CN114478795B - Fusion protein for improving stability of oral administration of polypeptide drugs and application thereof - Google Patents

Fusion protein for improving stability of oral administration of polypeptide drugs and application thereof Download PDF

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CN114478795B
CN114478795B CN202110188492.1A CN202110188492A CN114478795B CN 114478795 B CN114478795 B CN 114478795B CN 202110188492 A CN202110188492 A CN 202110188492A CN 114478795 B CN114478795 B CN 114478795B
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fusion protein
mannase
ser
polypeptide
glp
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CN114478795A (en
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徐冲
楼慧强
余卫雄
吴雷
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Anhui Xinximeng Biotechnology Co ltd
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Anhui Xinximeng Biotechnology Co ltd
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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
    • A—HUMAN NECESSITIES
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    • 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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    • A61K38/00—Medicinal preparations containing peptides
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    • 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
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    • 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
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
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    • C12N9/2477—Hemicellulases not provided in a preceding group
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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a fusion protein for improving the oral administration stability of polypeptide drugs and application thereof. The fusion protein sequentially comprises beta-mannase, connecting peptide and polypeptide from N end to C end, wherein the polypeptide comprises GLP-1, EPO, thymus hormone, cytokine, interferon, calcitonin, tumor necrosis factor and tumor marker molecules. The fusion protein overcomes the defects of poor self stability and easy degradation of polypeptide drugs, and has the characteristics of prolonging the half life of the drugs and improving the bioavailability.

Description

Fusion protein for improving stability of oral administration of polypeptide drugs and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a fusion protein for improving the oral administration stability of polypeptide drugs and application thereof.
Background
Most polypeptides have specific biological activities, and with the increasing maturation of biotechnology and polypeptide synthesis technology, the variety of polypeptide drugs which are developed and marketed is increasing, and more than one hundred of polypeptides are currently developed. The polypeptide medicine has wide application in the treatment field of diseases of an internal secretion system, an immune system, a digestive system, a cardiovascular system and the like, and has good treatment effect on chronic diseases such as tumors, diabetes, hepatitis and the like. However, most polypeptide drugs have a short half-life and are rapidly cleared by protease degradation and glomerular filtration. Therefore, in order to improve the drug effect of the polypeptide drug, the drug can only be administered by frequent injection, which brings inconvenience and pain to patients and greatly limits the clinical application.
When the polypeptide medicine is orally taken, the bioavailability is lower than 1%, which is mainly caused by the reasons of poor stability, small intestine mucosa epithelial barrier, malabsorption and the like of the polypeptide medicine. These factors are described below:
(1) Stability is poor: polypeptide drugs are affected by various factors such as protease, organic solvent, temperature, pH, microorganism, etc. in the gastrointestinal tract, and thus, polypeptide drugs are easily inactivated during absorption and release.
(2) Small intestine mucosal epithelial barrier: the transmembrane absorption of polypeptides is mainly mediated by receptor-mediated transport and cell gap diffusion. Receptor-mediated transport requires specific protein molecules, while polypeptides are polar molecules and do not readily pass through liposoluble vascular mucosa. Therefore, cell gap diffusion (transport through tight junctions between cells) becomes the primary route of absorption of polypeptide drugs. However, the aperture between the epithelial cells of the intestinal mucosa in the human body is 0.4nm, and the polypeptide medicine has larger molecular weight, poor fat solubility and difficult penetration into the blood circulation through the membrane hole only through amino acid, dipeptide and tripeptide.
(3) Absorption disorders: after the drug is administrated through the gastrointestinal tract, the drug is metabolized in intestinal mucosa and liver before being absorbed into the blood circulation, the bile content, the mucus layer on the surface of the gastrointestinal tract and the mechanical barrier effect of a non-flowing water layer and the instability of the self-conformation of the peptide drug lead to the reduction of the drug quantity entering the blood circulation, and the effective blood concentration can not be maintained, thus the bioavailability is low.
In order to overcome the problem of low oral bioavailability of polypeptide drugs, the prior art adopts a certain preparation process such as methods of enzyme inhibitors, absorption promoters, chemical modification and the like, and also carries out delivery through special systems such as systems of emulsion, liposome, microsphere, nanoparticle and the like. However, the above methods have drawbacks, for example, enzyme inhibitors have many side effects, which disturb the digestion and absorption of nutrients by the body, and the enzyme inhibitors must be released simultaneously with or earlier than the drug to exert the inhibitory effect. Absorption enhancers can reversibly remove or temporarily disrupt the barrier of the gastrointestinal tract with minimal damage to tissue, but oral administration is not possible for drugs with a short half-life. Glucagon-like peptide-1 (GLP-likepeptide-1) is a cytokine mimetic peptide, not only has excellent hypoglycemic effect, but also has the characteristics of controlling weight, regulating blood fat, bidirectionally regulating islet beta cell function and the like, but the half-life of natural GLP-1 is only 1.5-2.1 minutes, and the structure of GLP-1 is subjected to fatty acid side chain modification and fusion with macromolecular protein, so that the half-life of the macromolecular protein is prolonged. If the dolapride and the Abirudin are respectively fused with G4 immune albumin and serum albumin, the half life of drug metabolism is prolonged, and injection can be realized once a week, but adverse reaction at the injection site can occur after injection, and oral administration can not be realized yet.
Disclosure of Invention
The invention aims to provide a fusion protein for improving the stability of oral administration of polypeptide drugs.
It is still another object of the present invention to provide a recombinant expression vector containing the above fusion protein.
It is still another object of the present invention to provide a recombinant strain containing the above fusion protein.
It is a further object of the present invention to provide the use of the fusion protein described above.
It is a further object of the present invention to provide a fusion protein MANNase-GLP-1.
It is still another object of the present invention to provide a recombinant expression vector containing the above-mentioned fusion protein MANNase-GLP-1.
It is still another object of the present invention to provide a method for producing the above-mentioned fusion protein MANNase-GLP-1.
It is a further object of the present invention to provide the use of the above fusion protein MANNase-GLP-1.
According to the specific embodiment of the invention, the fusion protein for improving the oral administration stability of the polypeptide drug sequentially comprises beta-mannase, connecting peptide and polypeptide from N end to C end, wherein the polypeptide comprises anti-tumor polypeptide, antiviral polypeptide, polypeptide vaccine, cytokine mimic peptide and antibacterial active peptide.
Such polypeptides include interferons, insulin growth factors, interleukin-series, tumor necrosis factor, fibroblast growth factor, EPO (erythropoietin), adrenocorticotropic hormone (ACTH), calcitonin which promotes bone calcium production, teriparatide which stimulates bone formation and bone resorption, corticotropin Releasing Factor (CRF), erythropoietin (EPO) which stimulates and regulates the production and maturation of erythrocytes, granulocyte colony stimulating factor, nerve growth factor, human growth hormone for the treatment of senile diseases and dwarfism, luteinizing hormone for the treatment of prostate cancer and reproductive system tumors, vascular endothelial inhibin for the treatment of non-small cell lung cancer, and the like.
The amino acid sequence of the beta-mannanase is shown as SEQ ID No. 1.
SEQ ID No.1：
LPKASPAPSTSSSSASTSFASTSGLQFTIDGETGYFAGTNSYWIGFLTDDSDVDLVMSHLKSSGLKILRVWGFNDVTTQPSSGTVWYQLHQDGKSTINTGADGLQRLDYVVSSAEQHGIKLIINFVNYWTDYGGMSAYVSAYGGSDETDFYTSDTMQSAYQTYIKTVVERYSNSSAVFAWELANEPRCPSCDTTVLYDWIEKTSKFIKGLDADHMVCIGDEGFGLNTDSDGSYPYQFAEGLNFTMNLGIDTIDFATLHLYPDSWGTSDDWGNGWISAHGAACKAAGKPCLLEEYGVTSNHCSVESPWQQTALNTTGVSADLFWQYGDDLSTGESPDDGNTIYYGTSDYECLVTDHVAAIDSA
According to a specific embodiment of the invention, the amino acid of the β -mannanase is an active protein having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 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% of the amino acid sequence shown in SEQ ID No. 1; or the beta-mannanase may be a derivative having the amino acid sequence shown in SEQ ID No.1, obtained by substitution, deletion and/or insertion of one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9) amino acid residues, and still having beta-mannanase activity.
The nucleotide sequence of the gene of the beta-mannanase is shown as SEQ ID No.2 ：TTGCCAAAGGCTTCTCCAGCTCCATCTACTTCTTCTTCTTCTGCTTCTACTTCTTTTGCTTCTACTTCTGGTTTGCAATTTACTATTGATGGTGAAACTGGTTACTTTGCTGGTACTAACTCTTACTGGATTGGTTTTTTGACTGATGATTCTGATGTTGATTTGGTTATGTCTCATTTGAAGTCTTCTGGTTTGAAGATTTTGAGAGTTTGGGGTTTTAACGATGTTACTACTCAACCATCTTCTGGTACTGTTTGGTACCAATTGCATCAAGATGGTAAGTCTACTATTAACACTGGTGCTGATGGTTTGCAAAGATTGGATTACGTTGTTTCTTCTGCTGAACAACATGGTATTAAGTTGATTATTAACTTTGTTAACTACTGGACTGATTACGGTGGTATGTCTGCTTACGTTTCTGCTTACGGTGGTTCTGATGAAACTGATTTTTACACTTCTGATACTATGCAATCTGCTTACCAAACTTACATTAAGACTGTTGTTGAAAGATACTCTAACTCTTCTGCTGTTTTTGCTTGGGAATTGGCTAACGAACCAAGATGTCCATCTTGTGATACTACTGTTTTGTACGATTGGATTGAAAAGACTTCTAAGTTTATTAAGGGTTTGGATGCTGATCATATGGTTTGTATTGGTGATGAAGGTTTTGGTTTGAACACTGATTCTGATGGTTCTTACCCATACCAATTTGCTGAAGGTTTGAACTTTACTATGAACTTGGGTATTGATACTATTGATTTTGCTACTTTGCATTTGTACCCAGATTCTTGGGGTACTTCTGATGATTGGGGTAACGGTTGGATTTCTGCTCATGGTGCTGCTTGTAAGGCTGCTGGTAAGCCATGTTTGTTGGAAGAATACGGTGTTACTTCTAACCATTGTTCTGTTGAATCTCCATGGCAACAAACTGCTTTGAACACTACTGGTGTTTCTGCTGATTTGTTTTGGCAATACGGTGATGATTTGTCTACTGGTGAATCTCCAGATGATGGTAACACTATTTACTACGGTACTTCTGATTACGAATGTTTGGTTACTGATCATGTTGCTGCTATTGAT
Glucagon-like peptide-1 (glucon-likepeptide-1, GLP-1) with the nucleotide sequence of the coding gene shown in SEQ ID No. 3:
CACGCTGA AGGTACCTTC ACCTCTGACG TTTCTTCTTA CCTGGAAGGT CAGGCTGCTA AAGAATTCAT CGCTTGGCTG GTTCGTGGTC GTGG
According to the fusion protein of the specific embodiment of the invention, the amino acid sequence of the connecting peptide is DYKDDDDK; or the amino acid sequence of the connecting peptide is (GGGGS) n, n is=3 or 4; or the amino acid sequence of the connecting peptide is (EAAAK) n, and n is 2,3, 4 or 5. The connecting peptide to which the present invention is applicable is not limited to DYKDDDDK, (GGGGS) n and (EAAAK) n described above.
According to a specific embodiment of the invention, the fusion protein for improving the stability of oral administration of the polypeptide drugs comprises GLP-1, EPO, thymus hormone, cytokine, interferon, calcitonin, tumor necrosis factor or tumor marker molecules.
Interferon is a glycoprotein, and is mainly used for treating advanced hairy cell leukemia, renal carcinoma, melanoma, kaposi sarcoma, chronic granulocytic leukemia and medium-low malignant 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 loss of bone mass and increasing bone mass.
Thymic hormone refers to thymic peptide, and the thymic peptide commonly used clinically is a small molecule polypeptide which is discovered and purified from calf thymus and has a nonspecific immune effect. The thymus peptide can be used for treating various primary or secondary T cell deficiency diseases, certain autoimmune diseases, various diseases with low cellular immunity and adjuvant treatment of tumor.
Genetically engineered cytokine drugs are currently approved for marketing or clinical research: including interferons (α, β, γ), interleukin series, colony stimulating factors, insulin growth factors, tumor necrosis factors, erythropoietin, epidermal growth factors, platelet growth factors, fibroblast growth factors, nerve growth factors, connective tissue growth factors, and atrial natriuretic factors.
Recombinant expression vectors comprising genes encoding fusion proteins according to embodiments of the invention. The recombinant expression vector is any one of pPICZ alpha A, pPICZ alpha B, pPICZ alpha C.
Recombinant strains comprising genes encoding fusion proteins according to embodiments of the invention. Wherein the expression host can be selected from Escherichia coli, streptomyces, bacillus subtilis, yeast, mammalian cells, insect cells, and plant cells. Preferably, the expression host is Pichia pastoris, and the strain of Pichia pastoris can be any one of X-33, GS115, KM71, SMD1168 and SMD 1168H.
The fusion protein of the invention is composed of therapeutic polypeptide drugs, connecting peptides, beta-mannanase and homologues thereof, and can be fusion expressed in prokaryotic and eukaryotic expression systems. The beta-mannanase and the homologue thereof can hydrolyze the mannans into mannooligosaccharides, and the mannooligosaccharides serve as a prebiotic, can be absorbed and metabolized by probiotics in animals, and improve intestinal flora. The fusion protein can solve the common bottleneck that polypeptide medicines are intolerant to gastric acid and are easy to degrade by various digestive tract proteases, realizes oral administration, and can be used for developing long-acting oral preparations of various polypeptide medicines.
The fusion protein MANNase-GLP-1 according to the specific embodiment of the invention sequentially contains beta-mannase, connecting peptide (DYKDDDDK) and GLP-1 from the N end to the C end, and the amino acid sequence of the fusion protein is shown as SEQ ID No. 4:
LPKASPAPSTSSSSASTSFASTSGLQFTIDGETGYFAGTNSYWIGFLTDDSDVDLVMSHLKSSGLKILRVWGFNDVTTQPSSGTVWYQLHQDGKSTINTGADGLQRLDYVVSSAEQHGIKLIINFVNYWTDYGGMSAYVSAYGGSDETDFYTSDTMQSAYQTYIKTVVERYSNSSAVFAWELANEPRCPSCDTTVLYDWIEKTSKFIKGLDADHMVCIGDEGFGLNTDSDGSYPYQFAEGLNFTMNLGIDTIDFATLHLYPDSWGTSDDWGNGWISAHGAACKAAGKPCLLEEYGVTSNHCSVESPWQQTALNTTGVSADLFWQYGDDLSTGESPDDGNTIYYGTSDYECLVTDHVAAIDSADYKDDDDKHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG
A recombinant expression vector comprising a fusion protein MANNase-GLP-1 gene according to a specific embodiment of the invention is any one of ppiczα A, pPICZ α B, pPICZ αc.
A method for preparing a fusion protein MANNase-GLP-1 according to a specific embodiment of the invention, said method comprising the steps of:
(1) Transforming a host cell with a recombinant vector comprising a gene encoding fusion protein MANNase-GLP-1 to obtain a recombinant strain;
(2) Culturing the recombinant strain, and inducing expression of the fusion protein MANNase-GLP-1;
(3) Recovering and purifying the expressed fusion protein MANNase-GLP-1.
Specifically, genes encoding GLP-1 and beta-mannase are connected with pPICZ alpha A to obtain a recombinant expression vector; and electrically converting the recombinant plasmid into pichia pastoris X-33 competent cells to construct recombinant engineering bacteria, and carrying out induced expression. Fermenting and culturing the recombinant engineering bacteria, and inducing expression; and centrifuging the obtained fermentation liquor, and sequentially purifying, concentrating and drying the supernatant to obtain the fusion protein MANNase-GLP-1.
The invention has the beneficial effects that:
The fusion protein can obviously improve the stability of the polypeptide medicine in the gastrointestinal tract, namely the tolerance of the polypeptide medicine to pepsin, trypsin and gastric acid, and the adaptation temperature is 30-80 ℃, so that the half life of the polypeptide medicine in the human body is obviously prolonged, and the half life of the polypeptide medicine in the human body can be 10-5000 times of the natural half life; meanwhile, the beta-mannase and the homologue thereof can hydrolyze the mannans into mannooligosaccharides, and the mannooligosaccharides can be absorbed and metabolized by probiotics in animals, so that intestinal flora is improved, and the medicine absorption and utilization are promoted.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the result of pepsin tolerance of fusion protein MANNase-GLP-1;
FIG. 2 is a graph showing the results of the tolerance of fusion protein MANNase-GLP-1 to trypsin;
FIG. 3 is a pH stability result of fusion protein MANNase-GLP-1;
FIG. 4 is a thermal stability result of fusion protein MANNase-GLP-1;
FIG. 5 shows the results of constructing a mouse model of the high-fat high-sugar diet-induced metabolic syndrome, wherein A is the change of fasting blood glucose of the mice after continuous feeding with the high-fat high-sugar diet for 24 weeks; b is the weight change condition of mice after being continuously fed with high-fat and high-sugar (HFSD) feed for 24 weeks;
FIG. 6 is the results of oral glucose tolerance test for normal diet mice and high fat high sugar diet mice;
FIG. 7 shows the results of HE staining sections of liver and adipose tissue of high-fat high-sugar diet mice and normal diet mice;
FIG. 8 is a graph showing the effect of oral administration of fusion protein MANNase-GLP-1 on blood glucose and body weight in high-fat, high-sugar mice;
FIG. 9 shows the results of liver tissue staining sections after oral administration MANNase-GLP-1 to high-fat high-sugar mice;
FIG. 10 is a three-dimensional mimetic structure of fusion protein MANNase-GLP-1;
FIG. 11 shows the structure of residues at the GLP-1 end of fusion protein MANNase-GLP-1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
EXAMPLE 1 preparation of fusion protein MANNase-GLP-1
GLP-1 is connected with a connecting peptide (DYKDDDDK) of 8 amino acids to synthesize a coding gene of the connecting peptide GLP-1. The target fragments of beta-mannase and connecting peptide GLP-1 are cloned by adopting a primer pair, PCR amplification is respectively carried out, double enzyme digestion is carried out, and then the obtained gene sequences of GLP-1 and mannase are connected to pPICZ alpha A plasmid, so that a recombinant expression vector pPICZ alpha A-MANNase-GLP-1 is constructed.
Wherein, the primer pair for amplifying GLP-1 coding genes has the sequences shown in SEQ ID No.5 and SEQ ID No. 6:
SEQ ID No.5：5'-CGGGATCCGACTACAAGGACGACGACGAC-3'；
SEQ ID No.6：5'-GCTCTAGATTAACCTCTACCTCTAACCA-3'。
The primer pair for amplifying the coding gene sequence of the beta-mannanase has the sequences 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'。
And (3) electrically transforming the recombinant plasmid into pichia pastoris X-33 competent cells, constructing recombinant engineering bacteria MANNase-GLP-1-X-33, and carrying out induced expression. Inoculating single colony of recombinant engineering bacteria MANNase-GLP-1-X-33 into YPD liquid culture medium test tube containing bleomycin, and shake culturing at 30deg.C and 200rpm for 12 hr; pouring the bacterial liquid into YPD medium, culturing at 30deg.C and 200rpm for 12 hr to obtain first-stage seed liquid; inoculating the first-stage seed solution into YPD culture medium according to 10% of inoculation amount, and culturing at 30deg.C and 200rpm for 22 hr to obtain second-stage seed solution; the second-level seed liquid is inoculated into a 10L seed tank according to 10 percent of inoculation amount, then is inoculated into a 50L fermentation tank according to 10 percent, fermentation culture is carried out, an inducer is added for induction when the OD 600 of the fermentation liquid reaches over 60-120, and after the induction is finished, the seed liquid is placed into the tank, and thalli are centrifugally collected. 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). When fermentation culture was carried out, the initial fermentation temperature was 30℃and the stirring speed was 300rpm, the aeration rate was 4L/min and the pH was 5.5.
The specific steps of purifying, concentrating and drying the supernatant are as follows: filtering the supernatant with 0.8um filter membrane, filtering with 0.2um filter membrane, and collecting filtrate; concentrating the filtrate by ultrafiltration membrane bag for 10 times, adding deionized water, and concentrating for 10 times to obtain concentrated solution; and freeze-drying the concentrated solution to obtain the recombinant fusion protein MANNase-GLP-1.
Example 2 investigation of the Properties of fusion protein MANNase-GLP-1
2.1 Resistance to pepsin
Preparation of pepsin solution: 2.0g NaCl, 3.2g pepsin, 7mL concentrated hydrochloric acid, distilled water to 1000mL pH about 1.2. The formulation method refers to the artificial simulated gastric fluid formulation in the United states 1995 pharmacopoeia.
Enzymolysis reaction test: protein content 1:1, preparing an enzymolysis reaction system of the fusion protein MANNase-GLP-1 and pepsin, taking inactivated protease as a blank control, setting 4 gradients for reaction time, 0min, 30min, 60min and 120min, accurately timing, and immediately adding 0.05mL of 0.618mol/L sodium carbonate solution when the reaction is finished, and stopping the enzymolysis reaction. 50uL of enzymolysis reaction liquid is taken from each tube, the enzymolysis reaction liquid is treated for 5min at 70 ℃ in a 1.5mL centrifuge tube, and enzyme activity is measured after proper dilution.
As shown in FIG. 1, after 2 hours of treatment, more than 60% of the enzyme activity of the fusion protein MANNase-GLP-1 is measured, and the result shows that the fusion protein MANNase-GLP-1 has better tolerance to pepsin.
2.2 Trypsin tolerance
Preparing trypsin solution: 6.8g KH 2PO4 is dissolved in 250mL distilled water, 190mL of 0.2mol/L NaOH and 400mL distilled water are added after complete dissolution, 10.0g of trypsin is added, the mixture is uniformly mixed, the pH value is adjusted to 7.5+/-0.1 by using 0.2mol/L NaOH, the distilled water is used for fixing the volume to 1000mL, and the preparation method is referred to an artificial simulated intestinal juice formula in United states pharmacopoeia 1995.
Enzymolysis reaction test:
Protein content 1:1, preparing an enzymolysis reaction system of the fusion protein MANNase-GLP-1 and trypsin, taking inactivated protease as a blank control, setting 4 gradients for reaction time, and accurately timing 0min, 30min, 60min and 120min, and immediately adding 0.05mL of 30% glacial acetic acid solution when the reaction is finished, so as to terminate the enzymolysis reaction. 50uL of enzymolysis reaction liquid is taken from each tube, the enzymolysis reaction liquid is treated for 5min at 70 ℃ in a 1.5mL centrifuge tube, and enzyme activity is measured after proper dilution.
As shown in FIG. 2, after 2h treatment, more than 60% of the enzyme activity of the fusion protein MANNase-GLP-1 is measured, and the result shows that the fusion protein MANNase-GLP-1 has better tolerance to trypsin.
2.3PH stability
Respectively preparing 100mM buffers with different pH values, glycine-hydrochloric acid (pH 2.2-3.2), citric acid-disodium hydrogen phosphate (pH 3.2-6.2), disodium hydrogen phosphate-disodium hydrogen phosphate (pH 6.2-8.2), tris-HCl (pH 8.2-9.2) buffers, respectively preparing enzyme reaction substrates of 0.6% LBG and diluted enzyme solutions by using the buffers, respectively measuring the enzyme activities of the fusion protein MANNase-GLP-1 at different pH values at 50 ℃, and repeating each group of experiments three times.
As shown in FIG. 3, the optimal pH of fusion protein MANNase-GLP-1 is 3.2, which retains 80% of the enzyme activity under conditions of pH 2.
2.4 Thermal stability
The enzyme activity of the fusion protein MANNase-GLP-1 at 30-80 ℃ is determined by adopting a citric acid-disodium hydrogen phosphate buffer solution with the pH value of 3.2, and each experiment is repeated three times.
As shown in FIG. 4, the fusion protein MANNase-GLP-1 has the maximum enzyme activity at 60 ℃, and the enzyme activity is reduced to 0 after a few minutes; the preparation has strong tolerance under the condition of 40 ℃ (approaching the body temperature), and the enzyme activity is basically unchanged after 12 hours; at 50 ℃, the enzyme activity is about 80% after 1h, and the enzyme activity is basically reduced to 0 after 6 h.
Example 3 verification of the oral Effect of fusion protein MANNase-GLP-1
3.1 Construction of a mouse model for high-fat high-sugar diet-induced Metabolic syndrome
100C 57-6J mice (18-20 g) with age of 6 weeks (4-6 weeks old, male) are kept in separate cages, the temperature of the animal house is controlled to be 25+/-2 ℃, the humidity is controlled to be 50+/-10%, and the light is applied for 12 hours and the darkness is circulated for 12 hours, so that the environment is suitable for one week. Mice were randomly caged at 5-6 mice/group. All mice were fasted for 12h and body weight and fasting glucose (FBG) were measured, the control group was fed with standard diet and the model group was fed with high fat and high glucose (HFSD) diet for 24 weeks. After completion, the body weight and FBG of each group of mice were measured.
The weight of the high-fat high-sugar diet mice is about 42.5g, the weight of the normal diet mice is about 30g, the fasting blood glucose of the high-fat high-sugar diet mice is about 5.67, and the fasting blood glucose of the normal diet mice is 4.62, which have statistical differences.
As shown in figure 5A, B, C57-6J mice induced by the high-fat high-sugar diet had fasting blood glucose exceeding 22.73% of normal diet, with significant elevation of blood glucose; after 24 weeks of induction with a high fat and high sugar diet, the C57-6J mice had body weight exceeding 41.67% of normal diet, meeting the criteria for obesity model (20%) (*p<0.05,**p<0.01,*** p < 0.001). C57-6J mice induced by the high-fat high-sugar diet had fasting blood glucose exceeding that of the normal diet by 22.73%, and had significantly increased blood glucose.
After the ordinary diet mice and the high-fat and high-sugar mice were fasted overnight for 12 hours, 2g/kg (body weight) of gastric D-glucose was infused with blood glucose at 0, 0.5, 1.0, 1.5, 2.0, 2.5 hours, respectively, to obtain an oral glucose tolerance (OGTT) curve.
The results are shown in FIG. 6, and the glucose tolerance of the high-fat high-sugar mice was significantly impaired after 24 weeks of induction with the high-fat high-sugar diet.
HE staining section analysis was performed on liver and adipose tissue of high-fat high-sugar diet mice and normal diet mice, and as shown in fig. 7, model mice of high-fat high-sugar diet had significant fatty liver.
The experiment proves that the high-fat high-sugar diet induced metabolic syndrome mouse model is successfully constructed. After modeling was successful, the oral gavage experiments were performed with a random grouping of 11 animals/group.
3.2 Examination of the Effect of oral administration of fusion protein MANNase-GLP-1 on high-fat high-sugar diet-induced Metabolic syndrome
Mice were randomly divided into 5 groups: the high dose group (3.5 mg/kg.d) of the fusion protein, the low dose group (1.75 mg/kg.d) of the fusion protein and 30mg/kg of orlistat were used as positive control groups, and the normal feed control group and the high-fat and high-sugar feed negative control group were administered with the same volume of water, and all mice were orally administered with gastric lavage respectively without changing diet, and the change in body weight per week was measured.
The results are shown in fig. 8, where oral gavage fusion protein MANNase-GLP-1 high dose group significantly reduced body weight compared to negative control (water), and was close to the effect of commercially available orlistat (Oli).
Glucose tolerance by oral administration is an index of the glucose load degree of the organism, and impaired glucose tolerance means that the functions of islet beta cells and the regulating capacity of the organism on blood sugar are reduced, and the critical value of glucose tolerance is that the blood sugar is 7.8mmol/L after 2 hours of meal, and the condition that the glucose tolerance is higher than 7.8mmol/L is represented by impaired glucose tolerance of the organism. Mice with impaired glucose tolerance were subjected to different doses of MANNase-GLP-1 fusion protein dry prognosis, and their glucose tolerance levels were improved to different extents. The high dose group returns to normal level (7.5 mmol/L), and the blood sugar of the negative control group (water) is as high as 10mmol/L after 2 hours of meal, so that MANNase-GLP-1 fusion protein has obvious improvement effect on the sugar metabolism of organisms.
Therefore, under the condition of ensuring the safety of mice, MANNase-GLP-1 fusion protein has obvious weight reduction and glucose tolerance improvement effects.
The liver was taken after dissection and analyzed for HE staining sections, and the results are shown in FIG. 9, and the fusion protein MANNase-GLP-1 has the effect of obviously improving fatty liver.
EXAMPLE 4 analysis of the Structure of fusion protein MANNase-GLP-1
The present invention mimics the three-dimensional structure of fusion protein MANNase-GLP-1 as shown in FIG. 10. The structure of the GLP-1 moiety in the fusion protein MANNase-GLP-1 whole model was aligned with the B chain of 3L0L, and the main body portion (about 22/31) of GLP-1 was found to be alpha-helical with the B chain of 3 IOL.
The invention analyzes that the C-terminal 39 residues of the fusion protein MANNase-GLP-1 and the structural part of mannanase form an interaction interface. In the composition of the interface, the 39 residues at the GLP-1 end are about 23% of the solvent accessible area (ASA, 776.8) Takes part in the interface interactions. This interfacial interaction forms 12 hydrogen bonds and 7 salt bonds and results in the release of the-13.9 kcal mol-1 binding energy, indicating that the three-dimensional structure of fusion protein MANNase-GLP-1 is a lower energy steady state, and that its structure is stable.
As shown in FIG. 11, there is also a hydrophobic interaction with the 39 residues structure of the GLP-1 terminus.
The invention analyzes a plurality of pepsin specific hydrolysis recognition sites existing in GLP-1, such as residues of 6Phe, 13Tyr, 14Leu, 22Phe, 26Leu and the like, and forms internal hydrophobic interaction, thereby being capable of effectively resisting pepsin hydrolysis. In addition, some of the aromatic side chain amino acids in fusion protein MANNase-GLP-1 are also recognition sites for chymotrypsin, and also resist hydrolysis by pepsin to some extent. Therefore, the structural analysis of the fusion protein MANNase-GLP-1 is consistent with the experimental results of pepsin, trypsin and pH stability in the embodiment 2, and the function of the fusion protein MANNase-GLP-1 is verified from two angles of experimental and theoretical analysis.
Example 5 construction of various fusion proteins and verification of fusion protein Properties
The beta-mannase can be fused and expressed with GLP-1, and can also be fused and expressed with various polypeptide medicaments, such as anti-tumor polypeptides, antiviral polypeptides, polypeptide vaccines, cytokine mimetic peptides, antimicrobial active peptides and the like, so that the half-life period of the polypeptide medicaments is prolonged.
On one hand, the pepsin specific hydrolysis recognition site originally existing in the fusion-expressed polypeptide drug structure forms an internal hydrophobic structure to effectively resist pepsin hydrolysis; on the other hand, the specific protease recognition site on the amino acid side chain after fusion expression can also resist hydrolysis of pepsin to some extent.
This example will further take adrenocorticotropic hormone (ACTH), elcalcitonin (CCT), and ter Li Patai (TRP) as examples, construct a fusion expression process with β -mannase, and verify the effect of the fusion expression protein.
5.1 Fusion protein MANNase-ACTH
Adrenocorticotropic hormone (ACTH): has molecular weight 4541.1 and is composed of 39 amino acids, and can be used for treating rheumarthritis. The amino acid sequence is shown as SEQ ID No. 9:
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
Designing a primer pair, cloning target fragments for coding beta-mannase and connecting peptide (EAAAKEAAAK) -ACTH by using the primer pair, respectively carrying out PCR amplification, then carrying out double enzyme digestion, and then connecting the obtained gene sequences for coding the ACTH and the mannase to a pPICZ alpha A plasmid to construct a recombinant expression vector pPICZ alpha A-MANNase-ACTH.
And electrically transforming the recombinant plasmid into competent cells of Pichia pastoris X-33 to construct recombinant engineering bacteria MANNase-ACTH-X-33, performing induced expression, and purifying to obtain the fusion protein MANNase-ACTH.
The pepsin tolerance, trypsin tolerance, pH stability and thermostability of the fusion protein MANNase-ACTH were examined and the experimental procedure was as in example 2.
The results show that the fusion protein MANNase-ACTH has better tolerance to pepsin and trypsin.
5.2 Fusion proteins MANNase-CCT
Elytalciferin (CCT): comprises 32 amino acids, has molecular weight of 3363.77, is currently marketed as injection and nasal spray, and can promote bone calcium formation and treat osteoporosis.
The amino acid sequence of the elytalciferin is shown in SEQ ID No 10:
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
Designing a primer pair, cloning target fragments for encoding beta-mannase and connecting peptide (EAAAKEAAAK) -CCT by using the primer pair, respectively carrying out PCR amplification, then carrying out double enzyme digestion, and then connecting the obtained gene sequences for encoding the CCT and the mannase to pPICZ alpha A plasmid to construct and obtain a recombinant expression vector pPICZ alpha A-MANNase-CCT.
And (3) electrically converting the recombinant plasmid into pichia pastoris X-33 competent cells to construct recombinant engineering bacteria MANNase-CCT-X-33, performing induced expression, and purifying to obtain the fusion protein MANNase-CCT.
Pepsin tolerance, trypsin tolerance, pH stability and thermostability of the fusion protein MANNase-CCT were examined and the experimental procedure was as in example 2.
The result shows that the fusion protein MANNase-CCT has better tolerance to pepsin and trypsin.
5.3 Fusion protein MANNase-TRP
Japanese patent Li Patai (TRP) consisting of 34 amino acids, molecular weight: 4177.77 intravenous injection for the treatment of osteoporosis, stimulation of bone formation and bone resorption. The amino acid sequence is shown in SEQ ID No 11:
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
The preparation process of the fusion protein MANNase-TRP is the same as in example 2, and the experiments of pepsin tolerance, trypsin tolerance, pH stability and thermal stability of the fusion protein MANNase-CCT are the same as in example 2.
The results show that the fusion protein MANNase-TRP has better tolerance to pepsin and trypsin.
The invention provides a fusion protein for improving the stability of oral administration of polypeptide drugs and application thereof. The fusion protein sequentially comprises beta-mannase, connecting peptide and polypeptide from N end to C end, wherein the polypeptide comprises GLP-1, EPO, thymus hormone, cytokine, interferon, calcitonin, tumor necrosis factor and tumor marker molecules. The fusion protein overcomes the defects of poor self stability and easy degradation of polypeptide drugs, has the characteristics of prolonging the half life of the drugs and improving the bioavailability, and has better economic value and application prospect.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence listing
<110> Anhui Xinxi Biotech Co., ltd
<120> Fusion protein for improving stability of oral administration of polypeptide drugs and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
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Leu Pro Lys Ala Ser Pro Ala Pro Ser Thr Ser Ser Ser Ser Ala Ser
1 5 10 15
Thr Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu
20 25 30
Thr Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr
35 40 45
Asp Asp Ser Asp Val Asp Leu Val Met Ser His Leu Lys Ser Ser Gly
50 55 60
Leu Lys Ile Leu Arg Val Trp Gly Phe Asn Asp Val Thr Thr Gln Pro
65 70 75 80
Ser Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr
85 90 95
Ile Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser
100 105 110
Ser Ala Glu Gln His Gly Ile Lys Leu Ile Ile Asn Phe Val Asn Tyr
115 120 125
Trp Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala Tyr Gly Gly
130 135 140
Ser Asp Glu Thr Asp Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr
145 150 155 160
Gln Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr Ser Asn Ser Ser Ala
165 170 175
Val Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp
180 185 190
Thr Thr Val Leu Tyr Asp Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys
195 200 205
Gly Leu Asp Ala Asp His Met Val Cys Ile Gly Asp Glu Gly Phe Gly
210 215 220
Leu Asn Thr Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ala Glu Gly
225 230 235 240
Leu Asn Phe Thr Met Asn Leu Gly Ile Asp Thr Ile Asp Phe Ala Thr
245 250 255
Leu His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp Asp Trp Gly Asn
260 265 270
Gly Trp Ile Ser Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro
275 280 285
Cys Leu Leu Glu Glu Tyr Gly Val Thr Ser Asn His Cys Ser Val Glu
290 295 300
Ser Pro Trp Gln Gln Thr Ala Leu Asn Thr Thr Gly Val Ser Ala Asp
305 310 315 320
Leu Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr Gly Glu Ser Pro Asp
325 330 335
Asp Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr Glu Cys Leu Val
340 345 350
Thr Asp His Val Ala Ala Ile Asp Ser Ala
355 360
<210> 2
<211> 1080
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
ttgccaaagg cttctccagc tccatctact tcttcttctt ctgcttctac ttcttttgct 60
tctacttctg gtttgcaatt tactattgat ggtgaaactg gttactttgc tggtactaac 120
tcttactgga ttggtttttt gactgatgat tctgatgttg atttggttat gtctcatttg 180
aagtcttctg gtttgaagat tttgagagtt tggggtttta acgatgttac tactcaacca 240
tcttctggta ctgtttggta ccaattgcat caagatggta agtctactat taacactggt 300
gctgatggtt tgcaaagatt ggattacgtt gtttcttctg ctgaacaaca tggtattaag 360
ttgattatta actttgttaa ctactggact gattacggtg gtatgtctgc ttacgtttct 420
gcttacggtg gttctgatga aactgatttt tacacttctg atactatgca atctgcttac 480
caaacttaca ttaagactgt tgttgaaaga tactctaact cttctgctgt ttttgcttgg 540
gaattggcta acgaaccaag atgtccatct tgtgatacta ctgttttgta cgattggatt 600
gaaaagactt ctaagtttat taagggtttg gatgctgatc atatggtttg tattggtgat 660
gaaggttttg gtttgaacac tgattctgat ggttcttacc cataccaatt tgctgaaggt 720
ttgaacttta ctatgaactt gggtattgat actattgatt ttgctacttt gcatttgtac 780
ccagattctt ggggtacttc tgatgattgg ggtaacggtt ggatttctgc tcatggtgct 840
gcttgtaagg ctgctggtaa gccatgtttg ttggaagaat acggtgttac ttctaaccat 900
tgttctgttg aatctccatg gcaacaaact gctttgaaca ctactggtgt ttctgctgat 960
ttgttttggc aatacggtga tgatttgtct actggtgaat ctccagatga tggtaacact 1020
atttactacg gtacttctga ttacgaatgt ttggttactg atcatgttgc tgctattgat 1080
<210> 3
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<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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cacgctgaag gtaccttcac ctctgacgtt tcttcttacc tggaaggtca ggctgctaaa 60
gaattcatcg cttggctggt tcgtggtcgt gg 92
<210> 4
<211> 401
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 4
Leu Pro Lys Ala Ser Pro Ala Pro Ser Thr Ser Ser Ser Ser Ala Ser
1 5 10 15
Thr Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu
20 25 30
Thr Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr
35 40 45
Asp Asp Ser Asp Val Asp Leu Val Met Ser His Leu Lys Ser Ser Gly
50 55 60
Leu Lys Ile Leu Arg Val Trp Gly Phe Asn Asp Val Thr Thr Gln Pro
65 70 75 80
Ser Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr
85 90 95
Ile Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser
100 105 110
Ser Ala Glu Gln His Gly Ile Lys Leu Ile Ile Asn Phe Val Asn Tyr
115 120 125
Trp Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala Tyr Gly Gly
130 135 140
Ser Asp Glu Thr Asp Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr
145 150 155 160
Gln Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr Ser Asn Ser Ser Ala
165 170 175
Val Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp
180 185 190
Thr Thr Val Leu Tyr Asp Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys
195 200 205
Gly Leu Asp Ala Asp His Met Val Cys Ile Gly Asp Glu Gly Phe Gly
210 215 220
Leu Asn Thr Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ala Glu Gly
225 230 235 240
Leu Asn Phe Thr Met Asn Leu Gly Ile Asp Thr Ile Asp Phe Ala Thr
245 250 255
Leu His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp Asp Trp Gly Asn
260 265 270
Gly Trp Ile Ser Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro
275 280 285
Cys Leu Leu Glu Glu Tyr Gly Val Thr Ser Asn His Cys Ser Val Glu
290 295 300
Ser Pro Trp Gln Gln Thr Ala Leu Asn Thr Thr Gly Val Ser Ala Asp
305 310 315 320
Leu Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr Gly Glu Ser Pro Asp
325 330 335
Asp Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr Glu Cys Leu Val
340 345 350
Thr Asp His Val Ala Ala Ile Asp Ser Ala Asp Tyr Lys Asp Asp Asp
355 360 365
Asp Lys His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
370 375 380
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
385 390 395 400
Gly
<210> 5
<211> 29
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 5
cgggatccga ctacaaggac gacgacgac 29
<210> 6
<211> 28
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
gctctagatt aacctctacc tctaacca 28
<210> 7
<211> 28
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 7
cggaattctt gccaaaggct tctccagc 28
<210> 8
<211> 28
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 8
cgggatccag cagaatcaat agcagcaa 28

Claims (4)

1. The fusion protein for improving the oral administration stability of the polypeptide medicine is characterized in that the fusion protein sequentially comprises beta-mannase, connecting peptide and polypeptide from N end to C end, wherein the polypeptide is corticotropin, elvan and Li Patai; the amino acid sequence of beta-mannase is shown as SEQ ID No.1, the amino acid sequence of adrenocorticotropic hormone is shown as SEQ ID No.9, the amino acid sequence of elvan is shown as SEQ ID No.10, and the amino acid sequence of Li Patai is shown as SEQ ID No. 11.
2. The fusion protein for improving the stability of oral administration of polypeptide drugs according to claim 1, wherein the amino acid sequence of the connecting peptide is DYKDDDDK; or alternatively
The amino acid sequence of the connecting peptide is (GGGGS) n, n is=3 or 4; or alternatively
The amino acid sequence of the connecting peptide is (EAAAK) n, and n is2, 3,4 or 5.
3. A recombinant expression vector comprising a gene encoding the fusion protein of claim 1 that enhances the stability of oral administration of a polypeptide drug.
4. A recombinant strain comprising a gene encoding the fusion protein of claim 1 that enhances the stability of oral administration of a polypeptide drug.
CN202110188492.1A 2020-11-13 2021-02-19 Fusion protein for improving stability of oral administration of polypeptide drugs and application thereof Active CN114478795B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1845989A (en) * 2003-09-11 2006-10-11 诺和酶股份有限公司 Recombinant production of antimicrobial agents
CN111909955A (en) * 2020-08-10 2020-11-10 安徽新熙盟生物科技有限公司 Preparation and application of recombinant MANNase-GLP-1 and homologues

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1845989A (en) * 2003-09-11 2006-10-11 诺和酶股份有限公司 Recombinant production of antimicrobial agents
CN111909955A (en) * 2020-08-10 2020-11-10 安徽新熙盟生物科技有限公司 Preparation and application of recombinant MANNase-GLP-1 and homologues

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