4) part, underlined as the natural human IgG1 hinge region (hIgG1 hinge region) In part, the asterisk indicates the stop codon.
  • Figure 4 is a schematic view showing the structure of a recombinant expression plasmid of the high activity long-acting hypoglycemic fusion protein of the present invention.
  • A Schematic representation of recombinant plasmid pET-EX-mhIgG1Fc expressing the long-acting fusion protein EX-mhIgG1Fc of wild type exenatide (Exendin-4), T7promoter: T7 promoter; AP: ampicillin resistance gene; EX- mhIgG1Fc: a gene encoding the long-acting fusion protein EX-mhIgG1Fc of wild type exenatide (Exendin-4); Ori: pET21b plasmid origin of replication; lac I: lactose operon repressor protein.
  • EX-L21K Escherichia coli expressing high activity Schein peptide mutant (EX-L21K) long-acting fusion protein EX-L21K-mhIgG1Fc recombinant plasmid pET-EX-L21K-mhIgG1Fc schematic, T7 promoter: T7 promoter; AP: ampicillin resistance gene; EX-L21K- mhIgG1Fc: a gene encoding the long-acting fusion protein EX-L21K-mhIgG1Fc of the highly active exenatide mutant (EX-L21K); Ori: pET21b plasmid origin of replication; lac I: lactose operon repressor.
  • EX-L21K long-acting fusion protein EX-L21K-mhIgG1Fc recombinant plasmid pET-EX-L21K-mhIgG1Fc schematic,
  • Figure 5 is a result of electrophoresis of the highly active long-acting hypoglycemic fusion protein of the present invention.
  • EX- Sample purity of long-acting fusion protein EX-L21K-mhIgG1Fc of L21K M: low molecular weight protein Marker; Lane 1: cell supernatant; Lane 2 to 3: Protein A column through solution; Lane 4: Protein A pro And column purified sample; Lane 5: Superdex molecular sieve purified sample).
  • Figure 6 is a SEC-HPLC chromatographic method for detecting the molecular weight and purity results of the high activity long-acting hypoglycemic fusion protein of the present invention (column: Shodex PROTEIN KW-802.5, SHOWA DENKO KK, Japan.
  • the long-acting fusion protein EX-mhIgG1Fc of Exendin-4 has a retention time of 11.532 min, a corresponding retention volume of 8.07 ml, an estimated molecular weight of 54.62 kDa, and a purity of 98.296%.
  • Figure 7 is a long-acting fusion protein (EX) of wild type exenatide (Exendin-4, EX-4), wild type exenatide long-acting fusion protein (EX-mhIgG1Fc) and highly active exenatide mutant.
  • EX wild type exenatide
  • EX-mhIgG1Fc wild type exenatide long-acting fusion protein
  • Figure 8 shows wild type exenatide (Exendin-4, EX-4), wild type exenatide long-acting fusion protein (EX-mhIgG1Fc) and high activity exenatide mutant long-acting fusion protein (EX- Effect of L21K-mhIgG1Fc on blood glucose levels in BKS.Cg-Dock7 m +/+Lepr db /JNju mice after intraperitoneal administration.
  • Figure 10 shows wild type exenatide (Exendin-4, EX-4), wild type exenatide long-acting fusion protein (EX-mhIgG1Fc) and high activity exenatide mutant long-acting fusion protein (EX- Effect of L21K-mhIgG1Fc) on glucose tolerance in diabetic mice.
  • the amino acid sequence of the hinge region (mhIgG1 hinge region) of the human hIgG1 optimized for mutation is shown in SEQ. ID NO: 6, which is derived from the hinge region of the native human hIgG1 (amino acid sequence is shown in SEQ. ID NO: 5) (Edelman GM. Proc) Natl Acad Sci U S A. 1969; 63: 78-85) was mutated.
  • the amino acid of the N-terminal region of the hinge region (mhIgG1 hinge region) of the mutant human hIgG1 is rich in Gly and Ser, which enhances the N-terminus of the hinge region. Flexibility and hydrophilicity eliminate the possibility of the first Cys at the N-terminus of the original hinge region forming a mismatched disulfide bond during protein expression.
  • the underlined portion is the abrupt region.
  • VEPKSC DKTHTCPPCP SEQ. ID NO: 5
  • a linker peptide refers to a flexible peptide rich in Gly and/or Ala and/or Ser, between 1 and 50 amino acid residues in length, and the preferred linker peptide has the amino acid sequence of SEQ ID NO: 8 Shown.
  • the long-acting fusion protein EX-mhIgG1Fc of wild type exenatide Exendin-4 consists of the wild type exenatide Exendin-4, the linker peptide, and the hinge region of the optimized mutant human IgG1 (mhIgG1 hinge region) And the constant region CH2-CH3. Its structure is shown in Figure 1A.
  • the amino acid sequence (SEQ. ID NO: 9) and the coding gene sequence (SEQ. ID NO: 10) designed according to the codon preferred by E. coli are shown in Figure 1B.
  • the long-acting fusion protein EX-L21K-mhIgG1Fc of the highly active Exenatide mutant EX-L21K consists of the following components: the highly active Exenatide mutant EX-L21K, the linker peptide, and the optimized mutant human IgG1.
  • a schematic diagram of the structure is shown in Fig. 2A, and the amino acid sequence (SEQ. ID NO: 11) and the coding gene sequence (SEQ. ID NO: 12) designed according to the codon preferred by Escherichia coli are shown in Fig. 2B.
  • the long-acting fusion protein EX-hIgG1Fc of wild type exenatide Exendin-4 consists of the wild type exenatide Exendin-4, the linker peptide, the native human hIgG1 hinge region (hIgG1 hinge region) and Constant region CH2-CH3. Its structure is shown in Figure 3A.
  • the amino acid sequence (SEQ. ID NO: 13) and the coding gene sequence (SEQ. ID NO: 14) designed according to the codon preferred by E. coli are shown in Figure 3B.
  • the gene encoding the wild-type exenatide long-acting fusion protein EX-mhIgG1Fc (SEQ. ID NO: 10) was synthesized and cloned by Nanjing Jinsui Biotechnology Co., Ltd., and digested with Nde I and Hind III. It was cloned into the prokaryotic expression vector pET21b to construct the expression plasmid pET-EX-mhIgG1Fc (Fig. 4A). The sequence was verified by sequencing, and then transformed into E. coli BL21 (DE3) host strain by CaCl 2 method for expression. .
  • the colony was placed in 50 ml of LB liquid medium (containing 100 ⁇ g/ml ampicillin), and cultured at 200 rpm for 14 h at 37 °C with shaking. Transfer to 200 ml TB medium (tryptone 1.2%, yeast powder 2.4%, glycerol 0.4% (v/v), 17 mM KH 2 PO 4 , 72 mM K 2 HPO 4 ⁇ inoculated at 1% (v/v) 3H 2 O, 100 ⁇ g/ml ampicillin), cultured at 37 ° C with shaking to an OD 600nm of about 1.0, lactose was added to 1% (w/v), and induced to express for 15 h at 25 ° C with shaking at 200 rpm, and a negative control was established (ie no addition) Lactose induction).
  • LB liquid medium containing 100 ⁇ g/ml ampicillin
  • the fermentation broth was collected, centrifuged at 10,000 rpm for 10 min to collect the bacterial sludge, and the wet bacterial sludge was weighed.
  • the bacteria slurry was resuspended in PBS at a ratio of 1:15 (g/ml), and the homogenizer (AH100B, ATS Engineering Inc., Canada) was low temperature. Broken bacteria, the pressure of the homogenizer is maintained at 800-900 bar during the crushing process, and it is broken 3 times.
  • the cell disrupted solution was centrifuged at 1 ° C (12,000 rpm, 20 min), and the supernatant was subjected to 12% SDS-PAGE electrophoresis analysis. The results showed that there was a significant protein expression band at a molecular weight of about 30 KD (Fig. 5A). , the fusion protein was proved to be soluble expression.
  • Example 3 Cloning and expression of the long-acting fusion protein EX-L21K-mhIgG1Fc of the high-activity exenatide mutant
  • the gene encoding the long-acting fusion protein EX-mhIgG1Fc of wild type exenatide in Example 2 (SEQ. ID NO: 10) is a template, which is mutated by a method of overlap extension PCR site-directed mutagenesis (Ho SN. Gene. 1989; 77: 51-9) to obtain a long-acting fusion protein EX of a highly active exenatide mutant.
  • the gene encoding the L21K-mhIgG1Fc (SEQ. ID NO: 12).
  • EX-L21K-F 5'GGAAGAAGAAGCGGTTCGTAAATTCATCGAATGGCTGAAAAAC 3' (SEQ. ID NO: 17);
  • Amplification of upstream fragment A using the coding gene of EX-mhIgG1Fc (SEQ. ID NO: 10) as a template, The PCR reaction was carried out by the action of Fastpfu DNA Polymerase polymerase (TransGen Biotech product).
  • the composition of the 25 ⁇ l reaction system was as follows: outer forward primer (NdeI-EX-F) 10 pmol; reverse mutant primer (EX-L21K-R) 10 pmol; Fast pfu DNA Polymerase 2.5 units; 5 ⁇ reaction buffer 5 ⁇ l; dNTP (10 mM) Each) 0.5 ⁇ l; template plasmid DNA 0.5 ⁇ l (about 2.5 ng); made up to 25 ⁇ l with sterile water.
  • the PCR reaction conditions were as follows: denaturation at 95 ° C for 2 minutes; entering the circulation reaction: denaturation at 95 ° C for 20 seconds, annealing at 50 ° C for 20 seconds, extension at 72 ° C for 10 seconds, reaction for 30 cycles, and extension at 72 ° C for 5 minutes. After completion of the reaction, the product was identified by 2% agarose gel electrophoresis and recovered using a TaKaRa gel recovery kit.
  • Amplification of the downstream fragment B The PCR reaction was carried out under the action of Fastpfu DNA Polymerase polymerase (TransGen Biotech product) using the EX-mhIgG1Fc coding gene (SEQ. ID NO: 10) as a template.
  • the composition of the 25 ⁇ l reaction system was as follows: forward mutation primer (EX-L21K-F) 10 pmol; lateral reverse primer (HindIII-Fc-R) 10 pmol; Fast pfu DNA Polymerase 2.5 units; 5 ⁇ reaction buffer 5 ⁇ l; dNTP (10 mM) Each) 0.5 ⁇ l; template plasmid DNA 0.5 ⁇ l (about 2.5 ng); made up to 25 ⁇ l with sterile water.
  • the PCR reaction conditions were as follows: denaturation at 95 ° C for 2 minutes; entering the circulation reaction: denaturation at 95 ° C for 20 seconds, annealing at 50 ° C for 20 seconds, extension at 72 ° C for 30 seconds, reaction for 30 cycles, and extension at 72 ° C for 5 minutes.
  • the PCR product was identified by 1% agarose gel electrophoresis and recovered using a TaKaRa gel recovery kit.
  • PCR reaction was carried out under the action of Taq plus DNA Polymerase (product of TAKARA) using a mixture of upstream fragment A and downstream fragment B as a template.
  • the composition of the 50 ⁇ l reaction system was as follows: outer forward primer (NdeI-EX-F) 10 pmol; lateral reverse primer (HindIII-Fc-R) 10 pmol; Taq plus DNA Polymerase 2.5 units; 10 ⁇ Taq plus buffer (with MgCl 2 ) 5 ⁇ l; dNTP (10 mM each) 1 ⁇ l; 1 ⁇ l (about 5 ng) of a mixture of the upstream fragment A and the downstream fragment B; made up to 50 ⁇ l with sterile water.
  • the PCR reaction conditions were as follows: denaturation at 95 ° C for 3 minutes; entry into a circulation reaction: denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 1 minute, reaction for 30 cycles, and extension at 72 ° C for 5 minutes.
  • the PCR product was identified by 1% agarose gel electrophoresis and recovered using a TaKaRa gel recovery kit.
  • the obtained mutant gene was digested with Nde I and Hind III and cloned into the prokaryotic expression vector pET21b to construct the expression plasmid pET-EX-L21K-mhIgG1Fc (Fig. 4B).
  • the DNA sequence showed high activity with Esena.
  • the gene sequence (SEQ. ID NO: 12) of the long-acting fusion protein EX-L21K-mhIgG1Fc of the peptide mutant was identical.
  • the expression plasmid pET-EX-L21K-mhIgG1Fc was transformed into the E.
  • Example 2 the long-acting fusion protein EX-L21K-mhIgG1Fc of the high activity exenatide mutant was referred to the protein expression method of Example 2.
  • the cell disrupted solution was centrifuged at 4 ° C (12,000 rpm, 20 min), and the supernatant was subjected to 12% SDS-PAGE electrophoresis analysis. The results showed that there was a significant protein expression band at a molecular weight of about 30 KD (Fig. 5B), demonstrating the fusion. Protein is soluble Da.
  • the gene encoding the long-acting fusion protein EX-hIgG1Fc of wild type exenatide (SEQ. ID NO: 14) was synthesized and cloned by Nanjing Jinsui Biotechnology Co., Ltd., and digested with Nde I and Hind III.
  • the plasmid pET-EX-hIgG1Fc was cloned into the prokaryotic expression vector pET21b (Fig. 4C). After sequencing, it was transformed into E. coli BL21 (DE3) host strain by CaCl 2 method for expression.
  • the long-acting fusion protein EX-hIgG1Fc of wild type exenatide was expressed by referring to the protein expression method of Example 2. After expression, the cell disrupted solution was centrifuged at 4 ° C (12000 rpm, 20 min), and the supernatant was taken for 12% SDS-PAGE. By electrophoresis analysis, no expression protein expression band appeared at a molecular weight of about 30 KD, indicating that the fusion protein failed to be soluble.
  • the fermentation broth collects the bacterial sludge by centrifugation (10000 rpm, 10 min) at 4 ° C.
  • Wet bacteria are weighed and resuspended in PBS buffer according to the ratio of 1:15 (g/ml). And wash the slime 2 to 3 times. Resuspend the slime in 10% (w/v) ratio using a lysis buffer (PBS buffer (pH 7.4) containing 1 mM PMSF, 1 mM EDTA), ATS homogenizer (AH100B, ATS Engineering Inc., Canada)
  • the broken cells, the pressure of the homogenizer is maintained at 800-900 bar during the crushing process, and the crushing is performed 3 times.
  • the supernatant of the broken wall was collected by centrifugation (12,000 rpm, 20 min) at 4 °C.
  • Protein A affinity chromatography Balance buffer PBS (pH 7.4) fully balanced HiTrap rProtein A FF affinity chromatography prepacked column (GE products), the supernatant of the broken wall was filtered through a 0.22 ⁇ m aqueous filter. The sample was loaded at a flow rate of 0.5 ml/min. After the loading was completed, the column was washed with an equilibration buffer to wash away unbound heteroprotein. Then elute with elution buffer (0.1 M citric acid, NaOH adjusted to pH 4.0), add 450 ⁇ l of neutralization buffer per ml of collection solution (1 M Tris hydrochloric acid buffer (pH adjusted to 9.0) and glycerol 1:2 Mixed).
  • elution buffer 0.1 M citric acid, NaOH adjusted to pH 4.0
  • Ammonium sulfate precipitation After ice-cold saturated ammonium sulfate solution 0.22 ⁇ m membrane filtration, the above protein sample solution placed in an ice water bath is added dropwise at a flow rate of 1 ml/min to a concentration of 60% to precipitate the protein of interest, and magnetic stirring is performed throughout the process. Stir slowly. The precipitate was collected by centrifugation (10000 rpm, 20 min) at 4 ° C, and dissolved in an appropriate amount of PBS buffer (pH 7.4) to obtain a target protein concentrate.
  • PBS buffer pH 7.4
  • the analysis was performed on a HPLC system (LC-2010A HT, SHIMADZU Corp., Japan) by Size Exclusion Chromatography (SEC).
  • the column was Shodex PROTEIN KW-802.5 (SHOWA DENKO KK, Japan), and the mobile phase was 0.2 M phosphate buffer (pH 7.4) containing 0.1 M Na 2 SO 4 (added as required by the column instructions) at a flow rate of 0.7 ml/min and a detection wavelength of 280 nm.
  • the retention time of the wild type exenatide long-acting fusion protein EX-mhIgG1Fc was 11.532 min, as shown in (6B), the corresponding retention volume was 8.07 ml, and the molecular weight calculated according to the above formula was 54.62 kDa, with the ExPASy website ( Http://web.expasy.org/compute_pi/) The predicted dimer molecular weight is similar to 60.92 kDa.
  • the retention time of the long-acting fusion protein EX-L21K-mhIgG1Fc of the highly active exenatide mutant was 11.534 min, as shown in (6C), the corresponding retention volume was 8.07 ml, and the molecular weight calculated according to the above formula was 54.57 kDa.
  • the molecular weight of the dimer predicted by the ExPASy website (http://web.expasy.org/compute_pi/) is similar to 60.98 kDa.
  • the long-acting fusion protein EX-mhIgG1Fc of the purified wild type exenatide and the long-acting fusion protein EX-L21K-mhIgG1Fc of the high-activity exenatide mutant exist in the form of soluble dimer. .
  • Example 7 In vivo hypoglycemic test of C57BL/KsJ-db/db in type II diabetes model mice
  • mice Sixty-five-old male type 2 diabetes model mice, C57BL/KsJ-db/db (ie S.Cg-Dock7 m +/+Lepr db /JNju mice) and control mice, C57BLKS/JNju, were purchased from Nanjing. University-Nanjing Institute of Biomedical Research (Qualification No.: 201602819, License No.: SCXK (Su) 2015-0001). The mice were housed in an SPF animal room at room temperature of 25 ° C, humidity of 40-60%, and light and dark for 12 h. The experiment was carried out one week after adaptive feeding. Blood sugar concentration using Roche Performa blood glucose meter and blood glucose test strip were measured. The experimental grouping and administration methods are shown in Table 1.
  • NS physiological saline
  • Ex-4 is a positive drug exenatide acetate (Exendin-4), Shanghai Jill Biochemical Co., Ltd. product catalogue: 052143; batch number: P160102-CQ052143; molecular formula: C 184 H 282 N 50 O 60 S 1 ; purity 99.41%; molecular weight: 4186.66 Da)
  • Ex-4 and EX-L21K-mhIgG1Fc (5-20nmol/kg) group showed significant hypoglycemic effect, while EX-mhIgG1Fc showed hypoglycemic effect until 2h (P ⁇ 0.01vs model group). ), each drug group also showed strong hypoglycemic activity until 8 h of administration (P ⁇ 0.0001 vs model group).
  • the blood glucose of the mice gradually increased to the pre-dose level
  • the blood glucose of the EX-mhIgG1 Fc group gradually increased to the pre-dose level after 48 hours of administration.
  • the hypoglycemic effect of EX-L21K-mhIgG1Fc was longer lasting, and the hypoglycemic effect of 5nmol/kg EX-L21K-mhIgG1Fc continued until 72h after administration (P ⁇ 0.01vs model group), 10nmol/kg EX-L21K-mhIgG1Fc ( P ⁇ 0.01 vs model group) and 20 nmol/kg EX-L21K-mhIgG1Fc (P ⁇ 0.0001 vs model group) still had hypoglycemic effect after 96 h of administration.
  • hypoglycemic effect of Ex-4, EX-mhIgG1Fc and EX-L21K-mhIgG1Fc was evaluated by the area under the curve (AUC) method, as shown in Fig. 8, and normal control.
  • AUC area under the curve
  • the model groups AUC 0-24h , AUC 0-72h , and AUC 0-120h all increased significantly (p ⁇ 0.0001).
  • Ex-4 EX-mhIgG1Fc and EX-L21K-mhIgG1Fc all have hypoglycemic effects, of which Ex-4 has a rapid onset of action, and there is significant hypoglycemic effect after 1 hour of administration.
  • EX- L21K-mhIgG1Fc not only has hypoglycemic effect within 1 h of administration, but also lasts for a period of 96 h after administration.
  • mice in each group were fasted for 18 hours overnight.
  • intraperitoneal injection of normal saline, Ex-4, EX-mhIgG1Fc and EX-L21K-mhIgG1Fc were given.
  • each mouse was intraperitoneally injected with 1.5 g/kg glucose, tail vein. Blood was taken and blood glucose concentrations of -120, 0, 15, 30, 45, 60, 90, 120, and 180 min were recorded. The animals were fed a normal diet during the experiment.
  • the results of the IPGTT experiment are shown in Fig. 9.
  • the fasting blood glucose of the mice was significantly decreased (P ⁇ 0.0001vs model group).
  • the blood glucose of the model group was increased from 8.49 ⁇ 0.54 to 22.55 ⁇ 1.77mM and maintained at a high level, while the Ex-4, EX-mhIgG1Fc and EX-L21K-mhIgG1Fc groups were small.
  • the blood glucose increased, but quickly decreased to normal levels, and continued to maintain a low level until 180 minutes after administration did not rise to pre-sugar levels.

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Abstract

提供一种高活性长效降糖融合蛋白，由高活性艾塞那肽突变体通过连接肽或者直接与优化突变的人免疫球蛋白IgG1的Fc片段连接而成，所述优化突变的人免疫球蛋白IgG1的Fc片段包括：优化突变的人IgG1的铰链区、人IgG1恒定区CH2和CH3。

Description

一种高活性长效降糖融合蛋白及其制备方法与医药用途 技术领域
本发明属于生物技术制药领域，具体涉及一种高活性长效降糖融合蛋白及其在制备糖尿病治疗药物中的应用。
背景技术
糖尿病是一种因胰岛素分泌缺陷和/或胰岛素作用不足所致的以高血糖为主要特征的内分泌代谢紊乱性疾病，主要分为1型糖尿病和2型糖尿病。糖尿病患者中90％～95％属于2型糖尿病，又名非胰岛素依赖型糖尿病(non-insulin-dependent diabetes mellitus，NIDDM)，其主要病理表现为组织对胰岛素不敏感(胰岛素抵抗)和由于胰岛功能明显减退导致的胰岛素分泌相对不足，形成持久高血糖，并可能产生多种致命性并发症。
治疗2型糖尿病的药物主要有磺脲类、双胍类、格列奈类、噻唑烷二酮类、α-葡萄糖苷酶抑制剂、二肽基肽酶IV(DPP-4)抑制剂、胰高血糖素样肽-1(GLP-1)受体激动剂和钠–葡萄糖协同转运蛋白2(SGLT2)抑制剂等。其中，DPP-4抑制剂和GLP-1受体激动剂除具有降血糖、极少导致低血糖、安全性和耐受性较好等优点外，还对消化、中枢神经、心血管等多系统具有保护作用。
胰高血糖素样肽-1(GLP-1)是一种重要的内源性肠促胰岛素，由小肠Langerhans细胞合成分泌，与GLP-1受体结合后刺激胰岛β细胞分泌胰岛素、抑制胰高血糖素的分泌、增强组织对胰岛素的敏感性，从而降低血糖浓度。但GLP-1分泌进入血液后极易被二肽基肽酶Ⅳ(dipeptidylpeptidase 4,DPP-4)降解，半衰期不足2min(Vilsboll T.J Clin Endocrinol Metab.2003；88:220-4)。DPP-4是一种丝氨酸蛋白酶，可特异性切割GLP-1N端二肽使其失活。
艾塞那肽(Exendin-4)是一种从北美洲西北部钝尾毒蜥唾液中发现的外源性GLP-1受体多肽激动剂，由39个氨基酸组成，与GLP-1氨基酸序列具有约53％的同源性。它在哺乳动物体内的生理功能与GLP-1相似，能够刺激葡萄糖依赖性的胰岛素分泌，即只有在机体血糖浓度高时发挥作用，血糖正常或偏低时不发挥作用。
艾塞那肽于2005年4月由美国Amylin与Eli Lilly公司开发上市，并于2009年8月在中国上市，是首个上市的GLP-1受体激动剂药物。艾塞那肽对二肽基肽酶Ⅳ(dipeptidylpeptidase 4,DPP-4)不敏感，因此与GLP-1相比，其体内半衰期显著增长，达到3.3～4小时(Barnett AH.Drugs Today(Barc).2005；41:563-78；Kolterman OG.Am J Health Syst  Pharm.2005；62:173-81)。艾塞那肽在临床上与磺酰脲类药物、二甲双胍或噻唑烷二酮类药物合用，可有效控制2型糖尿病患者的血糖浓度，且在体内具有良好的安全性和耐受性，极少导致低血糖现象，目前已成为治疗2型糖尿病的一线治疗药物，但缺点是用药频率较高，病人一天需要注射不低于2次。
艾塞那肽是一种外源性的GLP-1受体激动肽，其分子结构及与GLP-1受体间的相互作用关系已研究比较清楚(Doyle ME.Regul Pept.2003；114:153-8；Al-Sabah S.Br J Pharmacol.2003；140:339-46；Donnelly D.Br J Pharmacol.2012；166:27-41.)。艾塞那肽N端是一段不规则卷曲，第7-28氨基酸残基形成α螺旋，C端是不规则亲水片断，其N端是激活受体信号传导途径的关键区域，中间部位和C端是受体结合区域。因此，基于艾塞那肽的分子结构及其与GLP-1受体之间的相互作用关系研究，对艾塞那肽结构进行进一步优化就可能获得药理活性更优的艾塞那肽突变体。
免疫球蛋白IgG是血液中最丰富的蛋白之一，其体内半衰期可长达21天。因此，在生物制药领域人的IgGFc片段(即人IgG的铰链区和恒定区CH2-CH3)已被用来与其它活性蛋白或多肽进行融合以延长其体内半衰期，从而减少用药频次，改善病人对药物治疗的依从性和耐受性。如，美国Amgen公司开发上市的Romiplostim(罗米司亭)，就是一种由血小板生成素受体(TPO)结合肽与人IgG1Fc组成的融合蛋白，它既保持了原有的结合并激活促血小板生成素(TPO)受体使血小板生成增加的功能，同时其体内半衰期也大大延长，在临床上用于治疗慢性免疫性血小板减少性紫癜(ITP)，再比如PTH-Fc、Trebananib(Amg386)、AMG819等也都是通过与人IgG的Fc片段进行融合以延长多肽药物的体内半衰期的(Kostenuik PJ.J Bone Miner Res.2007；22:1534-47；Shimamoto G.MAbs.2012；4:586-91)。由于动物细胞表达成本高、培养周期长且工艺放大困难，而大肠杆菌表达则具有表达产物均一、无糖基化修饰且发酵周期短、成本低的显著优点(Shimamoto G.MAbs.2012；4:586-91)，因此，上述Fc融合蛋白均采用大肠杆菌表达系统进行制备(Kostenuik PJ.J Bone Miner Res.2007；22:1534-47；Shimamoto G.MAbs.2012；4:586-91)。然而，由于这类Fc融合蛋白在大肠杆菌E.coli表达过程中会形成包涵体，而包涵体的下游处理工艺复杂，需要进行复杂的变性和复性过程才能形成有活性的可溶性蛋白，这给样品制备带来了较大困难(Kostenuik PJ.J Bone Miner Res.2007；22:1534-47；Shimamoto G.MAbs.2012；4:586-91；Baneyx F.Nat Biotechnol.2004；22:1399-408)。
发明内容
为了克服现有技术不足本发明提供了使用优化突变的人免疫球蛋白IgG1的Fc片段 修饰的高活性长效降糖融合蛋白，将艾塞那肽或其突变体通过连接肽或者直接与优化突变的人免疫球蛋白IgG1的Fc片段连接而成。本发明所述的融合蛋白不仅具有长效的降糖作用，还能够在大肠杆菌E.coli中进行可溶性表达，表达产物可直接从细菌破壁液上清中分离纯化得到，极大的简化了生产制备工艺，提高了产物收率，降低了生产成本。
本发明具体技术方案如下：
一种高活性长效降糖融合蛋白，由高活性艾塞那肽突变体通过连接肽或者直接与优化突变的人免疫球蛋白IgG1的Fc片段连接而成，所述Fc片段包括优化突变的人IgG1的铰链区和人IgG1恒定区的CH2和CH3，所述优化突变的人IgG1的铰链区(mutated human IgG1hinge,简称mhIgG1铰链区)的氨基酸序列为-SGGGGSDKTHTCPPCP-(SEQ ID NO:6)，由天然的人IgG1铰链区原形序列-VEPKSCDKTHTCPPCP-(SEQ ID NO:5)突变而成。所述的人IgG1恒定区的CH2和CH3序列如SEQ ID NO:7所示。
上述连接肽是指富含Gly和/或Ala和/或Ser的柔性肽，长度在1～50个氨基酸残基之间，优选连接肽的氨基酸序列如SEQ ID NO:8所示。
本发明所述高活性艾塞那肽突变体由野生型艾塞那肽(SEQ ID NO:1)的第21位Leu突变为Lys(EX-L21K，SEQ.ID NO:2)、Arg(SEQ.ID NO:3)或His(SEQ.ID NO:4)而成。
本发明的一个优选方案，该融合蛋白包括：药理活性增强的艾塞那肽突变体EX-L21K、富含Gly的连接肽、优化突变的人IgG1的铰链区(mutated human IgG1hinge,简称mhIgG1铰链区)及人IgG1恒定区CH2-CH3。该融合蛋白简称EX-L21K-mhIgG1Fc，其氨基酸序列如SEQ ID NO:11所示。
本发明的另一个优选方案，该融合蛋白包括：野生型艾塞那肽、富含Gly的连接肽、优化突变的人IgG1的铰链区(mutated human IgG1hinge,简称mhIgG1铰链区)及人IgG1恒定区CH2-CH3。简称EX-mhIgG1Fc，所述蛋白氨基酸序列如SEQ ID NO:9所示。
本发明提供的优化突变的人IgG1的铰链区-SGGGGSDKTHTCPPCP-(SEQ ID NO:6)由天然的人IgG1铰链区原形序列-VEPKSCDKTHTCPPCP-(SEQ ID NO:5)突变而成，突变后的人hIgG1的铰链区(mhIgG1铰链区)的N端区域富含Gly和Ser，这既增强了铰链区N-端的柔韧性和亲水性，且有利于保持融合蛋白的生物学活性及可溶性，同时又消除了原铰链区N端第1个Cys在蛋白表达过程中形成错配二硫键的可能性。
本发明的另一目的在于提供上述高活性长效降糖融合蛋白的制备方法，其特征在于包括如下步骤：
(1)设计合成并克隆本发明所述融合蛋白的编码基因；
(2)构建成表达质粒后转化大肠杆菌宿主菌中进行可溶性表达；
(3)收集菌泥及破壁，收集破壁液上清，分离纯化后得到如本发明所述可溶性融合蛋白。
本发明所述的长效融合蛋白EX-mhIgG1Fc和EX-L21K-mhIgG1Fc其体内降糖活性及降糖作用持续时间显著高于野生型艾塞那肽Exendin-4，其中以EX-L21K-mh IgG1Fc的作用更为优越。
本发明另一目的是提供本发明所述高活性长效降糖融合蛋白在制备降低血糖药物中的应用。
本发明所述的长效融合蛋白EX-mhIgG1Fc和EX-L21K-mhIgG1Fc可在大肠杆菌E.coli中进行可溶性表达，表达产物以可溶的二聚体形式存在，可直接从大肠杆菌E.coli破壁液上清中分离纯化得到，避免了由于形成包涵体而带来的复杂的下游变性和复性处理过程。而由野生型艾塞那肽与天然的人IgG1Fc(hIgG1Fc)组成的融合蛋白在大肠杆菌E.coli中以包涵体的形式表达，无法避免复杂的下游变性和复性处理过程。
本发明所述的长效融合蛋白EX-mhIgG1Fc和EX-L21K-mhIgG1Fc，具有降糖活性高和降糖作用持续时间长的显著优点，这将有利于降低获得治疗效果的用药剂量、减少给药频率、提高药物治疗的依从性，可用于治疗糖尿病及通过降低血糖而受益的其它疾病。
附图说明
图1为本发明所述高活性长效降糖融合蛋白EX-mhIgG1Fc的结构示意图。(A.野生型艾塞那肽(Exendin-4)的长效融合蛋白EX-mhIgG1Fc结构示意图,它主要包括：起始甲硫氨酸Met、野生型艾塞那肽(Exendin-4)、连接肽(-GGGG-)、优化突变的人IgG1的铰链区(mutated human IgG1hinge,简称mhIgG1铰链区)、恒定区CH2-CH3。B.完整的EX-mhIgG1Fc的氨基酸序列及其人工合成基因序列，阴影所示为野生型艾塞那肽(Exendin-4)部分，下划线所示为优化突变的人IgG1的铰链区(mutated human IgG1hinge,简称mhIgG1铰链区)部分，星号所示为终止密码子)。
图2为本发明所述高活性长效降糖融合蛋白EX-L21K-mhIgG1Fc的结构示意图。(A.高活性艾塞那肽突变体(EX-L21K)的长效融合蛋白EX-L21K-mhIgG1Fc结构示意图,它主要包括：起始甲硫氨酸Met、高活性艾塞那肽突变体(EX-L21K)、连接肽(-GGGG-)、(mutated human IgG1hinge,简称mhIgG1铰链区)、恒定区(CH2-CH3)。B.完整的EX-L21K-mhIgG1Fc的氨基酸序列及其人工合成基因序列，阴影所示为高活性艾塞那肽突变体(EX-L21K)部分，下划线所示为优化突变的人IgG1的铰链区(mutated human IgG1hinge, 简称mhIgG1铰链区)部分，星号为终止密码子)。
图3为：A.野生型艾塞那肽(Exendin-4)的长效融合蛋白EX-hIgG1Fc结构示意图,它主要包括：起始甲硫氨酸Met、野生型艾塞那肽(Exendin-4)、连接肽(-GGGG-)、天然的人IgG1的铰链区(hIgG1铰链区)、恒定区CH2-CH3。B.完整的EX-hIgG1Fc的氨基酸序列及其人工合成基因序列，阴影所示为野生型艾塞那肽(Exendin-4)部分，下划线所示为天然的人IgG1的铰链区(hIgG1铰链区)部分，星号所示为终止密码子。
图4为本发明所述高活性长效降糖融合蛋白的重组表达质粒结构示意图。(A.大肠杆菌表达野生型艾塞那肽(Exendin-4)的长效融合蛋白EX-mhIgG1Fc的重组质粒pET-EX-mhIgG1Fc示意图，T7promoter：T7启动子；AP：氨苄抗性基因；EX-mhIgG1Fc：野生型艾塞那肽(Exendin-4)的长效融合蛋白EX-mhIgG1Fc的编码基因；Ori：pET21b质粒复制起始点；lac I：乳糖操纵子阻遏蛋白。B.大肠杆菌表达高活性艾塞那肽突变体(EX-L21K)的长效融合蛋白EX-L21K-mhIgG1Fc的重组质粒pET-EX-L21K-mhIgG1Fc示意图，T7 promoter：T7启动子；AP：氨苄抗性基因；EX-L21K-mhIgG1Fc：高活性艾塞那肽突变体(EX-L21K)的长效融合蛋白EX-L21K-mhIgG1Fc的编码基因；Ori：pET21b质粒复制起始点；lac I：乳糖操纵子阻遏蛋白。C.大肠杆菌表达野生型艾塞那肽长效融合蛋白EX-hIgG1Fc的重组质粒pET-EX-hIgG1Fc示意图，T7promoter：T7启动子；AP：氨苄抗性基因；EX-hIgG1Fc：野生型艾塞那肽(Exendin-4)的长效融合蛋白EX-hIgG1Fc的编码基因；Ori：pET21b质粒复制起始点；lac I：乳糖操纵子阻遏蛋白)。
图5为本发明所述高活性长效降糖融合蛋白的电泳结果。(A.12％SDS-PAGE分析野生型艾塞那肽(Exendin-4)的长效融合蛋白EX-mhIgG1Fc的样品纯度，M：低分子量蛋白Marker；Lane 1：破菌液上清；Lane 2：Protein A柱穿过液；Lane 3：Protein A亲和柱纯化的样品；Lane 4～5：Superdex分子筛纯化的样品。B.12％SDS-PAGE分析高活性艾塞那肽突变体(EX-L21K)的长效融合蛋白EX-L21K-mhIgG1Fc的样品纯度，M：低分子量蛋白Marker；Lane 1：破菌液上清；Lane 2～3：Protein A柱穿过液；Lane 4：Protein A亲和柱纯化的样品；Lane 5：Superdex分子筛纯化的样品)。
图6为SEC-HPLC色谱检测本发明所述高活性长效降糖融合蛋白分子量和纯度结果(色谱柱：Shodex PROTEIN KW-802.5，SHOWA DENKO K.K.，Japan。A.分析四种标准蛋白：BSA(MW＝67kDa)、鸡卵清蛋白(MW＝43kDa)、胰凝乳蛋白酶原(MW＝25kDa)和溶菌酶(MW＝14.4kDa)，它们的保留时间分别是11.15min，11.94min，13.28min，14.31min，所对应的保留体积分别为7.81ml，8.36ml，9.30ml，10.02ml。B.分析纯 化的艾塞那肽(Exendin-4)长效融合蛋白EX-mhIgG1Fc，其保留时间为11.532min，对应的保留体积为8.07ml，推算的分子量为54.62kDa，纯度达98.296％。C.分析纯化的高活性艾塞那肽突变体(EX-L21K)长效融合蛋白EX-L21K-mhIgG1Fc，其保留时间为11.534min，对应的保留体积为8.07ml，推算的分子量为54.57kDa，纯度达99.642％)。
图7为野生型艾塞那肽(Exendin-4,EX-4)、野生型艾塞那肽长效融合蛋白(EX-mhIgG1Fc)及高活性艾塞那肽突变体的长效融合蛋白(EX-L21K-mhIgG1Fc)腹腔给药后对BKS.Cg-Dock7m+/+Leprdb/JNju小鼠血糖水平的影响(n＝8,means±SEM)。
图8为野生型艾塞那肽(Exendin-4,EX-4)、野生型艾塞那肽长效融合蛋白(EX-mhIgG1Fc)及高活性艾塞那肽突变体长效融合蛋白(EX-L21K-mhIgG1Fc)腹腔给药后对BKS.Cg-Dock7m+/+Leprdb/JNju小鼠血糖水平的影响。AUC0-120h如图所示。####表示与正常组比较p<0.0001；*、**和****表示与模型组比较P<0.05,P<0.01及P<0.0001；ΔΔ和ΔΔΔΔ表示与Ex-4组比较P<0.01及P<0.0001(n＝8,means±SEM)。
图9为野生型艾塞那肽(Exendin-4,EX-4)、野生型艾塞那肽长效融合蛋白(EX-mhIgG1Fc)及高活性艾塞那肽突变体长效融合蛋白(EX-L21K-mhIgG1Fc)对BKS.Cg-Dock7m+/+Leprdb/JNju小鼠IPGTT的影响(n＝8,means±SEM)。
图10为野生型艾塞那肽(Exendin-4,EX-4)、野生型艾塞那肽长效融合蛋白(EX-mhIgG1Fc)及高活性艾塞那肽突变体长效融合蛋白(EX-L21K-mhIgG1Fc)对糖尿病小鼠糖耐量的影响。如图所示0-180min的血糖曲线下面积AUC0-180min，####表示与正常组比较P<0.0001；****表示与模型组比较P<0.0001(n＝8,means±SEM)。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例限制。
在本发明中所使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解，该实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
下面结合具体实施例对本发明进一步说明。
下述实例中所用的材料、试剂等，如无特殊说明，均可从商业途径获得。
实施例1野生型艾塞那肽长效融合蛋白及高活性艾塞那肽突变体长效融合蛋白的设计
1、野生型艾塞那肽Exendin-4氨基酸序列(SEQ ID NO:1)，将其第21位Leu突变成Lys便得到高活性艾塞那肽突变体(EX-L21K)的氨基酸序列(SEQ ID NO:2)。
野生型艾塞那肽Exendin-4氨基酸序列：HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO:1)。
高活性艾塞那肽突变体EX-L21K氨基酸序列：HGEGTFTSDLSKQMEEEAVRKFIEWLKNGGPSSGAPPPS(SEQ ID NO:2)。
2、优化突变的人hIgG1的铰链区(mhIgG1铰链区)氨基酸序列见SEQ.ID NO:6，它由天然的人hIgG1的铰链区(氨基酸序列见SEQ.ID NO:5)(Edelman GM.Proc Natl Acad Sci U S A.1969；63:78-85)突变而成。与天然的人hIgG1的铰链区(hIgG1铰链区)相比，突变后的人hIgG1的铰链区(mhIgG1铰链区)的N端区域的氨基酸富含Gly和Ser，这样既增强了铰链区N-端的柔韧性和亲水性，又消除了原铰链区N端第1个Cys在蛋白表达过程中形成错配二硫键的可能性。下划线部分为突变区域。
天然的人hIgG1铰链区(hIgG1铰链区)氨基酸序列：VEPKSCDKTHTCPPCP(SEQ.ID NO:5)
优化突变的人hIgG1铰链区(mutated human IgG1 hinge,简称mhIgG1铰链区)氨基酸序列：SGGGGSDKTHTCPPCP(SEQ.ID NO:6)
连接肽序列的设计：连接肽是指富含Gly和/或Ala和/或Ser的柔性肽，长度在1～50个氨基酸残基之间，优选的连接肽的氨基酸序列如SEQ ID NO:8所示。
3、野生型艾塞那肽Exendin-4的长效融合蛋白EX-mhIgG1Fc由以下几部分组成：野生型艾塞那肽Exendin-4、连接肽、优化突变的人IgG1的铰链区(mhIgG1铰链区)和恒定区CH2-CH3。其结构示意图见图1A。其氨基酸序列(SEQ.ID NO:9)及根据大肠杆菌偏爱的密码子设计的编码基因序列(SEQ.ID NO:10)见图1B。
4、高活性艾塞那肽突变体EX-L21K的长效融合蛋白EX-L21K-mhIgG1Fc由以下几部分组成：高活性的艾塞那肽突变体EX-L21K、连接肽、优化突变的人IgG1的铰链区(mhIgG1铰链区)和恒定区CH2-CH3。其结构示意图见图2A，其氨基酸序列(SEQ.ID NO:11)及根据大肠杆菌偏爱的密码子设计的编码基因序列(SEQ.ID NO:12)见图2B。
5.野生型艾塞那肽Exendin-4的长效融合蛋白EX-hIgG1Fc由以下几部分组成：野生型艾塞那肽Exendin-4、连接肽、天然的人hIgG1铰链区(hIgG1铰链区)和恒定区CH2-CH3。其结构示意图见图3A。其氨基酸序列(SEQ.ID NO:13)及根据大肠杆菌偏爱的密码子设计的编码基因序列(SEQ.ID NO:14)见图3B。
实施例2野生型艾塞那肽长效融合蛋白EX-mhIgG1Fc的克隆与表达
野生型艾塞那肽的长效融合蛋白EX-mhIgG1Fc的编码基因(SEQ.ID NO:10)由南京金斯瑞生物科技有限公司合成并克隆，用Nde I和Hind III双酶切后再亚克隆到原核表达载体pET21b中，构建成表达质粒pET-EX-mhIgG1Fc(图4A)，测序验证其序列正确性，再用CaCl2法将之转化大肠杆菌E.coliBL21(DE3)宿主菌中进行表达。
接单菌落于50ml LB液体培养基(含100μg/ml氨苄青霉素)，200rpm于37℃振荡培养14h。按1％(V/V)的接种量转接至200ml TB培养基(胰蛋白胨1.2％，酵母粉2.4％，甘油0.4％(v/v)，17mM KH2PO4，72mM K2HPO4·3H2O，100μg/ml氨苄青霉素)，37℃振荡培养至OD600nm达1.0左右，加入乳糖至1％(w/v)，200rpm于25℃振荡诱导表达15h，同时设立阴性对照(即不加乳糖诱导)。
收集发酵液，10000rpm离心10min收集菌泥，湿菌泥称重，按照1：15(g/ml)的比例，用PBS重悬菌泥，均质机(AH100B，ATS Engineering Inc.，Canada)低温破碎细菌，破碎过程中保持均质机的压力在800～900bar，破碎3次。菌体破碎完成后，将细胞破碎液于4℃离心(12000rpm，20min)，取上清进行12％SDS-PAGE电泳分析，结果表明在分子量约30KD处有明显的蛋白表达条带(图5A)，证明该融合蛋白为可溶性表达。
实施例3高活性艾塞那肽突变体的长效融合蛋白EX-L21K-mhIgG1Fc的克隆与表达以实施例2中野生型艾塞那肽的长效融合蛋白EX-mhIgG1Fc的编码基因(SEQ.ID NO:10)为模板，采用重叠延伸PCR定点突变的方法(Ho SN.Gene.1989；77:51-9)对之进行突变，得到高活性艾塞那肽突变体的长效融合蛋白EX-L21K-mhIgG1Fc的编码基因(SEQ.ID NO:12)。
由南京金斯瑞生物科技有限公司合成以下引物：
(1)正向突变引物(EX-L21K-F):5'GGAAGAAGAAGCGGTTCGTAAATTCATCGAATGGCTGAAAAAC 3'(SEQ.ID NO:17)；
(2)反向突变引物(EX-L21K-R):5'GTTTTTCAGCCATTCGATGAATTTACGAACCGCTTCTTCTTCC 3'(SEQ.ID NO:18)；
(3)外侧正向引物(NdeI-EX-F):5'AGATATACATATGCACGGTGAAGGTACCTTCACCTCTGAC 3'(SEQ.ID NO:19)；
(4)外侧反向引物(HindIII-Fc-R):5'CGTCGACAAGCTTCTATTATTTACCCGGAGACAGAGACAGAG 3'(SEQ.ID NO:20)。
扩增上游片段A：以EX-mhIgG1Fc的编码基因(SEQ.ID NO:10)为模板，在 Fastpfu DNA Polymerase聚合酶(TransGen Biotech产品)的作用下进行PCR反应。25μl反应体系组成如下：外侧正向引物(NdeI-EX-F)10pmol；反向突变引物(EX-L21K-R)10pmol；Fast pfu DNA Polymerase 2.5个单位；5×反应缓冲液5μl；dNTP(10mM each)0.5μl；模板质粒DNA 0.5μl(约2.5ng)；用无菌水补足到25μl。PCR反应条件如下：95℃变性2分钟；进入循环反应:95℃变性20秒，50℃退火20秒，72℃延伸10秒，反应30个循环；72℃延伸5分钟。反应结束后，产物用2％琼脂糖凝胶电泳进行鉴定，并用TaKaRa的胶回收试剂盒进行回收。
扩增下游片段B：以EX-mhIgG1Fc编码基因(SEQ.ID NO:10)为模板，在Fastpfu DNA Polymerase聚合酶(TransGen Biotech产品)的作用下进行PCR反应。25μl反应体系组成如下：正向突变引物(EX-L21K-F)10pmol；外侧反向引物(HindIII-Fc-R)10pmol；Fast pfu DNA Polymerase 2.5个单位；5×反应缓冲液5μl；dNTP(10mM each)0.5μl；模板质粒DNA 0.5μl(约2.5ng)；用无菌水补足到25μl。PCR反应条件如下：95℃变性2分钟；进入循环反应:95℃变性20秒，50℃退火20秒，72℃延伸30秒，反应30个循环；72℃延伸5分钟。PCR产物用1％琼脂糖凝胶电泳进行鉴定，并用TaKaRa的胶回收试剂盒进行回收。
重叠延伸PCR扩增获得完整的突变体基因：以上游片段A和下游片段B的混合液为模板，在Taq plus DNA Polymerase(TAKARA公司产品)的作用下进行PCR反应。50μl反应体系组成如下：外侧正向引物(NdeI-EX-F)10pmol；外侧反向引物(HindIII-Fc-R)10pmol；Taq plus DNA Polymerase 2.5个单位；10×Taq plus buffer(with MgCl2)5μl；dNTP(10mM each)1μl；上游片段A和下游片段B的混合液1μl(约5ng)；用无菌水补足到50μl。PCR反应条件如下：95℃变性3分钟；进入循环反应:94℃变性30秒，55℃退火30秒，72℃延伸1分钟，反应30个循环；72℃延伸5分钟。PCR产物用1％琼脂糖凝胶电泳鉴定，并用TaKaRa的胶回收试剂盒进行回收。
将获得的突变体基因用Nde I和Hind III双酶切后克隆到原核表达载体pET21b中，构建成表达质粒pET-EX-L21K-mhIgG1Fc(图4B)，DNA序列结果表明与高活性艾塞那肽突变体的长效融合蛋白EX-L21K-mhIgG1Fc的基因序列(SEQ.ID NO:12)一致。将该表达质粒pET-EX-L21K-mhIgG1Fc转化至大肠杆菌E.coliBL21(DE3)宿主中，参照实例2的蛋白表达方法对高活性艾塞那肽突变体的长效融合蛋白EX-L21K-mhIgG1Fc进行表达。表达后将细胞破碎液于4℃离心(12000rpm，20min)，取上清进行12％SDS-PAGE电泳分析，结果表明在分子量约30KD处有明显的蛋白表达条带(图5B)，证明该融合蛋白为可溶性表 达。
实施例4野生型艾塞那肽长效融合蛋白EX-hIgG1Fc的克隆与表达
野生型艾塞那肽的长效融合蛋白EX-hIgG1Fc的编码基因(SEQ.ID NO:14)由南京金斯瑞生物科技有限公司合成并克隆，用Nde I和Hind III双酶切后再亚克隆到原核表达载体pET21b中，构建成表达质粒pET-EX-hIgG1Fc(图4C),测序验证后用CaCl2法将之转化大肠杆菌E.coliBL21(DE3)宿主菌中进行表达。
参照实例2的蛋白表达方法对野生型艾塞那肽的长效融合蛋白EX-hIgG1Fc进行表达，表达后将细胞破碎液于4℃离心(12000rpm，20min)，取上清进行12％SDS-PAGE电泳分析，结果在分子量约30KD处未出现目的蛋白表达条带，说明该融合蛋白未能可溶性表达。
实施例5融合蛋白EX-mhIgG1Fc和EX-L21K-mhIgG1Fc的分离纯化
1、收集菌泥及破壁：发酵液于4℃离心(10000rpm，10min)收集菌泥，湿菌泥称重，按照1：15(g/ml)的比例，用PBS缓冲液重悬菌泥，并洗涤菌泥2到3次。再用破菌缓冲液(PBS缓冲液(pH 7.4)含1mM的PMSF，1mM的EDTA)按10％(w/v)比例重悬菌泥，ATS均质机(AH100B，ATS Engineering Inc.，Canada)破碎细胞，破碎过程中保持均质机的压力在800～900bar，破碎3次。4℃离心(12000rpm，20min)收集破壁液上清。
2、Protein A亲和层析：平衡缓冲液PBS(pH 7.4)充分平衡HiTrap rProtein A FF亲和色谱预装柱(GE公司产品)，破壁液上清经0.22μm水相滤膜抽滤后上样，流速0.5ml/min，上样完成后用平衡缓冲液冲洗色谱柱，洗去未结合的杂蛋白。然后用洗脱缓冲液(0.1M柠檬酸，NaOH调pH至4.0)进行洗脱，每毫升收集液加入450μl中和缓冲液(1M Tris盐酸缓冲液(pH调至9.0)与甘油按1:2混合而成)。
3、硫酸铵沉淀：冰冷饱和硫酸铵溶液0.22μm膜抽滤后，按1ml/min流速滴加至置于冰水浴的上述蛋白样品溶液，至60％浓度，沉淀目的蛋白，整个过程中磁力搅拌器缓慢搅拌。4℃离心(10000rpm，20min)收集沉淀，并用适量PBS缓冲液(pH 7.4)溶解，得到目的蛋白浓缩液。
4、凝胶色谱层析：PBS缓冲液(pH 7.4)充分平衡Superdex 200Increase 10/300GL凝胶过滤预装柱(GE公司产品)。取500μl硫酸铵沉淀的目的蛋白浓缩液，4℃离心(10000rpm，10分钟)取上清，上样至已平衡的色谱柱，流速为0.4ml/min。收集目的蛋白洗脱峰，并在超净台上用0.22μm滤膜过滤除菌后分装，-70℃保存。
纯化各步收集的样品经12％SDS-PAGE电泳分析，结果见图5A和5B。
实施例6融合蛋白EX-mhIgG1Fc和EX-L21K-mhIgG1Fc分子量检测及纯度分析
利用分子排阻法(Size Exclusion Chromatography，SEC)在HPLC系统(LC-2010A HT，SHIMADZU Corp.，Japan)上进行分析，色谱柱为Shodex PROTEIN KW-802.5(SHOWA DENKO K.K.，Japan)，流动相为0.2M磷酸缓冲液(pH 7.4)，含0.1M Na2SO4(按层析柱说明书要求添加)，流速为0.7ml/min，检测波长为280nm。
对上海源叶生物科技公司购买的四种标准蛋白进行单独分析和混合分析，结果表明，四种标准蛋白BSA(MW＝67kDa)、鸡卵清蛋白(MW＝43kDa)、胰凝乳蛋白酶原(MW＝25kDa)和溶菌酶(MW＝14.4kDa)的保留时间分别是11.15 2min，11.938min，13.284min，14.31min(图6A)，所对应的保留体积分别为7.81ml，8.36ml，9.30ml，10.02ml。根据各标准蛋白的分子量和保留体积做出标准曲线，得到分子量计算公式为lgMW＝-0.2965×Ve+4.1308。
野生型艾塞那肽长效融合蛋白EX-mhIgG1Fc的保留时间为11.532min，如图(6B)所示，对应的保留体积为8.07ml，根据上述公式计算的分子量为54.62kDa，与ExPASy网站(http://web.expasy.org/compute_pi/)预测的二聚体分子量60.92kDa相近。
高活性艾塞那肽突变体的长效融合蛋白EX-L21K-mhIgG1Fc的保留时间为11.534min，如图(6C)所示，对应的保留体积为8.07ml，根据上述公式计算的分子量为54.57kDa，与ExPASy网站(http://web.expasy.org/compute_pi/)预测的二聚体分子量60.98kDa相近。
由此可知，纯化得到的野生型艾塞那肽的长效融合蛋白EX-mhIgG1Fc和高活性艾塞那肽突变体的长效融合蛋白EX-L21K-mhIgG1Fc均以可溶的二聚体形式存在。
此外，采用峰面积比较法对上述两种蛋白的SEC-HPLC分析结果进行纯度分析，结果显示，纯化的EX-mhIgG1Fc样品的纯度为98.296％(图6B)，纯化的EX-L21K-mhIgG1Fc样品的纯度为99.642％(图6C)。
实施例7II型糖尿病模型小鼠C57BL/KsJ-db/db体内降糖试验
六周龄雄性II型糖尿病模型小鼠C57BL/KsJ-db/db(即S.Cg-Dock7m+/+Leprdb/JNju小鼠)50只及对照小鼠C57BLKS/JNju 10只均购自南京大学—南京生物医药研究院(合格证号：201602819，许可证号：SCXK(苏)2015-0001)。小鼠饲养于SPF级动物房，室温25℃，湿度40-60％，明暗各12h，适应性饲养一周后进行实验。血糖浓度采用Roche
Figure PCTCN2017093699-appb-000001
Performa血糖仪及血糖试纸进行测定。实验分组及给药方式见表1。
表1实验动物分组及给药情况
组别 小鼠 n 药物 给药剂量 给药方式
1 C57BLKS/JNju 8 NS - ip.
2 BKS.Cg-Dock7m+/+Leprdb/JNju 8 NS - ip.
3 BKS.Cg-Dock7m+/+Leprdb/JNju 8 Ex-4 10nmol/kg ip.
4 BKS.Cg-Dock7m+/+Leprdb/JNju 8 EX-mhIgG1Fc 10nmol/kg ip.
5 BKS.Cg-Dock7m+/+Leprdb/JNju 8 EX-L21K-mhIgG1Fc 5nmol/kg ip.
6 BKS.Cg-Dock7m+/+Leprdb/JNju 8 EX-L21K-mhIgG1Fc 10nmol/kg ip.
7 BKS.Cg-Dock7m+/+Leprdb/JNju 8 EX-L21K-mhIgG1Fc 20nmol/kg ip.
注：NS，为生理盐水；Ex-4为阳性药醋酸艾塞那肽(Exendin-4)，上海吉尔生化有限公司产品(目录号:052143；批号：P160102-CQ052143；分子式：C184H282N50O60S1；纯度99.41％；分子量：4186.66Da)
1.急性降糖试验
给药前检测各组小鼠血糖水平。如表2、图7所示，模型组、Ex-4、EX-mhIgG1Fc及EX-L21K-mhIgG1Fc(5-20nmol/kg)分别为：24.16±0.81、24.53±1.20、23.31±1.05、23.04±1,23、22.41±1.51、24.99±1.43mM，与正常对照组8.58±0.25mM比较，差异极显著(P<0.0001)，模型组与各药物组之间无差异，说明BKS.Cg-Dock7m+/+Leprdb/JNju已经形成糖尿病，且药物组与模型组之间无差异，可以进行后续实验。
小鼠给药物后，连续监测给药后1、2、4、8、12、24、36、48、60、72、96、120h血糖水平。结果如表2及图7所示。给药1h后，Ex-4、EX-L21K-mhIgG1Fc(5-20nmol/kg)组即有较显著降糖作用，而EX-mhIgG1Fc直至2h才表现出其降糖作用(P<0.01vs模型组)，各药物组至给药8h还表现出很强的降糖活性(P<0.0001vs模型组)。Ex-4组给药12h后小鼠血糖即逐渐回升至给药前水平，EX-mhIgG1Fc组小鼠直至给药48h后血糖才逐渐升至给药前水平。EX-L21K-mhIgG1Fc各剂量组降糖作用更持久，其中5nmol/kg EX-L21K-mhIgG1Fc降糖作用持续至给药后72h(P<0.01vs模型组)，10nmol/kg EX-L21K-mhIgG1Fc(P<0.01vs模型组)和20nmol/kg EX-L21K-mhIgG1Fc(P<0.0001vs模型组)直至给药96h后仍有降糖作用。
表2.Ex-4/FC及EX-L21K/FC对BKS.Cg-Dock7m+/+Leprdb/JNju小鼠的降糖活性
Figure PCTCN2017093699-appb-000002
注：####表示与正常组比较P<0.0001；*、**、***和****表示与模型组比较P<0.05、P<0.01、P<0.001及P<0.0001；Δ、ΔΔ、ΔΔΔ及ΔΔΔΔ表示与Ex-4组比较P<0.05、P<0.01、P<0.001及P<0.0001；(n＝8,means±SEM)。
进一步用曲线下面积(area under the curve,AUC)方法分析评价Ex-4、EX-mhIgG1Fc及EX-L21K-mhIgG1Fc(5-20nmol/kg)的降糖作用，如图8所示，与正常对照组比较，模型组AUC0-24h、AUC0-72h、AUC0-120h均显著增大(p<0.0001)。给予Ex-4、EX-mhIgG1Fc及EX-L21K-mhIgG1Fc(5-20nmol/kg)后，AUC0-24h分别降至374.46±6.20mM·h(P<0.05vs模型组)、337.76±13.35mM·h(P<0.0001vs模型组)、296.69±34.39mM·h(P<0.0001vs模型组)、237.34±16.87mM·h(P<0.0001vs模型组)、230.80±20.09mM·h(P<0.0001vs模型组)。与Ex-4比较，EX-mhIgG1Fc及EX-L21K-mhIgG1Fc(5-20nmol/kg)各组药物的AUC0-24h无显著差异。EX-mhIgG1Fc及EX-L21K-mhIgG1Fc(5-20nmol/kg)各组AUC0-72h、AUC0-120h与模型组比较均显著降低，而Ex-4组与模型组比较无显著变化。
综上所述，Ex-4、EX-mhIgG1Fc及EX-L21K-mhIgG1Fc 3个药物均有降糖作用，其中Ex-4起效较快，给药1h后即有显著降糖作用。EX-mhIgG1Fc降糖作用起效相对较慢，给药2h后才表现出降糖作用，但是其降糖作用持续时间较长，可延长至给药后48h。EX- L21K-mhIgG1Fc不仅给药1h内即有降糖作用，而且持续时间更延长至给药后96h。
2.腹腔注射葡萄糖耐量实验(IPGTT)
实验前各组动物过夜禁食18h，首先分别腹腔注射生理盐水、Ex-4、EX-mhIgG1Fc及EX-L21K-mhIgG1Fc，给药2h后，每只小鼠腹腔注射1.5g/kg葡萄糖，尾静脉取血，记录-120、0、15、30、45、60、90、120、180min血糖浓度。实验期间动物正常饮食供水。
IPGTT实验结果见图9，给10nmol/kg Ex-4、10nmol/kg EX-mhIgG1Fc及5-20nmol/kg EX-L21K-mhIgG1Fc后，小鼠空腹血糖均显著降低(P<0.0001vs模型组)。腹腔注射1.5g/kg葡萄糖15min后，模型组小鼠血糖由8.49±0.54升至22.55±1.77mM并且一直维持较高水平，而Ex-4、EX-mhIgG1Fc及EX-L21K-mhIgG1Fc各药物组小鼠腹腔注射葡萄糖后，血糖虽有升高，但很快降低至正常水平，并持续维持较低水平,直至给药后180min还未升至给糖前水平。
进一步应用曲线线下面积分析，结果如图10所示，与正常对照组比较，模型组AUC0-180min显著增大(p<0.0001)。首先给10nmol/kg Ex-4、10nmol/kg EX-mhIgG1Fc及5-20nmol/kg EX-L21K-mhIgG1Fc预作用120min，然后再腹腔注射1.5g/kg葡萄糖，AUC0-180min分别降至2058±109.5(P<0.0001vs模型组)、2397±62.49(P<0.0001vs模型组)、2338±181.2(P<0.0001vs模型组)、2035±169.6(P<0.0001vs模型组)、2082±119.9mM·min(P<0.0001vs模型组)。由此可见，Ex-4、EX-mhIgG1Fc及EX-L21K-mhIgG1Fc均可显著增强糖尿病小鼠对于葡萄糖的耐受性。

Claims (9)

  1. 一种高活性长效降糖融合蛋白，其特征在于由高活性艾塞那肽突变体通过连接肽或者直接与优化突变的人免疫球蛋白IgG1的Fc片段连接而成。
  2. 根据权利要求1所述的高活性长效降糖融合蛋白，其特征在于所述优化突变的人免疫球蛋白IgG1的Fc片段包括：优化突变的人IgG1的铰链区、人IgG1恒定区CH2和CH3，所述优化突变的人IgG1的铰链区的氨基酸序列如SEQ ID NO:6所示。
  3. 根据权利要求2所述的高活性长效降糖融合蛋白，其特征在于所述的人IgG1恒定区的CH2和CH3的氨基酸序列如SEQ ID NO:7所示。
  4. 根据权利要求1所述的高活性长效降糖融合蛋白，其特征在于所述连接肽是指富含Gly和/或Ala和/或Ser的柔性肽，长度在1～50个氨基酸残基之间。
  5. 根据权利要求4所述的高活性长效降糖融合蛋白，其特征在于所述连接肽的氨基酸序列如SEQ ID NO:8所示。
  6. 根据权利要求1所述的高活性长效降糖融合蛋白，其特征在于所述高活性艾塞那肽突变体的氨基酸序列如SEQ ID NO:2-4所示。
  7. 根据权利要求1所述的高活性长效降糖融合蛋白，其特征在于所述融合蛋白的氨基酸序列如SEQ ID NO:11、15、16所示。
  8. 根据权利要求1-7之一项所述的高活性长效降糖融合蛋白的制备方法，其特征在于所述融合蛋白可在大肠杆菌中进行可溶性表达，具体包括如下步骤：
    (1)设计合成并克隆得到根据权利要求1-7之一项所述融合蛋白的编码基因；
    (2)构建成表达质粒后转化大肠杆菌宿主菌中进行表达；
    (3)收集菌泥及破壁，收集破壁液上清，分离纯化后得到如权利要求1-7之一项所述的可溶性融合蛋白。
  9. 根据权利要求1-7之一项所述的高活性长效降糖融合蛋白在制备降低血糖药物中的应用。
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WO2021216205A1 (en) * 2020-04-21 2021-10-28 Kentucky Bioprocessing, Inc. Vaccines formed by virus and antigen conjugation
US11690907B2 (en) * 2018-06-12 2023-07-04 Kbio Holdings Limited Vaccines formed by virus and antigen conjugation
US11696948B2 (en) * 2018-06-12 2023-07-11 Kbio Holdings Limited Vaccines formed by virus and antigen conjugation
CN110151980B (zh) * 2019-06-30 2022-12-09 中国药科大学 Glp-1受体激动剂融合蛋白在制备预防或治疗高血脂药物中的应用
CN113150172B (zh) * 2021-04-28 2023-09-22 中国药科大学 Glp-1r/gipr双靶点激动剂融合蛋白及其制备方法与应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1483041A (zh) * 2000-12-07 2004-03-17 Glp-1融合蛋白
US20050186662A1 (en) * 2004-01-28 2005-08-25 Syntonix Pharmaceuticals, Inc. Heterodimeric follicle stimulating hormone-Fc (FSH-Fc) fusion proteins for the treatment of infertility
WO2011153965A1 (zh) * 2010-06-11 2011-12-15 北京精益泰翔技术发展有限公司 Exendin-4及其类似物的融合蛋白，其制备和应用
CN102311501A (zh) * 2010-07-08 2012-01-11 天津药物研究院 含有glp-1或其类似物的融合蛋白、制备方法及其应用
CN102453094A (zh) * 2010-11-01 2012-05-16 天津药物研究院 含有Exendin-4的融合蛋白、制备方法及其应用
CN102558362A (zh) * 2012-02-17 2012-07-11 东莞金朗生物科技有限公司 一种用于治疗糖尿病的融合蛋白及其制备方法
CN102952192A (zh) * 2011-08-19 2013-03-06 天津拓飞生物科技有限公司 一种含有Exendin-4的融合蛋白及其用途
CN105753963A (zh) * 2016-04-13 2016-07-13 中国药科大学 高活性艾塞那肽类似物及其医药应用
CN106046176A (zh) * 2016-08-16 2016-10-26 中国药科大学 一种高活性长效降糖融合蛋白及其制备方法与医药用途

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6924264B1 (en) * 1999-04-30 2005-08-02 Amylin Pharmaceuticals, Inc. Modified exendins and exendin agonists
CN1935846A (zh) * 2005-09-14 2007-03-28 王庆华 一种用于治疗糖尿病的融合蛋白及其制备方法和应用
CN101875700B (zh) * 2010-04-09 2012-09-26 无锡和邦生物科技有限公司 一种增加促胰岛素分泌肽融合蛋白生物活性的方法
CN102585013B (zh) * 2011-01-07 2014-04-23 中国人民解放军军事医学科学院生物工程研究所 一种含有ω干扰素的融合蛋白及制备方法
AU2012336069A1 (en) * 2011-11-07 2014-05-22 Medimmune, Llc Multispecific and multivalent binding proteins and uses thereof

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1483041A (zh) * 2000-12-07 2004-03-17 Glp-1融合蛋白
US20050186662A1 (en) * 2004-01-28 2005-08-25 Syntonix Pharmaceuticals, Inc. Heterodimeric follicle stimulating hormone-Fc (FSH-Fc) fusion proteins for the treatment of infertility
WO2011153965A1 (zh) * 2010-06-11 2011-12-15 北京精益泰翔技术发展有限公司 Exendin-4及其类似物的融合蛋白，其制备和应用
CN102311501A (zh) * 2010-07-08 2012-01-11 天津药物研究院 含有glp-1或其类似物的融合蛋白、制备方法及其应用
CN102453094A (zh) * 2010-11-01 2012-05-16 天津药物研究院 含有Exendin-4的融合蛋白、制备方法及其应用
CN102952192A (zh) * 2011-08-19 2013-03-06 天津拓飞生物科技有限公司 一种含有Exendin-4的融合蛋白及其用途
CN102558362A (zh) * 2012-02-17 2012-07-11 东莞金朗生物科技有限公司 一种用于治疗糖尿病的融合蛋白及其制备方法
CN105753963A (zh) * 2016-04-13 2016-07-13 中国药科大学 高活性艾塞那肽类似物及其医药应用
CN106046176A (zh) * 2016-08-16 2016-10-26 中国药科大学 一种高活性长效降糖融合蛋白及其制备方法与医药用途

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