WO2022099963A1 - 提高多肽类药物口服给药稳定性的融合蛋白及其应用 - Google Patents

提高多肽类药物口服给药稳定性的融合蛋白及其应用 Download PDF

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WO2022099963A1
WO2022099963A1 PCT/CN2021/081484 CN2021081484W WO2022099963A1 WO 2022099963 A1 WO2022099963 A1 WO 2022099963A1 CN 2021081484 W CN2021081484 W CN 2021081484W WO 2022099963 A1 WO2022099963 A1 WO 2022099963A1
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fusion protein
glp
mannase
polypeptide
stability
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PCT/CN2021/081484
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French (fr)
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徐冲
楼慧强
余卫雄
吴雷
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安徽新熙盟生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
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    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/695—Corticotropin [ACTH]
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    • C07—ORGANIC CHEMISTRY
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the invention belongs to the technical field of genetic engineering, and particularly relates to a fusion protein for improving the stability of oral administration of polypeptide drugs and its application.
  • peptides have specific biological activities.
  • Polypeptide drugs are widely used in the treatment of endocrine system, immune system, digestive system, cardiovascular system and other diseases, and have good therapeutic effect on chronic diseases such as tumor, diabetes and hepatitis.
  • the half-life of most peptide drugs is very short, and the peptide drugs are quickly cleared by protease degradation and glomerular filtration. Therefore, in order to improve the efficacy of polypeptide drugs, it can only be administered through frequent injections, which brings inconvenience and pain to patients and greatly limits clinical applications.
  • the polypeptide drug When the polypeptide drug is administered orally, its bioavailability is less than 1%, which is mainly caused by the poor stability of the polypeptide drug, the epithelial barrier of the small intestinal mucosa and the absorption disorder. These factors are described below:
  • Small intestinal mucosal epithelial barrier The transmembrane absorption of polypeptide drugs is mainly through receptor-mediated transport and intercellular diffusion. Receptor-mediated transport requires specific protein molecules, while polypeptide drugs are polar molecules that cannot easily pass through the lipid-soluble vascular mucosa. Therefore, intercellular diffusion (transport through tight junctions between cells) becomes the main route of peptide drug absorption.
  • the pore size between the small intestinal mucosa epithelial cells in the human body is 0.4 nm, and only amino acids, dipeptides, and tripeptides can pass through.
  • the prior art adopts certain preparation techniques such as enzyme inhibitors, absorption enhancers, chemical modification and other methods, and also delivers them through special systems, such as emulsions, liposomes, Microspheres, Nanoparticles and other systems.
  • enzyme inhibitors have many side effects, which can disrupt the body's digestion and absorption of nutrients, and the enzyme inhibitors must be released at the same time as the drug or earlier than the drug in order to exert an inhibitory effect.
  • Absorption enhancers can reversibly remove or temporarily disrupt the barrier of the gastrointestinal tract with minimal tissue damage, but oral administration of drugs with short half-lives is still not possible.
  • Glucagon-like peptide-1 (glucagon-likepeptide-1, GLP-1) is a cytokine mimetic peptide, which not only has excellent hypoglycemic effect, but also has the characteristics of weight control, blood lipid regulation, and bidirectional regulation of islet ⁇ -cell function. However, the half-life of natural GLP-1 is only 1.5-2.1 minutes. The structure of GLP-1 is modified with fatty acid side chains and fused with macromolecular proteins to prolong its half-life.
  • dulaglutide and albiglutide are respectively fused with G4 immune albumin and serum albumin, which prolongs the half-life of drug metabolism and can be injected once a week, but there will be adverse reactions at the injection site after injection, and it is still impossible to achieve Oral administration.
  • the purpose of the present invention is to provide a fusion protein that improves the stability of oral administration of polypeptide drugs.
  • Another object of the present invention is to provide a recombinant expression vector containing the above fusion protein.
  • Another object of the present invention is to provide a recombinant strain containing the above fusion protein.
  • Another object of the present invention is to provide the application of the above fusion protein.
  • Another object of the present invention is to provide a fusion protein MANNase-GLP-1.
  • Another object of the present invention is to provide a recombinant expression vector containing the above fusion protein MANNase-GLP-1.
  • Another object of the present invention is to provide a method for preparing the above fusion protein MANNase-GLP-1.
  • Another object of the present invention is to provide the application of the above fusion protein MANNase-GLP-1.
  • the fusion protein contains ⁇ -mannanase, a linking peptide and a polypeptide in sequence from the N-terminus to the C-terminus, wherein the polypeptide comprises Antitumor peptides, antiviral peptides, peptide vaccines, cytokine mimetic peptides, antibacterial active peptides.
  • polypeptides include interferon, insulin growth factor, interleukin series, tumor necrosis factor, fibroblast growth factor, EPO (erythropoietin), adrenocorticotropic hormone (ACTH), calcitonin that promotes bone calcium production, Stimulates bone formation and bone resorption Teriparatide, corticotropin-releasing factor (CRF), erythropoietin (EPO) that stimulates and regulates erythropoiesis and maturation, granulocyte colony-stimulating factor, nerve growth factor, treats aging Human growth hormone for disease and dwarfism, luteinizing hormone-releasing hormone for prostate cancer and reproductive system tumors, endostatin for non-small cell lung cancer, etc.
  • EPO erythropoietin
  • ACTH adrenocorticotropic hormone
  • calcitonin that promotes bone calcium production
  • CRF corticotropin-releasing factor
  • EPO erythropoietin
  • amino acid sequence of ⁇ -mannanase is shown in SEQ ID No.1.
  • the amino acid of ⁇ -mannanase is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% of the amino acid sequence shown in SEQ ID No. 1 %, 88%, 89%, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 98.1%, 98.2%, 98.3%, 98.4%, 98.5 %, 98.6%, 98.7%, 98.8%, 98.9%, or 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% active protein; or,
  • the ⁇ -mannanase can have the amino acid sequence shown in SEQ ID No. 1 through one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9) amino acid residues Derivatives obtained by substitution, deletion and/or insertion of ⁇ -mannanase and
  • nucleotide sequence of the gene of ⁇ -mannanase is shown in SEQ ID No.2:
  • Glucagon-like peptide-1 (glucagon-likepeptide-1, GLP-1), the nucleotide sequence of its encoding gene is shown in SEQ ID No.3:
  • the linker peptide applicable to the present invention is not limited to the above-mentioned DYKDDDDK, (GGGGS) n and (EAAAK) n .
  • the polypeptide includes GLP-1, EPO, thymus hormone, cytokine, interferon, calcitonin, tumor necrosis factor or tumor marker molecular.
  • Interferons are a class of glycoproteins that are mainly used in the treatment of advanced hairy cell leukemia, renal cancer, melanoma, Kaposi's sarcoma, chronic myeloid leukemia, and low- and moderate-grade non-Hodgkin's lymphoma.
  • Calcitonin is a calcium-regulating hormone drug that inhibits the biological activity of osteoclasts and reduces the number of osteoclasts, thereby preventing bone loss and increasing bone mass.
  • Thymus hormone refers to thymosin, and thymosin commonly used in clinic is a small molecule polypeptide with non-specific immune effect found and purified from calf thymus. Thymosin can be used to treat various primary or secondary T cell deficiency diseases, certain autoimmune diseases, various diseases with low cellular immune function and adjuvant therapy for tumors.
  • cytokine drugs that have been approved for marketing or undergoing clinical research include interferon ( ⁇ , ⁇ , ⁇ ), interleukin series, colony stimulating factor, insulin growth factor, tumor necrosis factor, erythropoietin, epidermal growth factor Growth factor, platelet growth factor, fibroblast growth factor, nerve growth factor, connective tissue growth factor and atrial natriuretic hormone, etc.
  • a recombinant expression vector comprising a gene encoding a fusion protein according to a specific embodiment of the present invention.
  • the recombinant expression vector is any one of pPICZ ⁇ A, pPICZ ⁇ B, and pPICZ ⁇ C.
  • a recombinant strain comprising a gene encoding a fusion protein according to a specific embodiment of the present invention.
  • the expression host can be selected from Escherichia coli, Streptomyces, Bacillus subtilis, yeast, mammalian cells, insect cells, and plant cells.
  • the expression host is Pichia pastoris, and the strain of Pichia pastoris can be any one of X-33, GS115, KM71, SMD1168, and SMD1168H.
  • the fusion protein of the present invention is composed of a therapeutic polypeptide drug, a linker peptide, ⁇ -mannanase and its homologues, which can be fused and expressed in prokaryotic and eukaryotic expression systems.
  • ⁇ -Mannanase and its homologues can hydrolyze mannan into mannose oligosaccharides, which, as prebiotics, can be absorbed and metabolized by probiotics in animals to improve intestinal flora.
  • the fusion protein of the invention can solve the common bottleneck that polypeptide drugs are intolerant to gastric acid and easily degraded by various digestive tract proteases, realize oral administration, and can be used for developing long-acting oral preparations of various polypeptide drugs.
  • the fusion protein MANNase-GLP-1 contains ⁇ -mannanase, connecting peptide (DYKDDDDK) and GLP-1 in sequence from the N-terminus to the C-terminus, and its amino acid sequence is as shown in SEQ ID No.4 shown:
  • the recombinant expression vector comprising the fusion protein MANNase-GLP-1 gene according to a specific embodiment of the present invention
  • the recombinant expression vector is any one of pPICZ ⁇ A, pPICZ ⁇ B, and pPICZ ⁇ C.
  • described method comprises the following steps:
  • the genes encoding GLP-1 and ⁇ -mannanase were connected to pPICZ ⁇ A to obtain a recombinant expression vector; the recombinant plasmid was electro-transformed into Pichia pastoris X-33 competent cells to construct a recombinant engineered bacterium and induce expression.
  • the recombinant engineered bacteria are fermented and cultured to induce expression; the obtained fermentation broth is centrifuged, and the supernatant is taken for purification, concentration and drying in sequence to obtain the fusion protein MANNase-GLP-1.
  • the fusion protein of the present invention can significantly improve the stability of polypeptide drugs in the gastrointestinal tract, that is, improve their tolerance to pepsin, trypsin and gastric acid, and adapt to a temperature range of 30-80° C., thereby significantly prolonging the life of polypeptide drugs in human beings.
  • the half-life in the body, its half-life in the human body can be 10 to 5000 times the natural half-life; at the same time, ⁇ -mannanase and its homologues can hydrolyze mannan into mannose oligosaccharides, which can be used in animals.
  • the absorption and metabolism of probiotics can improve the intestinal flora and promote the absorption and utilization of drugs.
  • Fig. 1 is the result of pepsin resistance of fusion protein MANNase-GLP-1;
  • Figure 2 is the result of trypsin resistance of fusion protein MANNase-GLP-1
  • Fig. 3 is the pH stability result of fusion protein MANNase-GLP-1
  • Fig. 4 is the thermostability result of fusion protein MANNase-GLP-1
  • FIG 5 shows the construction results of the high-fat and high-sugar diet-induced metabolic syndrome mouse model, in which, A is the change of fasting blood glucose of mice after continuous feeding with high-fat and high-sugar diet for 24 weeks; B is the high-fat and high-sugar diet. Changes in body weight of mice after continuous feeding of (HFSD) feed for 24 weeks;
  • Fig. 6 shows the results of oral glucose tolerance test in normal diet mice and high-fat and high-sugar diet mice
  • Figure 7 shows the results of HE staining of the liver and adipose tissue of mice on a high-fat and high-sugar diet and mice on a normal diet
  • Figure 8 shows the effect of oral administration of fusion protein MANNase-GLP-1 on blood glucose and body weight in high-fat and high-glucose mice
  • Figure 9 shows the results of liver tissue staining sections after oral administration of MANNase-GLP-1 to high-fat and high-glucose mice;
  • Fig. 10 is the three-dimensional simulation structure of fusion protein MANNase-GLP-1;
  • Figure 11 shows the structure of the residues at the GLP-1 end of the fusion protein MANNase-GLP-1.
  • GLP-1 was linked with an 8-amino acid linker peptide (DYKDDDDK) to synthesize the linker peptide-GLP-1-encoding gene.
  • the target fragments encoding ⁇ -mannanase and linker peptide-GLP-1 were cloned using primer pairs, PCR amplification was performed, and then double-enzyme digestion was performed, and then the obtained gene sequences encoding GLP-1 and mannanase were obtained.
  • the recombinant expression vector pPICZ ⁇ A-MANNase-GLP-1 was constructed by connecting to pPICZ ⁇ A plasmid.
  • sequence of the primer pair of amplifying GLP-1 encoding gene is shown as SEQ ID No.5, SEQ ID No.6:
  • SEQ ID No. 5 5'-CGGGATCCGACTACAAGGACGACGACGAC-3';
  • SEQ ID No. 6 5'-GCTCTAGATTAACCTCTACCTCTAACCA-3'.
  • sequences of the primer pairs for amplifying the coding gene sequence of ⁇ -mannanase are shown in SEQ ID No.7 and SEQ ID No.8:
  • SEQ ID No. 7 5'-CGGAATTCTTGCCAAAGGCTTCTCCAGC-3';
  • SEQ ID No. 8 5'-CGGGATCCAGCAGAATCAATAGCAGCAA-3'.
  • the recombinant plasmid was electro-transformed into Pichia pastoris X-33 competent cells to construct a recombinant engineering strain MANNase-GLP-1-X-33, and induce expression.
  • a single colony of recombinant engineered bacteria MANNase-GLP-1-X-33 was inoculated into a YPD liquid medium test tube containing bleomycin, and cultured with shaking at 30 °C and 200 rpm for 12 h; In the medium, culture at 30°C and 200rpm for 12h to obtain first-class seed solution; inoculate the first-class seed solution into YPD medium at 10% of the inoculum, and cultivate at 30°C and 200rpm for 22h, namely Obtain secondary seed liquid; insert the secondary seed liquid into a 10L seed tank according to 10% of the inoculation amount, and then into a 50L fermenter according to 10%, and carry out fermentation culture.
  • the OD 600 of the fermentation liquid reaches 60-120 or more Add an inducer for induction, put it in a tank after induction, and collect the cells by centrifugation.
  • the fermentation culture is high-density fermentation culture, the inducer is methanol, and the addition amount of the inducer is 0.2%-3% (V/V).
  • the initial fermentation temperature was 30° C.
  • the stirring speed was 300 rpm
  • the aeration rate was 4 L/min
  • the pH was 5.5.
  • the specific steps of purifying, concentrating and drying the supernatant are as follows: take the supernatant, filter it with a 0.8um filter membrane, then filter it with a 0.2um filter membrane, and collect the filtrate; first, concentrate the filtrate with an ultrafiltration membrane bag 10 times, adding deionized water and then concentrating 10 times to obtain a concentrated solution; freeze-drying the concentrated solution to obtain a recombinant fusion protein MANNase-GLP-1.
  • Embodiment 2 investigates the characteristic of fusion protein MANNase-GLP-1
  • pepsin solution 2.0g NaCl, 3.2g pepsin, 7mL concentrated hydrochloric acid, distilled water to 1000mL, pH is about 1.2.
  • the preparation method refers to the artificial simulated gastric juice formula in the US 1995 Pharmacopoeia.
  • Enzymatic hydrolysis reaction test The enzymatic hydrolysis reaction system of fusion protein MANNase-GLP-1 and pepsin was prepared in the ratio of protein content of 1:1. The inactivated protease was used as a blank control, and the reaction time was set to 4 gradients, 0 min, 30min, 60min, and 120min were accurately timed. When the reaction was over, 0.05 mL of 0.618 mol/L sodium carbonate solution was immediately added to terminate the enzymatic hydrolysis reaction. Take 50uL of the enzymatic hydrolysis reaction solution from each tube into a 1.5mL centrifuge tube, treat at 70°C for 5min to inactivate the enzyme, and measure the enzyme activity after appropriate dilution.
  • the enzymatic hydrolysis reaction system of fusion protein MANNase-GLP-1 and trypsin was prepared at the ratio of protein content of 1:1.
  • the inactivated protease was used as the blank control, and the reaction time was set to 4 gradients, 0min, 30min, 60min, and 120min. , accurate timing, when the reaction is over, immediately add 0.05 mL of 30% glacial acetic acid solution to stop the enzymatic hydrolysis reaction.
  • the fusion protein MANNase-GLP-1 still had more than 60% enzymatic activity, and the results showed that the fusion protein MANNase-GLP-1 had good resistance to trypsin.
  • the optimum pH of the fusion protein MANNase-GLP-1 is 3.2, and it still retains 80% of the enzymatic activity under the condition of pH 2.
  • the enzymatic activity of fusion protein MANNase-GLP-1 was measured at a temperature of 30-80 °C using citric acid-disodium hydrogen phosphate buffer with pH 3.2, and each experiment was repeated three times.
  • the fusion protein MANNase-GLP-1 has the maximum enzymatic activity at 60 °C, and the enzyme activity drops to 0 after a few minutes; it has a strong tolerance at 40 °C (close to human body temperature), After 12h, the enzyme activity basically did not change; under the condition of 50°C, about 80% of the enzyme activity remained after 1h, and the enzyme activity basically decreased to 0 after 6h.
  • Embodiment 3 Verify the oral effect of fusion protein MANNase-GLP-1
  • mice 100 6-week-old (18-20g) C57-6J mice (4-6 weeks old, male) were kept in separate cages, the temperature of the animal room was controlled at 25 ⁇ 2°C, the humidity was 50 ⁇ 10%, and the light was dark for 12 hours. 12h cycle, adapt to the environment for a week. Mice were randomly divided into cages of 5-6 mice/group. All mice were fasted for 12 h and their body weight and fasting blood-glucose (FBG) were measured. The control group was fed with standard feed, and the model group was fed with high-fat and high-sugar (HFSD) feed for 24 weeks. After the end, the body weight and FBG of the mice in each group were measured.
  • FBG blood-glucose
  • mice on high-fat and high-sugar diet is about 42.5g, and the weight of mice on normal diet is about 30g. .
  • HE staining was performed on the liver and adipose tissue of mice on a high-fat and high-sugar diet and mice on a normal diet. As shown in Figure 7, the model mice on a high-fat and high-sugar diet had obvious fatty liver.
  • mice were randomly divided into 5 groups: fusion protein high-dose group (3.5 mg/kg d), fusion protein low-dose group (1.75 mg/kg d), 30 mg/kg orlistat as the positive control group, normal diet
  • the control group and the high-fat and high-sugar diet negative control group were given the same volume of water. All mice were given oral gavage respectively without changing their diet, and the weekly body weight changes were measured.
  • Oral glucose tolerance is an indicator of the body's level of glucose load. Impaired glucose tolerance means that the function of islet ⁇ cells and the body's ability to regulate blood sugar are reduced.
  • the critical value of glucose tolerance is 2 hours after a meal. 7.8mmol/L represents impaired glucose tolerance of the body.
  • the high-dose group returned to the normal level (7.5mmol/L), and the blood glucose of the negative control group (water) was as high as 10mmol/L 2 hours after meals.
  • MANNase-GLP-1 fusion protein has obvious weight loss and glucose tolerance improvement effects.
  • the fusion protein MANNase-GLP-1 has the effect of significantly improving fatty liver.
  • the present invention simulates the three-dimensional structure of fusion protein MANNase-GLP-1, as shown in FIG. 10 .
  • the structure of part of GLP-1 in the overall model of fusion protein MANNase-GLP-1 was compared with the B chain of 310L, and it was found that the main part (about 22/31) of GLP-1 and the B chain of 310L were the same ⁇ -helix.
  • the present invention analyzes that the C-terminal 39 residues of fusion protein MANNase-GLP-1 and the structural part of mannanase form an interaction interface.
  • the composition of the interface about 23% of the solvent accessible area of the 39 residues at the GLP-1 end (ASA, ) are involved in the interface interaction.
  • This interfacial interaction formed 12 hydrogen bonds and 7 salt bonds, and resulted in the release of the binding energy of -13.9kcal mol-1, indicating that the three-dimensional structure of the fusion protein MANNase-GLP-1 is a stable state with lower energy, Its structure is stable.
  • the structure of the 39 residues at the GLP-1 end also has hydrophobic interactions.
  • the present invention analyzes several pepsin-specific hydrolysis recognition sites in GLP-1, such as residues such as 6Phe, 13Tyr, 14Leu, 22Phe and 26Leu, and forms internal hydrophobic interactions, thereby effectively resisting pepsin hydrolysis .
  • residues such as 6Phe, 13Tyr, 14Leu, 22Phe and 26Leu
  • some aromatic ring side chain amino acids in the fusion protein MANNase-GLP-1 are also the recognition sites of chymotrypsin, which can also resist the hydrolysis of pepsin to a certain extent. Therefore, the structural analysis of the fusion protein MANNase-GLP-1 in the present invention is consistent with the experimental results of pepsin, trypsin and pH stability in Example 2, and the fusion protein MANNase-GLP is verified from both experimental and theoretical analysis. -1 function.
  • ⁇ -mannanase can also be fused and expressed with a variety of polypeptide drugs, such as anti-tumor polypeptides, anti-viral polypeptides, polypeptide vaccines, cytokine mimetic peptides, and antibacterial active peptides. Extend the half-life of peptide drugs.
  • the pepsin-specific hydrolysis recognition site originally existing in the polypeptide drug structure after fusion expression forms an internal hydrophobic structure, which effectively resists pepsin hydrolysis; on the other hand, the amino acid side chain after fusion expression
  • the specific protease recognition site on the pepsin can also resist the hydrolysis of pepsin to a certain extent.
  • adrenocorticotropic hormone (ACTH), calcitonin (CCT), and teriparatide (TRP) will be used as examples to construct the fusion expression process with ⁇ -mannanase, and verify the fusion expression. effect of protein.
  • ACTH adrenocorticotropic hormone
  • CCT calcitonin
  • TRP teriparatide
  • Adrenocorticotropic hormone molecular weight 4541.1, composed of 39 amino acids, for the treatment of rheumatoid arthritis.
  • the amino acid sequence is shown in SEQ ID No.9:
  • the recombinant plasmid was electrotransformed into Pichia pastoris X-33 competent cells to construct a recombinant engineering strain MANNase-ACTH-X-33, which was induced to express and purified to obtain a fusion protein MANNase-ACTH.
  • Calcitonin (CCT): It is composed of 32 amino acids and has a molecular weight of 3363.77.
  • the currently marketed drugs are injections and nasal sprays, which can promote bone calcium production and treat osteoporosis.
  • Design primer pairs use the primer pairs to clone the target fragments encoding ⁇ -mannanase and connecting peptide (EAAAKEAAAK)-CCT, respectively carry out PCR amplification, and then carry out double digestion, and then the obtained encoding CCT and mannanase.
  • the gene sequence was connected to the pPICZ ⁇ A plasmid, and the recombinant expression vector pPICZ ⁇ A-MANNase-CCT was constructed.
  • the recombinant plasmid was electro-transformed into Pichia pastoris X-33 competent cells to construct a recombinant engineering strain MANNase-CCT-X-33, which was induced to express and purified to obtain a fusion protein MANNase-CCT.
  • TRP Teriparatide
  • the preparation process of the fusion protein MANNase-TRP is the same as that in Example 2, and the experiments on the pepsin resistance, trypsin resistance, pH stability and thermal stability of the fusion protein MANNase-CCT are the same as those in Example 2.
  • the invention provides a fusion protein for improving the stability of oral administration of polypeptide drugs and its application.
  • the fusion protein of the present invention contains ⁇ -mannanase, connecting peptide and polypeptide in sequence from the N-terminus to the C-terminus, wherein the polypeptide includes GLP-1, EPO, thymus hormone, cytokine, interferon, calcitonin, Tumor necrosis factor, tumor marker molecule.
  • the fusion protein of the invention overcomes the defects of poor stability and easy degradation of polypeptide drugs, has the characteristics of prolonging the half-life of the drug and improving the bioavailability, and has good economic value and application prospect.
  • the invention provides a fusion protein for improving the stability of oral administration of polypeptide drugs and its application.
  • the fusion protein of the present invention contains ⁇ -mannanase, connecting peptide and polypeptide in sequence from the N-terminus to the C-terminus, wherein the polypeptide includes GLP-1, EPO, thymus hormone, cytokine, interferon, calcitonin, Tumor necrosis factor, tumor marker molecule.
  • the fusion protein of the invention overcomes the defects of poor stability and easy degradation of polypeptide drugs, has the characteristics of prolonging the half-life of the drug and improving the bioavailability, and has good economic value and application prospect.

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Abstract

本发明提供了提高多肽类药物口服给药稳定性的融合蛋白及其应用。本发明的融合蛋白从N端到C端依次含有β-甘露聚糖酶、连接肽和多肽，其中，所述多肽包括GLP-1、EPO、胸腺激素、细胞因子、干扰素、降钙素、肿瘤坏死因子、肿瘤标志物分子。本发明的融合蛋白克服了多肽类药物自身稳定性差、容易降解的缺陷，具有延长药物半衰期和提高生物利用度的特点。

Description

提高多肽类药物口服给药稳定性的融合蛋白及其应用
交叉引用
本申请要求2020年11月13日提交的专利名称为“提高多肽类药物口服给药稳定性的融合蛋白及其应用”的第202011272849.6号中国专利申请以及2021年2月19日提交的专利名称为“提高多肽类药物口服给药稳定性的融合蛋白及其应用”的第202110188492.1号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明属于基因工程技术领域，具体涉及提高多肽类药物口服给药稳定性的融合蛋白及其应用。
背景技术
大多数多肽具有特定的生物活性，随着生物技术与多肽合成技术日益成熟，已开发上市的多肽药物种类不断增多，目前已达一百多种。多肽药物在内分泌系统、免疫系统、消化系统、心血管系统等疾病的治疗领域应用广泛，对肿瘤、糖尿病、肝炎等慢性病具有良好的治疗效果。但是大多数多肽药物的半衰期很短，经蛋白酶降解和肾小球的过滤作用，多肽药物被快速清除。因此，为了提高多肽药物药效，只能通过频繁的注射给药，给患者带来不便和痛苦，极大的限制了临床应用。
多肽药物采用口服给药方式时，其生物利用度低于1％，这主要由多肽药物的稳定性差、小肠粘膜上皮屏障和吸收障碍等原因引起的。下面分别对这些因素进行阐述：
(1)稳定性差：多肽类药物在胃肠道内受各种因素的影响，例如蛋白酶、有机溶剂、温度、pH、微生物等，因此，多肽药物在吸收和释放过程中很容易失活。
(2)小肠粘膜上皮屏障：多肽类药物跨膜吸收主要通过受体介导的转运和细胞间隙扩散。受体介导的转运需要特定的蛋白分子，而多肽类药 物为极性分子，不易通过脂溶性的血管粘膜。因此，细胞间隙扩散(通过细胞间的紧密连接转运)成为多肽类药物吸收的主要途径。然而人体内小肠粘膜上皮细胞间孔径为0.4nm，只能通过氨基酸、二肽、三肽，多肽药物分子量较大、脂溶性差、难于透过膜孔进入血液循环。
(3)吸收障碍：药物经胃肠道给药后，在尚未吸收进入血循环之前，在肠粘膜和肝脏被代谢，胆汁内容物、胃肠道表面的黏液层和不流动水层的机械屏障作用以及肽类药物自身构象的不稳定性，使得进入血循环的药量减少，而不能维持有效的血药浓度，从而导致生物利用度低。
为了克服多肽类药物口服生物利用度低的问题，现有技术采用一定的制剂工艺如酶抑制剂、吸收促进剂、化学修饰等方法，还通过特殊系统进行递送，如通过乳剂、脂质体、微球、纳米粒子等系统。但是上述方法仍存在一定缺陷，例如，酶抑制剂有很多副作用，其会扰乱机体对营养物质的消化吸收，且酶抑制剂必须与药物同时释放或早于药物释放，才能发挥抑制作用。吸收促进剂可以在对组织最小损害的情况下可逆的去除或暂时破坏胃肠道的屏障，但是对于半衰期较短药物仍无法实现口服给药。胰高血糖素样肽-1(glucagon-likepeptide-1,GLP-1)为细胞因子模拟肽，不仅具有优异的降糖效果，还有控制体重、调节血脂、双向调节胰岛β细胞功能等特点，但是天然GLP-1半衰期仅为1.5-2.1分钟，对GLP-1的结构进行脂肪酸侧链修饰和融合大分子蛋白，延长其半衰期。如度拉糖肽和阿必鲁肽分别融合了G4免疫白蛋白和血清白蛋白，延长了药物代谢的半衰期，可实现每周注射一次，但注射后会出现注射部位不良反应，且仍无法实现口服给药。
发明内容
本发明的目的在于提供一种提高多肽类药物口服给药稳定性的融合蛋白。
本发明的再一目的在于提供含有上述融合蛋白的重组表达载体。
本发明的再一目的在于提供含有上述融合蛋白的重组菌株。
本发明的再一目的在于提供上述融合蛋白的应用。
本发明的再一目的在于提供一种融合蛋白MANNase-GLP-1。
本发明的再一目的在于提供含有上述融合蛋白MANNase-GLP-1的重组表达载体。
本发明的再一目的在于提供上述融合蛋白MANNase-GLP-1的制备方法。
本发明的再一目的在于提供上述融合蛋白MANNase-GLP-1的应用。
根据本发明具体实施方式的提高多肽类药物口服给药稳定性的融合蛋白，所述融合蛋白从N端到C端依次含有β-甘露聚糖酶、连接肽和多肽，其中，所述多肽包括抗肿瘤多肽、抗病毒多肽、多肽疫苗、细胞因子模拟肽、抗菌活性肽。
上述多肽包括干扰素、胰岛素生长因子、白细胞介素系列、肿瘤坏死因子、成纤维细胞生长因子、EPO(促红细胞生成素)、促肾上腺皮质激素(ACTH)、促进骨钙生成的降钙素、刺激骨形成和骨吸收特立帕肽、促肾上腺皮质激素释放因子(CRF)、刺激和调节红细胞的生成和成熟的促红细胞生成素(EPO)、粒细胞集落刺激因子、神经生长因子、治疗老年疾病和侏儒症的人生长激素、治疗前列腺癌和生殖系统肿瘤黄体激素释放激素、治疗非小细胞肺癌的血管内皮抑制素等。
β-甘露聚糖酶的氨基酸序列如SEQ ID No.1所示。
Figure PCTCN2021081484-appb-000001
Figure PCTCN2021081484-appb-000002
根据本发明的具体实施方式，β-甘露聚糖酶的氨基酸为与SEQ ID No.1所示氨基酸序列具有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％，或者90％、91％、92％、93％、94％、95％、96％、97％、98％，或98.1％、98.2％、98.3％、98.4％、98.5％、98.6％、98.7％、98.8％、98.9％，或99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％的活性蛋白；或者，所述β-甘露聚糖酶可以为具有SEQ ID No.1所示的氨基酸序列经过一个或多个(例如，1、2、3、4、5、6、7、8、9)氨基酸残基的取代、缺失和/或插入获得，并仍然具有β-甘露聚糖酶活性的衍生物。
β-甘露聚糖酶的基因的核苷酸序列如SEQ ID No.2所示：
Figure PCTCN2021081484-appb-000003
Figure PCTCN2021081484-appb-000004
胰高血糖素样肽-1(glucagon-likepeptide-1,GLP-1)，其编码基因的核苷酸序列如SEQ ID No.3所示：
Figure PCTCN2021081484-appb-000005
根据本发明具体实施方式的融合蛋白，所述连接肽的氨基酸序列为DYKDDDDK；或所述连接肽的氨基酸序列为(GGGGS) n，n为＝3或4；或所述连接肽的氨基酸序列为(EAAAK) n，n为2、3、4或5。本发明所适用连接肽并不限于上述DYKDDDDK、(GGGGS) n及(EAAAK) n。
根据本发明具体实施方式的提高多肽类药物口服给药稳定性的融合蛋白，所述多肽包括GLP-1、EPO、胸腺激素、细胞因子、干扰素、降钙素、肿瘤坏死因子或肿瘤标志物分子。
干扰素是一类糖蛋白，主要用于治疗晚期毛细胞白血病、肾癌、黑色素瘤、Kaposi肉瘤、慢性粒细胞性白血病和中低度恶性非霍奇金淋巴瘤。
降钙素是一种钙调节激素药物，能抑制破骨细胞的生物活性和减少破骨细胞的数量，从而阻止骨量丢失并增加骨量。
胸腺激素指胸腺肽，临床上常用的胸腺肽是从小牛胸腺发现并提纯的有非特异性免疫效应的小分子多肽。胸腺肽可用于治疗各种原发性或继发性T细胞缺陷病，某些自身免疫性疾病，各种细胞免疫功能低下的疾病及肿瘤的辅助治疗。
目前发已批准上市或进行临床研究的基因工程细胞因子药物：包括干扰素(α、β、γ)、白细胞介素系列、集落刺激因子、胰岛素生长因子、肿瘤坏死因子、促红细胞生成素、表皮生长因子、血小板生长因子、成纤维细胞生长因子在、神经生长因子、结缔组织生长因子及心钠素等。
根据本发明具体实施方式的包含编码融合蛋白的基因的重组表达载体。所述重组表达载体为pPICZαA、pPICZαB、pPICZαC中的任一种。
根据本发明具体实施方式的包含编码融合蛋白的基因的重组菌株。其中，表达宿主可以选择大肠杆菌、链霉菌、枯草杆菌、酵母、哺乳动物细胞、昆虫细胞、植物细胞。优选的，表达宿主为毕赤酵母，毕赤酵母的菌株可以为X-33、GS115、KM71、SMD1168、SMD1168H中的任一种。
本发明的融合蛋白由治疗性多肽药物、连接肽、β-甘露聚糖酶及其同源物构成，其可以在原核和真核表达系统中融合表达。β-甘露聚糖酶及其同源物可将甘露聚糖水解成甘露寡糖，甘露寡糖作为一种益生元，能够被动物体内的益生菌吸收代谢，改善肠道菌群。本发明的融合蛋白可以解决多肽类药物不耐胃酸，且易被各种消化道蛋白酶降解的共性瓶颈，实现口服给药，可用于开发多种多肽药物的长效口服制剂。
根据本发明具体实施方式的融合蛋白MANNase-GLP-1，其从N端到C端依次含有β-甘露聚糖酶、连接肽(DYKDDDDK)和GLP-1，其氨基酸序列如SEQ ID No.4所示：
Figure PCTCN2021081484-appb-000006
Figure PCTCN2021081484-appb-000007
根据本发明具体实施方式的包含融合蛋白MANNase-GLP-1基因的重组表达载体，所述重组表达载体为pPICZαA、pPICZαB、pPICZαC中的任一种。
根据本发明具体实施方式的融合蛋白MANNase-GLP-1的制备方法，所述方法包括以下步骤：
(1)用包含融合蛋白MANNase-GLP-1编码基因的重组载体转化宿主细胞，获得重组菌株；
(2)培养重组菌株，诱导融合蛋白MANNase-GLP-1表达；
(3)回收并纯化所表达的融合蛋白MANNase-GLP-1。
具体地，将编码GLP-1、β-甘露聚糖酶基因连接pPICZαA，得到重组表达载体；将重组质粒电转化到毕赤酵母X-33感受态细胞中构建得到重组工程菌，进行诱导表达。将所述重组工程菌进行发酵培养，诱导表达；将得到发酵液进行离心，取上清液依次进行纯化、浓缩、干燥后，即得融合蛋白MANNase-GLP-1。
本发明的有益效果：
本发明的融合蛋白能够显著提高多肽类药物在胃肠道的稳定性，即提高其对胃蛋白酶、胰蛋白酶、胃酸的耐受性，适应温度范围30～80℃，从而显著延长多肽药物在人体内的半衰期，其在人体中半衰期可为自然半衰期的10～5000倍；同时，β-甘露聚糖酶及其同源物可将甘露聚糖水解成甘露寡糖，甘露寡糖能够被动物体内的益生菌吸收代谢，改善肠道菌群，对药物吸收利用起到促进作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附 图。
图1是融合蛋白MANNase-GLP-1对胃蛋白酶耐受性结果；
图2是融合蛋白MANNase-GLP-1对胰蛋白酶耐受性结果；
图3是融合蛋白MANNase-GLP-1的pH稳定性结果；
图4是融合蛋白MANNase-GLP-1的热稳定性结果；
图5为高脂高糖饮食诱导代谢综合征小鼠模型的构建结果，其中，A为以高脂高糖饲料持续饲喂24周后小鼠的空腹血糖变化情况；B为以高脂高糖(HFSD)饲料持续饲喂24周后小鼠的体重变化情况；
图6是普通饮食小鼠和高脂高糖饮食小鼠口服葡萄糖耐量测试结果；
图7为高脂高糖饮食小鼠和正常饮食小鼠的肝脏和脂肪组织的HE染色切片结果；
图8为融合蛋白MANNase-GLP-1口服给药对高脂高糖小鼠血糖和体重的影响结果；
图9为高脂高糖小鼠口服给药MANNase-GLP-1后肝脏组织染色切片结果；
图10为融合蛋白MANNase-GLP-1的三维模拟结构；
图11为融合蛋白MANNase-GLP-1的GLP-1端的残基的结构。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
实施例1制备融合蛋白MANNase-GLP-1
将GLP-1与8个氨基酸的连接肽(DYKDDDDK)进行连接，合成连接肽-GLP-1的编码基因。采用引物对克隆编码β-甘露聚糖酶和连接肽-GLP-1的目的片段，分别进行PCR扩增，然后进行双酶切，之后将得到 的编码GLP-1与甘露聚糖酶的基因序列连接到pPICZαA质粒上，构建得到重组表达载体pPICZαA-MANNase-GLP-1。
其中，扩增GLP-1编码基因的引物对的序列如SEQ ID No.5、SEQ ID No.6所示：
SEQ ID No.5：5'-CGGGATCCGACTACAAGGACGACGACGAC-3'；
SEQ ID No.6：5'-GCTCTAGATTAACCTCTACCTCTAACCA-3'。
扩增β-甘露聚糖酶的编码基因序列的引物对的序列如SEQ ID No.7、SEQ ID No.8所示：
SEQ ID No.7：5'-CGGAATTCTTGCCAAAGGCTTCTCCAGC-3'；
SEQ ID No.8：5'-CGGGATCCAGCAGAATCAATAGCAGCAA-3'。
将重组质粒电转化到毕赤酵母X-33感受态细胞中，构建得到重组工程菌MANNase-GLP-1-X-33，进行诱导表达。将重组工程菌MANNase-GLP-1-X-33的单菌落接种于含有博来霉素的YPD液体培养基试管中，在30℃、200rpm条件下振荡培养12h；将菌液倒入装有YPD培养基中，在30℃、200rpm条件下培养12h，即得一级种子液；将一级种子液按10％的接种量接种于YPD培养基中，在30℃、200rpm条件下培养22h，即得二级种子液；将二级种子液按10％的接种量接入10L种子罐中，再按10％接入50L发酵罐中，进行发酵培养，当发酵液OD 600达到60-120以上时加入诱导剂进行诱导，诱导完成后放罐，离心收集菌体。发酵培养为高密度发酵培养，诱导剂为甲醇，所述诱导剂的加入量为0.2％-3％(V/V)。进行发酵培养时，初始发酵温度为30℃，搅拌速度为300rpm，通气量为4L/min，pH为5.5。
对上清液进行纯化、浓缩、干燥的具体步骤如下：取上清液，先用0.8um的滤膜过滤，再用0.2um的滤膜过滤，收集滤液；将滤液先用超滤膜包浓缩10倍，加去离子水后再浓缩10倍，得到浓缩液；将浓缩液进行冷冻干燥，即得重组融合蛋白MANNase-GLP-1。
实施例2考察融合蛋白MANNase-GLP-1的特性
2.1对胃蛋白酶耐受性
胃蛋白酶溶液的配制：2.0g NaCl、3.2g胃蛋白酶、7mL浓盐酸，蒸馏水定容于1000mL，pH约为1.2。配制方法参照美国1995药典中的人工模拟胃液配方。
酶解反应试验：以蛋白含量1：1的比例配制成融合蛋白MANNase-GLP-1与胃蛋白酶的酶解反应体系，以灭活的蛋白酶作为空白对照，将反应时间设置4个梯度，0min、30min、60min、120min，准确计时，反应结束时，立即加入0.618mol/L的碳酸钠溶液0.05mL，终止酶解反应。每管取50uL酶解反应液于1.5mL离心管中，70℃处理5min灭活酶，适当稀释后测定酶活力。
如图1所示，处理2h后，测得融合蛋白MANNase-GLP-1仍有60％以上酶活，结果表明，融合蛋白MANNase-GLP-1对胃蛋白酶具有较好的耐受性。
2.2对胰蛋白酶耐受性
胰蛋白酶溶液的配制：6.8g KH 2PO 4溶于250mL蒸馏水，完全溶解后加入190mL 0.2mol/L NaOH和400mL的蒸馏水，加胰蛋白酶10.0g，混匀，用0.2mol/L NaOH调pH值到7.5±0.1，蒸馏水定容1000mL，配置方法参见美国1995药典中的人工模拟肠液配方。
酶解反应试验：
以蛋白含量1：1的比例配制成融合蛋白MANNase-GLP-1与胰蛋白酶的酶解反应体系，以灭活的蛋白酶作为空白对照，将反应时间设置4个梯度，0min、30min、60min、120min，准确计时，反应结束时，立即加入30％的冰乙酸溶液0.05mL，终止酶解反应。每管取50uL酶解反应液于1.5mL离心管中，70℃处理5min灭活酶，适当稀释后测定酶活力。
如图2所示，处理2h后，测得融合蛋白MANNase-GLP-1仍有60％以上酶活，结果表明，融合蛋白MANNase-GLP-1对胰蛋白酶具有较好的耐受性。
2.3 pH稳定性
分别配制100mM不同pH的缓冲液，甘氨酸-盐酸(pH2.2～3.2)，柠檬酸-磷酸氢二钠(pH3.2～6.2)，磷酸氢二钠-磷酸二氢那(pH6.2～8.2)，Tris-HCl(pH8.2～9.2)缓冲液，分别用各缓冲液配制酶反应底物0.6％LBG及稀释酶液，50℃时分别测定融合蛋白MANNase-GLP-1在不同pH下的酶活，每组实验三次重复。
如图3所示，融合蛋白MANNase-GLP-1的最适pH为3.2，其在pH为2的条件下中仍保有80％酶活性。
2.4热稳定性
采用pH为3.2的柠檬酸-磷酸氢二钠缓冲液，分别测定融合蛋白MANNase-GLP-1在温度为30～80℃条件下的酶活，每组实验重复三次。
如图4所示，融合蛋白MANNase-GLP-1在60℃具有最大的酶活，数分钟后酶活降为0；其在40℃(接近人体体温)条件下具有很强的耐受性，12h后酶活基本没有变化；50℃条件下，1h后酶活还剩余80％左右，6h后酶活基本降为0。
实施例3验证融合蛋白MANNase-GLP-1的口服效果
3.1构建高脂高糖饮食诱导代谢综合征小鼠模型
100只6周龄(18-20g)C57-6J小鼠(4-6周龄，雄性)，将小鼠分笼饲养，控制动物房温度25±2℃，湿度50±10％，光照12h黑暗12h循环，适应环境一周。将小鼠以5-6只/组随机分笼。所有小鼠禁食12h后测定体重及空腹血糖(Fasting blood-glucose，FBG)，对照组以标准饲料继续饲喂，模型组以高脂高糖(HFSD)饲料持续饲喂24周。结束后，测定各组小鼠体重及FBG。
高脂高糖饮食小鼠的体重约为42.5g左右，正常饮食小鼠体重约30g左右，高脂高糖饮食小鼠的空腹血糖约5.67，正常饮食小鼠的空腹血糖4.62，具有统计学差异。
如图5中A、B所示，经过高脂高糖饮食诱导的C57-6J小鼠，空腹 血糖超过正常饮食22.73％，血糖显著升高；C57-6J小鼠经过高脂高糖饮食诱导24周后，体重超过正常饮食41.67％，符合肥胖模型的标准(20％)( *p<0.05, **p<0.01, ***p<0.001)。经过高脂高糖饮食诱导的C57-6J小鼠，空腹血糖超过正常饮食22.73％，血糖显著升高。
普通饮食小鼠和高脂高糖小鼠过夜禁食12h后，按2g/kg(体重)灌胃D-葡萄糖，分别于0、0.5、1.0、1.5、2.0、2.5h的血糖，得到口服葡萄糖耐量(OGTT)曲线。
结果如图6所示，经过高脂高糖饮食诱导24周后，高脂高糖小鼠的葡萄糖耐量明显受损。
对高脂高糖饮食小鼠和正常饮食小鼠的肝脏和脂肪组织进行HE染色切片分析，如图7所示，高脂高糖饮食的模型小鼠有明显的脂肪肝。
上述实验证明，高脂高糖饮食诱导代谢综合征小鼠模型构建成功。建模成功后，以11只/组进行随机分组，进行口服灌胃实验。
3.2考察融合蛋白MANNase-GLP-1口服给药对高脂高糖饮食诱导的代谢综合症的影响
将小鼠随机分成5组：融合蛋白高剂量组(3.5mg/kg·d)、融合蛋白低剂量组(1.75mg/kg·d)、30mg/kg奥利司他作为正对照组，正常饲料对照组和高脂高糖饲料负对照组均给予相同体积的水，所有小鼠在不改变饮食的情况下，分别进行口服灌胃，测定每周体重变化。
结果如图8所示，与负对照(水)相比，口服灌胃融合蛋白MANNase-GLP-1高剂量组显著降低了体重，与市售奥利司他(Oli)效果接近。
口服葡萄糖耐量是机体对葡萄糖负荷程度的指标，糖耐量受损意味着胰岛β细胞功能和机体对血糖的调节能力下降，糖耐量的临界值是餐后2小时血糖为7.8mmol/L，高于7.8mmol/L代表机体的糖耐量受损。对糖耐量受损小鼠进行不同剂量的MANNase-GLP-1融合蛋白干预后，小鼠的糖耐量水平有了不同程度的改善。高剂量组恢复到正常水平(7.5mmol/L)， 负对照组(水)的餐后2小时血糖高达10mmol/L，可见MANNase-GLP-1融合蛋白对机体的糖代谢有明显的改善作用。
因此，在保证小鼠安全的情况下，MANNase-GLP-1融合蛋白具有明显的减重和改善糖耐量效果。
解剖后取肝脏进行HE染色切片分析，结果如图9所示，融合蛋白MANNase-GLP-1具有明显改善脂肪肝的效果。
实施例4分析融合蛋白MANNase-GLP-1的结构
本发明模拟了融合蛋白MANNase-GLP-1的三维结构，如图10所示。将融合蛋白MANNase-GLP-1整体模型中GLP-1部分结构与3l0L的B链比对，发现GLP-1的主体部分(约22/31)与3IOL的B链同为α-螺旋。
本发明分析融合蛋白MANNase-GLP-1的C-端39个残基与甘露聚糖酶的结构部分形成了相互作用界面。界面的组成中，GLP-1端的39个残基约23％的溶剂可接近面积(ASA，
Figure PCTCN2021081484-appb-000008
)参与了界面相互作用。这种界面相互作用形成了12个氢键及7个盐键，并造成-13.9kcal mol-1结合能的释放，表明融合蛋白MANNase-GLP-1的三维结构是一个能量较低的稳定态，其结构是稳定存在的。
如图11所示，GLP-1端的39个残基的结构还存在着疏水相互作用。
本发明分析GLP-1中存在的若干个胃蛋白酶特异性水解识别位点，如6Phe、13Tyr、14Leu、22Phe及26Leu等残基，形成了内部的疏水相互作用，从而，可有效对抗胃蛋白酶水解。此外，融合蛋白MANNase-GLP-1中的一些芳香环侧链氨基酸也是胰凝乳蛋白酶的识别位点，同样可在一定程度上对抗胃蛋白酶的水解。因此，本发明对融合蛋白MANNase-GLP-1的结构分析与实施例2中对于胃蛋白酶、胰蛋白酶及pH稳定性实验结果相吻合，从实验与理论分析两个角度验证了融合蛋白MANNase-GLP-1的功能。
实施例5构建多种融合蛋白并验证融合蛋白特性
β-甘露聚糖酶除了可与GLP-1融合表达外，还可与多种多肽药物，如抗肿瘤多肽、抗病毒多肽、多肽疫苗、细胞因子模拟肽、抗菌活性肽等进行融合表达，从而延长多肽药物的半衰期。
从三维结构角度分析，一方面，融合表达后的多肽药物结构中原本存在的胃蛋白酶特异性水解识别位点形成了内部疏水结构，有效对抗胃蛋白酶水解；另一方面，融合表达后氨基酸侧链上的特异性蛋白酶识别位点，同样可在一定程度上对抗胃蛋白酶的水解。
本实施例将进一步以促肾上腺皮质激素(ACTH)、依降钙素(CCT)、特利帕肽(TRP)为例，构建其与β-甘露聚糖酶的融合表达过程，并验证融合表达蛋白的效果。
5.1融合蛋白MANNase-ACTH
促肾上腺皮质激素(ACTH)：分子量4541.1，由39个氨基酸构成，治疗风湿性关节炎。氨基酸序列如SEQ ID No.9所示：
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe
设计引物对，采用引物对克隆编码β-甘露聚糖酶和连接肽(EAAAKEAAAK)-ACTH的目的片段，分别进行PCR扩增，然后进行双酶切，之后将得到的编码ACTH与甘露聚糖酶的基因序列连接到pPICZαA质粒上，构建得到重组表达载体pPICZαA-MANNase-ACTH。
将重组质粒电转化到毕赤酵母X-33感受态细胞中构建得到重组工程菌MANNase-ACTH-X-33，进行诱导表达，纯化，即得融合蛋白MANNase-ACTH。
对融合蛋白MANNase-ACTH的胃蛋白酶耐受性、胰蛋白酶耐受性、pH稳定性和热稳定性进行考察，实验方法同实施例2。
结果表明，融合蛋白MANNase-ACTH对胃蛋白酶和胰蛋白酶均具有较好的耐受性。
5.2融合蛋白MANNase-CCT
依降钙素(CCT)：由32个氨基酸构成，分子量为3363.77,目前上市药物为注射液和鼻喷剂，促进骨钙生成，治疗骨质疏松症。
依降钙素的氨基酸序列如SEQ ID No 10所示：
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gin-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro
设计引物对，采用引物对克隆编码β-甘露聚糖酶和连接肽(EAAAKEAAAK)-CCT的目的片段，分别进行PCR扩增，然后进行双酶切，之后将得到的编码CCT与甘露聚糖酶的基因序列连接到pPICZαA质粒上，构建得到重组表达载体pPICZαA-MANNase-CCT。
将重组质粒电转化到毕赤酵母X-33感受态细胞中构建得到重组工程菌MANNase-CCT-X-33，进行诱导表达，纯化，即得融合蛋白MANNase-CCT。
对融合蛋白MANNase-CCT的胃蛋白酶耐受性、胰蛋白酶耐受性、pH稳定性和热稳定性进行考察，实验方法同实施例2。
结果表明，融合蛋白MANNase-CCT对胃蛋白酶和胰蛋白酶均具有较好的耐受性。
5.3融合蛋白MANNase-TRP
特利帕肽(TRP):由34个氨基酸组成，分子量：4177.77，静脉注射治疗骨质疏松症，刺激骨形成和骨吸收。氨基酸序列如SEQ ID No 11所示：
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe
融合蛋白MANNase-TRP的制备过程同实施例2，融合蛋白MANNase-CCT对胃蛋白酶耐受性、胰蛋白酶耐受性、pH稳定性和热稳定性实验同实施例2。
结果表明，融合蛋白MANNase-TRP对胃蛋白酶和胰蛋白酶均具有较好的耐受性。
本发明提供一种提高多肽类药物口服给药稳定性的融合蛋白及其应用。本发明的融合蛋白从N端到C端依次含有β-甘露聚糖酶、连接肽和多肽，其中，所述多肽包括GLP-1、EPO、胸腺激素、细胞因子、干扰素、降钙素、肿瘤坏死因子、肿瘤标志物分子。本发明的融合蛋白克服了多肽类药物自身稳定性差、容易降解的缺陷，具有延长药物半衰期和提高生物利用度的特点，具有较好的经济价值和应用前景。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
工业实用性
本发明提供一种提高多肽类药物口服给药稳定性的融合蛋白及其应用。本发明的融合蛋白从N端到C端依次含有β-甘露聚糖酶、连接肽和多肽，其中，所述多肽包括GLP-1、EPO、胸腺激素、细胞因子、干扰素、降钙素、肿瘤坏死因子、肿瘤标志物分子。本发明的融合蛋白克服了多肽类药物自身稳定性差、容易降解的缺陷，具有延长药物半衰期和提高生物利用度的特点，具有较好的经济价值和应用前景。

Claims (10)

  1. 提高多肽类药物口服给药稳定性的融合蛋白，其特征在于，所述融合蛋白从N端到C端依次含有β-甘露聚糖酶、连接肽和多肽，其中，所述多肽包括抗肿瘤多肽、抗病毒多肽、细胞因子模拟肽、多肽疫苗、抗菌活性肽；β-甘露聚糖酶的氨基酸序列如SEQ ID No.1所示。
  2. 根据权利要求1所示的提高多肽类药物口服给药稳定性的融合蛋白，其特征在于，所述连接肽的氨基酸序列为DYKDDDDK；或者
    所述连接肽的氨基酸序列为(GGGGS)n，n为＝3或4；或者
    所述连接肽的氨基酸序列为(EAAAK)n，n为2、3、4或5。
  3. 根据权利要求1所示的提高多肽类药物口服给药稳定性的融合蛋白，其特征在于，所述多肽包括GLP-1、促红细胞生成素、胸腺激素、细胞因子、干扰素、降钙素、肿瘤坏死因子或肿瘤标志物分子。
  4. 包含编码权利要求1所述的提高多肽类药物口服给药稳定性的融合蛋白的基因的重组表达载体。
  5. 包含编码权利要求1所述的提高多肽类药物口服给药稳定性的融合蛋白的基因的重组菌株。
  6. 权利要求1所述的提高多肽类药物口服给药稳定性的融合蛋白在制备口服剂型药物方面的应用。
  7. 融合蛋白MANNase-GLP-1，其特征在于，其氨基酸序列如SEQ ID No.4所示。
  8. 包含编码权利要求7所述的融合蛋白MANNase-GLP-1基因的重组表达载体。
  9. 权利要求7所述的融合蛋白MANNase-GLP-1的制备方法，其特征在于，所述方法包括以下步骤：
    (1)用包含融合蛋白MANNase-GLP-1编码基因的重组载体转化宿主细胞，获得重组菌株；
    (2)培养重组菌株，诱导融合蛋白MANNase-GLP-1表达；
    (3)回收并纯化所表达的融合蛋白MANNase-GLP-1。
  10. 权利要求7所述的融合蛋白MANNase-GLP-1在制备GLP-1口服药物方面的应用。
PCT/CN2021/081484 2020-11-13 2021-03-18 提高多肽类药物口服给药稳定性的融合蛋白及其应用 WO2022099963A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1845989A (zh) * 2003-09-11 2006-10-11 诺和酶股份有限公司 抗微生物剂的重组产生
CN111909955A (zh) * 2020-08-10 2020-11-10 安徽新熙盟生物科技有限公司 重组MANNase-GLP-1及同源物的制备及应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1845989A (zh) * 2003-09-11 2006-10-11 诺和酶股份有限公司 抗微生物剂的重组产生
CN111909955A (zh) * 2020-08-10 2020-11-10 安徽新熙盟生物科技有限公司 重组MANNase-GLP-1及同源物的制备及应用

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