CN114478795A - Fusion protein for improving oral administration stability of polypeptide medicine and application thereof - Google Patents
Fusion protein for improving oral administration stability of polypeptide medicine and application thereof Download PDFInfo
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- CN114478795A CN114478795A CN202110188492.1A CN202110188492A CN114478795A CN 114478795 A CN114478795 A CN 114478795A CN 202110188492 A CN202110188492 A CN 202110188492A CN 114478795 A CN114478795 A CN 114478795A
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- fusion protein
- mannase
- glp
- polypeptide
- ser
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- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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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 contains beta-mannase, connecting peptide and polypeptide from N end to C end, wherein the polypeptide comprises GLP-1, EPO, thymic hormone, cytokine, interferon, calcitonin, tumor necrosis factor and tumor marker molecules. The fusion protein overcomes the defects of poor stability and easy degradation of polypeptide drugs, and has the characteristics of prolonging the half-life period of the drugs and improving the bioavailability.
Description
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 of polypeptides have specific biological activity, and as biotechnology and polypeptide synthesis technology are mature, the types of polypeptide drugs developed and marketed are increasing, and currently, more than one hundred kinds of polypeptide drugs are developed and marketed. The polypeptide medicine has wide application in the treatment field of diseases of endocrine system, immune system, digestive system, cardiovascular system and the like, and has good treatment effect on chronic diseases such as tumor, 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 be only administered by frequent injection, which brings inconvenience and pain to patients and greatly limits clinical application.
When the polypeptide medicament is orally administered, the bioavailability is lower than 1 percent, which is mainly caused by the reasons of poor stability of the polypeptide medicament, epithelial barrier of small intestinal mucosa, malabsorption and the like. These factors are illustrated separately below:
(1) poor stability: polypeptide drugs are affected by various factors in the gastrointestinal tract, such as proteases, organic solvents, temperature, pH, microorganisms, etc., and thus, polypeptide drugs are easily inactivated during absorption and release.
(2) Small intestine mucosal epithelial barrier: the transmembrane uptake of polypeptide drugs is mainly through receptor-mediated transport and intercellular diffusion. The receptor-mediated transport needs specific protein molecules, while the polypeptide drugs are polar molecules and are not easy to pass through fat-soluble vascular mucosa. Therefore, intercellular diffusion (transport through tight junctions between cells) is the major route for polypeptide drug absorption. However, in human body, the pore diameter of epithelial cells of small intestine mucosa is 0.4nm, and only amino acid, dipeptide and tripeptide can pass through, and polypeptide drugs have large molecular weight and poor fat solubility and are difficult to penetrate through membrane pores to enter blood circulation.
(3) Malabsorption: after the drug is administrated through the gastrointestinal tract, before the drug is absorbed and enters the blood circulation, the drug is metabolized in intestinal mucosa and liver, the mechanical barrier action of bile contents, mucus layers and non-flowing water layers on the surface of the gastrointestinal tract and the instability of the self conformation of the peptide drug reduce the dosage entering the blood circulation, and the effective blood concentration can not be maintained, thereby causing the low bioavailability.
In order to overcome the problem of low oral bioavailability of polypeptide drugs, the prior art adopts certain preparation processes such as methods of enzyme inhibitors, absorption promoters, chemical modification and the like, and also delivers the polypeptide drugs through special systems such as emulsion, liposome, microspheres, nanoparticles and the like. However, the above methods have drawbacks, for example, the enzyme inhibitor has many side effects which disturb the digestive absorption of nutrients in the body, and the enzyme inhibitor 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 the tissue, but oral administration of drugs with short half-lives is not possible. Glucagon-like peptide-1 (GLP-1) is cytokine mimic peptide, has the characteristics of excellent blood sugar reduction effect, weight control, blood fat regulation, two-way regulation of islet beta cell functions and the like, but has the half-life of only 1.5-2.1 minutes in natural GLP-1, and fatty acid side chain modification and macromolecular protein fusion are carried out on the structure of GLP-1 to prolong the half-life of the GLP-1. For example, the dulaglutide and the albiglutide are respectively fused with G4 immune albumin and serum albumin, so that the half-life period of drug metabolism is prolonged, the injection once per week can be realized, but adverse reaction of an injection part can occur after the injection, and the oral administration can not be realized.
Disclosure of Invention
The invention aims to provide a fusion protein for improving the oral administration stability 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.
The invention further aims to provide application of the fusion protein.
Still another object of the present invention is to provide a fusion protein MANNase-GLP-1.
Still another object of the present invention is to provide a recombinant expression vector containing the above fusion protein MANNase-GLP-1.
The invention also aims to provide a preparation method of the fusion protein MANNase-GLP-1.
The invention further aims to provide application of the fusion protein MANNase-GLP-1.
According to the fusion protein for improving the oral administration stability of the polypeptide drug, the fusion protein sequentially contains beta-mannanase, connecting peptide and polypeptide from the N end to the C end, wherein the polypeptide comprises antitumor polypeptide, antiviral polypeptide, polypeptide vaccine, cytokine mimic peptide and antibacterial active peptide.
The above polypeptides include interferon, insulin growth factor, interleukin series, tumor necrosis factor, fibroblast growth factor, EPO (erythropoietin), adrenocorticotropic hormone (ACTH), calcitonin for promoting bone calcium production, teriparatide for stimulating bone formation and bone resorption, Corticotropin Releasing Factor (CRF), Erythropoietin (EPO) for stimulating and regulating red blood cell production and maturation, granulocyte colony stimulating factor, nerve growth factor, human growth hormone for treating senile diseases and dwarfism, luteinizing hormone releasing hormone for treating prostate cancer and reproductive system tumors, vascular endothelial inhibin for treating non-small cell lung cancer, etc.
The amino acid sequence of the beta-mannase is shown in SEQ ID No. 1.
SEQ ID No.1:
LPKASPAPSTSSSSASTSFASTSGLQFTIDGETGYFAGTNSYWIGFLTDDSDVDLVMSHLKSSGLKILRVWGFNDVTTQPSSGTVWYQLHQDGKSTINTGADGLQRLDYVVSSAEQHGIKLIINFVNYWTDYGGMSAYVSAYGGSDETDFYTSDTMQSAYQTYIKTVVERYSNSSAVFAWELANEPRCPSCDTTVLYDWIEKTSKFIKGLDADHMVCIGDEGFGLNTDSDGSYPYQFAEGLNFTMNLGIDTIDFATLHLYPDSWGTSDDWGNGWISAHGAACKAAGKPCLLEEYGVTSNHCSVESPWQQTALNTTGVSADLFWQYGDDLSTGESPDDGNTIYYGTSDYECLVTDHVAAIDSA
According to a particular 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% to the amino acid sequence shown in SEQ ID No. 1; alternatively, the β -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 β -mannanase activity.
The nucleotide sequence of the gene of the beta-mannase is shown as SEQ ID No. 2: TTGCCAAAGGCTTCTCCAGCTCCATCTACTTCTTCTTCTTCTGCTTCTACTTCTTTTGCTTCTACTTCTGGTTTGCAATTTACTATTGATGGTGAAACTGGTTACTTTGCTGGTACTAACTCTTACTGGATTGGTTTTTTGACTGATGATTCTGATGTTGATTTGGTTATGTCTCATTTGAAGTCTTCTGGTTTGAAGATTTTGAGAGTTTGGGGTTTTAACGATGTTACTACTCAACCATCTTCTGGTACTGTTTGGTACCAATTGCATCAAGATGGTAAGTCTACTATTAACACTGGTGCTGATGGTTTGCAAAGATTGGATTACGTTGTTTCTTCTGCTGAACAACATGGTATTAAGTTGATTATTAACTTTGTTAACTACTGGACTGATTACGGTGGTATGTCTGCTTACGTTTCTGCTTACGGTGGTTCTGATGAAACTGATTTTTACACTTCTGATACTATGCAATCTGCTTACCAAACTTACATTAAGACTGTTGTTGAAAGATACTCTAACTCTTCTGCTGTTTTTGCTTGGGAATTGGCTAACGAACCAAGATGTCCATCTTGTGATACTACTGTTTTGTACGATTGGATTGAAAAGACTTCTAAGTTTATTAAGGGTTTGGATGCTGATCATATGGTTTGTATTGGTGATGAAGGTTTTGGTTTGAACACTGATTCTGATGGTTCTTACCCATACCAATTTGCTGAAGGTTTGAACTTTACTATGAACTTGGGTATTGATACTATTGATTTTGCTACTTTGCATTTGTACCCAGATTCTTGGGGTACTTCTGATGATTGGGGTAACGGTTGGATTTCTGCTCATGGTGCTGCTTGTAAGGCTGCTGGTAAGCCATGTTTGTTGGAAGAATACGGTGTTACTTCTAACCATTGTTCTGTTGAATCTCCATGGCAACAAACTGCTTTGAACACTACTGGTGTTTCTGCTGATTTGTTTTGGCAATACGGTGATGATTTGTCTACTGGTGAATCTCCAGATGATGGTAACACTATTTACTACGGTACTTCTGATTACGAATGTTTGGTTACTGATCATGTTGCTGCTATTGAT
The nucleotide sequence of the encoding gene of the glucagon-like peptide-1 (GLP-1) is shown in SEQ ID No. 3:
CACGCTGA AGGTACCTTC ACCTCTGACG TTTCTTCTTA CCTGGAAGGT CAGGCTGCTA AAGAATTCAT CGCTTGGCTG GTTCGTGGTC GTGG
according to the fusion protein of the 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)nN is 3 or 4; or the amino acid sequence of the connecting peptide is (EAAAK)nAnd n is 2, 3, 4 or 5. The linker peptide to which the present invention is applicable is not limited to the above-mentioned DYKDDDDK, (GGGGS)nAnd (EAAAK)n。
The fusion protein for improving the oral administration stability of the polypeptide drug according to the embodiment of the invention comprises GLP-1, EPO, thymic hormone, cytokine, interferon, calcitonin, tumor necrosis factor or tumor marker molecules.
Interferons are a class of glycoproteins that are used mainly in the treatment of advanced hairy cell leukemia, renal carcinoma, melanoma, Kaposi's sarcoma, chronic myelogenous leukemia, and low 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 mass loss and increasing bone mass.
The thymic hormone refers to thymic peptide, and the thymic peptide which is commonly used clinically is small molecular polypeptide which is found and purified from calf thymus and has nonspecific immune effect. The thymosin can be used for the adjuvant treatment of various primary or secondary T cell deficiency diseases, some autoimmune diseases, various diseases with low cellular immune function and tumors.
Currently, genetically engineered cytokine drugs approved for marketing or clinical research are developed: including interferon (alpha, beta, gamma), interleukin series, colony stimulating factor, insulin growth factor, tumor necrosis factor, erythropoietin, epidermal growth factor, platelet growth factor, fibroblast growth factor, nerve growth factor, connective tissue growth factor, atrial natriuretic peptide, etc.
A recombinant expression vector according to an embodiment of the present invention comprises a gene encoding a fusion protein. The recombinant expression vector is any one of pPICZ alpha A, pPICZ alpha B, pPICZ alpha C.
A recombinant strain comprising a gene encoding a fusion protein according to embodiments of the present invention. Wherein the expression host can be selected from Escherichia coli, Streptomyces, Bacillus subtilis, yeast, mammalian cell, insect cell, and plant cell. Preferably, the expression host is pichia pastoris, and the pichia pastoris strain 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 peptide, beta-mannanase and homologues thereof, and can be expressed in a prokaryotic and eukaryotic expression system in a fusion manner. The beta-mannase and the congeners thereof can hydrolyze mannan into mannan oligosaccharide which is taken as a prebiotic and can be absorbed and metabolized by probiotics in animal bodies to improve intestinal flora. The fusion protein can solve the common bottleneck that polypeptide drugs are not resistant 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 drugs.
The fusion protein MANNase-GLP-1 according to the embodiment of the invention sequentially comprises beta-mannanase, 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
the recombinant expression vector containing the fusion protein MANNase-GLP-1 gene according to the embodiment of the invention is any one of pPICZ alpha A, pPICZ alpha B, pPICZ alpha C.
A method for the preparation of the fusion protein MANNase-GLP-1 according to a particular embodiment of the invention, said method comprising the steps of:
(1) transforming host cells by using a recombinant vector containing a fusion protein MANNase-GLP-1 coding gene to obtain a recombinant strain;
(2) culturing the recombinant strain, and inducing the expression of the fusion protein MANNase-GLP-1;
(3) recovering and purifying the expressed fusion protein MANNase-GLP-1.
Specifically, GLP-1 and beta-mannase coding genes are connected with pPICZ alpha A to obtain a recombinant expression vector; and (3) electrically transforming the recombinant plasmid into a pichia pastoris X-33 competent cell to construct a recombinant engineering bacterium, and performing induced expression. Carrying out fermentation culture on the recombinant engineering bacteria, and carrying out induced 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 drug in the gastrointestinal tract, namely improve the tolerance of the polypeptide drug to pepsin, trypsin and gastric acid, and adapt to the temperature range of 30-80 ℃, so that the half-life period of the polypeptide drug in a human body is obviously prolonged, and the half-life period in the human body can be 10-5000 times of the natural half-life period; meanwhile, the beta-mannase and the congeners thereof can hydrolyze mannan into mannan-oligosaccharides, and the mannan-oligosaccharides can be absorbed and metabolized by probiotics in animal bodies, improve intestinal flora and play a role in promoting drug absorption and utilization.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the results of the pepsin resistance of the fusion protein MANNase-GLP-1;
FIG. 2 shows the results of the trypsin resistance of the fusion protein MANNase-GLP-1;
FIG. 3 shows the results of pH stability of the fusion protein MANNase-GLP-1;
FIG. 4 is the results of the thermostability of the fusion protein MANNase-GLP-1;
FIG. 5 shows the result of constructing a mouse model of metabolic syndrome induced by high-fat and high-sugar diet, wherein A is the fasting blood-glucose change of the mouse after being continuously fed with high-fat and high-sugar diet for 24 weeks; b is the body weight change of the mice after 24 weeks of continuous feeding with high fat high sugar (HFSD) diet;
FIG. 6 is the results of oral glucose tolerance tests in normal diet mice and high fat high sugar diet mice;
FIG. 7 shows the results of HE stained sections of liver and adipose tissues of a high-fat high-sugar diet mouse and a normal diet mouse;
FIG. 8 shows the effect of oral administration of MANNase-GLP-1 on blood glucose and body weight in high-fat and high-sugar mice;
FIG. 9 shows the result of stained liver tissue sections after oral administration of MANNase-GLP-1 to high-fat and high-sugar mice;
FIG. 10 is a three-dimensional mimic structure of the fusion protein MANNase-GLP-1;
FIG. 11 is the structure of the GLP-1 terminal residue of the 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 is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
EXAMPLE 1 preparation of the fusion protein MANNase-GLP-1
GLP-1 is connected with a connecting peptide (DYKDDDDK) with 8 amino acids to synthesize a coding gene of the connecting peptide-GLP-1. Cloning target fragments of the coding beta-MANNase and the connecting peptide-GLP-1 by adopting a primer pair, respectively carrying out PCR amplification, then carrying out double enzyme digestion, connecting the obtained gene sequences of the coding GLP-1 and the MANNase to pPICZ alpha A plasmid, and constructing a recombinant expression vector pPICZ alpha A-MANNase-GLP-1.
Wherein the sequence of the primer pair for amplifying the GLP-1 encoding gene is shown as SEQ ID No.5 and SEQ ID No. 6:
SEQ ID No.5:5'-CGGGATCCGACTACAAGGACGACGACGAC-3';
SEQ ID No.6:5'-GCTCTAGATTAACCTCTACCTCTAACCA-3'。
the sequences of the primer pair for amplifying the coding gene sequence of the beta-mannase are shown as 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 a pichia pastoris X-33 competent cell to construct and obtain a recombinant engineering bacterium MANNase-GLP-1-X-33 for induced expression. Inoculating a single colony of the recombinant engineering bacterium MANNase-GLP-1-X-33 into a YPD liquid culture medium test tube containing bleomycin, and carrying out shake culture for 12h at 30 ℃ and 200 rpm; pouring the bacterial liquid into YPD culture medium, culturing at 30 deg.C and 200rpm for 12 hr to obtain first-stage seed liquid; inoculating the first-stage seed solution into YPD culture medium at an inoculum size of 10%, and culturing at 30 deg.C and 200rpm for 22h to obtain second-stage seed solution; inoculating the second-stage seed solution into 10L seed tank according to 10% of inoculum size, inoculating into 50L fermentation tank according to 10%, performing fermentation culture when fermentation liquid OD 600Adding inducer for induction when the induction reaches above 60-120, and finishing the inductionThen placing the mixture into a tank, and centrifugally collecting thalli. 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 is performed, the initial fermentation temperature is 30 ℃, the stirring speed is 300rpm, the aeration rate is 4L/min, and the pH is 5.5.
The specific steps of purifying, concentrating and drying the supernatant fluid are as follows: taking supernatant, filtering with 0.8um filter membrane, filtering with 0.2um filter membrane, and collecting filtrate; concentrating the filtrate by 10 times with ultrafiltration membrane, adding deionized water, and concentrating by 10 times to obtain concentrated solution; and (4) freeze-drying the concentrated solution to obtain the recombinant fusion protein MANNase-GLP-1.
Example 2 examination of the characteristics of the fusion protein MANNase-GLP-1
2.1 resistance to pepsin
Preparing a 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 juice formula in the us 1995 pharmacopoeia.
And (3) enzymolysis reaction test: the protein content is 1: 1, taking inactivated protease as blank control, setting 4 gradients for reaction time, 0min, 30min, 60min and 120min, accurately timing, immediately adding 0.05mL of 0.618mol/L sodium carbonate solution when the reaction is finished, and stopping the enzymolysis reaction. 50uL of the enzymolysis reaction solution is taken from each tube and is put into a centrifugal tube with the volume of 1.5mL, the enzyme is inactivated after being treated for 5min at the temperature of 70 ℃, and the enzyme activity is measured after the enzyme is properly diluted.
As shown in figure 1, after 2h of treatment, the fusion protein MANNase-GLP-1 is still measured to have more than 60% of enzyme activity, and the result shows that the fusion protein MANNase-GLP-1 has better tolerance to pepsin.
2.2 Trypsin resistance
Preparing a trypsin solution: 6.8g KH2PO4Dissolving in 250mL of distilled water, adding 190mL of 0.2mol/L NaOH and 400mL of distilled water after completely dissolving, adding 10.0g of trypsin, mixing, adjusting pH to 7.5 +/-0.1 with 0.2mol/L NaOH, and fixing volume of 1000mL of distilled water, wherein the preparation method is described in the human pharmacopoeia of American 1995And (5) simulating an intestinal juice formula.
And (3) enzymolysis reaction test:
the protein content is 1: 1, taking the inactivated protease as a blank control, setting 4 gradients for reaction time, 0min, 30min, 60min and 120min, accurately timing, immediately adding 0.05mL of 30% glacial acetic acid solution when the reaction is finished, and stopping the enzymolysis reaction. 50uL of the enzymolysis reaction solution is taken from each tube and is put into a centrifugal tube with the volume of 1.5mL, the enzyme is inactivated after being treated for 5min at the temperature of 70 ℃, and the enzyme activity is measured after the enzyme is properly diluted.
As shown in figure 2, after 2h of treatment, the fusion protein MANNase-GLP-1 is still measured to have more than 60% of enzyme activity, and the result shows that the fusion protein MANNase-GLP-1 has better tolerance to trypsin.
2.3pH stability
Respectively preparing 100mM buffer solutions with different pH values, glycine-hydrochloric acid (pH value is 2.2-3.2), citric acid-disodium hydrogen phosphate (pH value is 3.2-6.2), disodium hydrogen phosphate-dihydrogena phosphate (pH value is 6.2-8.2) and Tris-HCl (pH value is 8.2-9.2), respectively preparing enzyme reaction substrates 0.6% LBG and diluted enzyme solutions by using the buffer solutions, respectively measuring the enzyme activity of the fusion protein MANNase-GLP-1 under different pH values at 50 ℃, and repeating the experiment for three times in each group.
As shown in FIG. 3, the fusion protein MANNase-GLP-1 has an optimum pH of 3.2, and retains 80% of its enzymatic activity at pH2.
2.4 thermal stability
And respectively measuring the enzyme activity of the fusion protein MANNase-GLP-1 at the temperature of 30-80 ℃ by adopting a citric acid-disodium hydrogen phosphate buffer solution with the pH value of 3.2, and repeating each group of experiments for 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 several minutes; the enzyme has strong tolerance under the condition of 40 ℃ (close to the body temperature of a human body), and the enzyme activity is basically not changed after 12 hours; at 50 ℃, about 80% of enzyme activity is remained after 1 hour, and the enzyme activity is basically reduced to 0 after 6 hours.
Example 3 demonstration of the oral Effect of the fusion protein MANNase-GLP-1
3.1 construction of mouse model of high fat and high sugar diet induced metabolic syndrome
100C 57-6J mice (4-6 weeks old, male) with age of 6 weeks are raised in cages, the temperature of the animal room is controlled to be 25 +/-2 ℃, the humidity is controlled to be 50 +/-10%, the illumination is 12h, the dark is 12h, and the environment is adapted for one week. Mice were randomized into cages at 5-6/group. All mice were fasted for 12h and then body weight and Fasting Blood Glucose (FBG) were measured, the control group was continuously fed with standard diet and the model group was continuously fed with high fat high sugar (HFSD) diet for 24 weeks. After completion, the body weight and FBG of each group of mice were measured.
The body weight of the mice on high-fat and high-sugar diet is about 42.5g, the body weight of the mice on normal diet is about 30g, the fasting blood sugar of the mice on high-fat and high-sugar diet is about 5.67, and the fasting blood sugar of the mice on normal diet is 4.62, which have statistical differences.
As shown in A, B in fig. 5, in C57-6J mice induced by high-fat and high-sugar diet, fasting blood sugar is 22.73% higher than normal diet, and blood sugar is significantly increased; after 24 weeks of induction of C57-6J mice on a high fat and high sugar diet, the body weight of the mice exceeded the normal diet by 41.67%, and the mice met the standard of an obesity model (20%) (*p<0.05,**p<0.01,***p<0.001). The fasting blood sugar of the C57-6J mouse induced by high-fat and high-sugar diet is 22.73 percent higher than that of the normal diet, and the blood sugar is obviously increased.
After fasting overnight for 12h, general diet mice and high-fat and high-sugar mice were gavaged with D-glucose at 2g/kg (body weight) for 0, 0.5, 1.0, 1.5, 2.0, and 2.5h, respectively, to obtain an oral glucose tolerance (OGTT) curve.
As shown in fig. 6, after 24 weeks of induction by high-fat and high-sugar diet, the glucose tolerance of the high-fat and high-sugar mice was significantly impaired.
HE stained sections of liver and adipose tissues of high-fat high-sugar diet mice and normal diet mice were analyzed, and as shown in fig. 7, model mice on high-fat high-sugar diet had significant fatty liver.
The experiment proves that the mouse model of the metabolic syndrome induced by high-fat and high-sugar diet is successfully constructed. After successful modeling, the samples were randomly grouped into 11 samples/group for oral gavage.
3.2 investigation of the Effect of oral administration of the fusion protein MANNase-GLP-1 on high-fat high-sugar diet-induced Metabolic syndrome
Mice were randomized into 5 groups: the fusion protein high dose group (3.5 mg/kg. d), the fusion protein low dose group (1.75 mg/kg. d), and 30mg/kg orlistat were used as positive control groups, and the normal diet control group and the high-fat high-sugar diet negative control group were administered with the same volume of water, and all mice were orally gavaged without changing diet, respectively, to measure the weekly weight change.
The results are shown in fig. 8, and compared with the negative control (water), the oral gavage fusion protein MANNase-GLP-1 high dose group significantly reduced body weight, which is close to the effect of orlistat (Oli) on the market.
Oral glucose tolerance is an index of the body on the degree of glucose load, impaired glucose tolerance means a decrease in the function of islet beta cells and the body's ability to regulate blood glucose, and the critical value of glucose tolerance is that blood glucose is 7.8 mmol/L2 hours after a meal, and a value higher than 7.8mmol/L represents an impaired glucose tolerance of the body. After different doses of MANNase-GLP-1 fusion protein are performed on the mice with impaired glucose tolerance, the glucose tolerance level of the mice is improved to different degrees. The high-dose group is restored to a normal level (7.5mmol/L), the postprandial 2-hour blood sugar of the negative control group (water) is up to 10mmol/L, and the MANNase-GLP-1 fusion protein can be seen to have obvious improvement effect on the carbohydrate metabolism of the organism.
Therefore, under the condition of ensuring the safety of mice, the MANNase-GLP-1 fusion protein has obvious weight loss and glucose tolerance improvement effects.
After dissection, the liver was taken out and analyzed by HE stained section, and the result is 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 the fusion protein MANNase-GLP-1
The present invention mimics the three-dimensional structure of the fusion protein MANNase-GLP-1, as shown in FIG. 10. The structure of the GLP-1 portion of the fusion protein MANNase-GLP-1 in the bulk model was aligned with the B chain of 3l0L, and the main portion of GLP-1 (about 22/31) was found to be alpha-helical with the B chain of 3 IOL.
The invention analyzes 39C-terminal residues of the fusion protein MANNase-GLP-1The group forms an interaction interface with a structural part of the mannanase. The composition of the interface is such that about 23% of the solvent accessible area (ASA, 776.8 +) is present at the 39 residues of the GLP-1 terminus) Participate in interfacial interactions. This interfacial interaction forms 12 hydrogen bonds and 7 salt bonds and results in the release of-13.9 kcal mol-1 binding energy, indicating that the three-dimensional structure of the fusion protein MANNase-GLP-1 is a stable state with low energy, and the structure is stable.
As shown in FIG. 11, hydrophobic interactions also exist for the 39 residue structure at the GLP-1 terminus.
The invention analyzes a plurality of pepsin specific hydrolysis recognition sites in GLP-1, such as residues of 6Phe, 13Tyr, 14Leu, 22Phe, 26Leu and the like, and forms internal hydrophobic interaction, thereby effectively resisting pepsin hydrolysis. In addition, some aromatic ring side chain amino acids in the fusion protein MANNase-GLP-1 are recognition sites of chymotrypsin, and can also resist the hydrolysis of pepsin to a certain extent. Therefore, the structural analysis of the fusion protein MANNase-GLP-1 of the invention is identical with the experimental results of pepsin, trypsin and pH stability in example 2, and the function of the fusion protein MANNase-GLP-1 is verified from two aspects 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 medicines, such as antitumor polypeptide, antiviral polypeptide, polypeptide vaccine, cytokine mimic peptide, antibacterial active peptide, etc., so as to prolong the half-life of the polypeptide medicines.
From the perspective of a three-dimensional structure, on one hand, the original pepsin specific hydrolysis recognition sites in the polypeptide drug structure after fusion expression form an internal hydrophobic structure, so that pepsin hydrolysis is effectively resisted; on the other hand, specific protease recognition sites on the amino acid side chains after fusion expression may also be somewhat resistant to hydrolysis by pepsin.
In this example, adrenocorticotropic hormone (ACTH), elcatonin (CCT), and Teriparatide (TRP) were further taken as examples to construct a fusion expression process with β -mannanase, and to verify the effect of the fusion expressed protein.
5.1 fusion protein MANNase-ACTH
Adrenocorticotropic hormone (ACTH): 4541.1 molecular weight, is composed of 39 amino acids, and can be used for treating rheumatic arthritis. 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 of coding beta-MANNase and connecting peptide (EAAAKEAAAK) -ACTH by adopting the primer pair, respectively carrying out PCR amplification, then carrying out double enzyme digestion, connecting the obtained gene sequences of coding ACTH and MANNase to pPICZ alpha A plasmid, and constructing a recombinant expression vector pPICZ alpha A-MANNase-ACTH.
And (3) electrically transforming the recombinant plasmid into a pichia pastoris X-33 competent cell to construct a recombinant engineering bacterium MANNase-ACTH-X-33, and carrying out induced expression and purification to obtain the fusion protein MANNase-ACTH.
The fusion protein MANNase-ACTH was examined for pepsin, trypsin, pH and thermostability, and the 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 protein MANNase-CCT
Elcatonin (CCT): the medicine is composed of 32 amino acids, has molecular weight of 3363.77, is an injection and a nasal spray in the current market, promotes the generation of bone calcium and treats osteoporosis.
The amino acid sequence of elcatonin 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 of coding beta-MANNase and connecting peptide (EAAAKEAAAK) -CCT by adopting the primer pair, respectively carrying out PCR amplification, then carrying out double enzyme digestion, connecting the obtained gene sequences of coding CCT and MANNase to pPICZ alpha A plasmid, and constructing to obtain a recombinant expression vector pPICZ alpha A-MANNase-CCT.
And (3) electrically transforming the recombinant plasmid into pichia pastoris X-33 competent cells to construct recombinant engineering bacteria MANNase-CCT-X-33, and performing induced expression and purification to obtain the fusion protein MANNase-CCT.
The fusion protein MANNase-CCT was examined for pepsin resistance, trypsin resistance, pH stability and thermostability as in example 2.
The results show that the fusion protein MANNase-CCT has better tolerance to pepsin and trypsin.
5.3 fusion protein MANNase-TRP
Teriparatide (TRP) consisting of 34 amino acids, molecular weight: 4177.77, intravenous injection for osteoporosis, stimulation of bone formation and bone resorption. The amino acid sequence is shown as 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 procedure for preparing the fusion protein MANNase-TRP was the same as in example 2, and the experiments on the pepsin resistance, trypsin resistance, pH stability and thermostability of the fusion protein MANNase-CCT were 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 oral administration stability of polypeptide drugs and application thereof. The fusion protein sequentially contains beta-mannase, connecting peptide and polypeptide from N end to C end, wherein the polypeptide comprises GLP-1, EPO, thymic hormone, cytokine, interferon, calcitonin, tumor necrosis factor and tumor marker molecules. The fusion protein overcomes the defects of poor stability and easy degradation of polypeptide drugs, has the characteristics of prolonging the half-life period of the drugs and improving the bioavailability, and has better economic value and application prospect.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily think of the changes or substitutions within the technical scope of the present invention, and shall cover 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 New Xian Union Biotechnology Limited
<120> fusion protein for improving oral administration stability of polypeptide drug and application thereof
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<170> SIPOSequenceListing 1.0
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Leu Pro Lys Ala Ser Pro Ala Pro Ser Thr Ser Ser Ser Ser Ala Ser
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Thr Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu
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Thr Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr
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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
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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
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<213> Artificial Sequence (Artificial Sequence)
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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
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cacgctgaag gtaccttcac ctctgacgtt tcttcttacc tggaaggtca ggctgctaaa 60
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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 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)
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cgggatccag cagaatcaat agcagcaa 28
Claims (10)
1. The fusion protein for improving the oral administration stability of the polypeptide drug is characterized by comprising beta-mannase, a connecting peptide and polypeptide from the N end to the C end in sequence, wherein the polypeptide comprises antitumor polypeptide, antiviral polypeptide, cytokine mimic peptide, polypeptide vaccine and antibacterial active peptide; the amino acid sequence of the beta-mannase is shown in SEQ ID No. 1.
2. The fusion protein for improving the oral administration stability of polypeptide drugs as claimed in claim 1, wherein the amino acid sequence of the linker peptide is DYKDDDDK; or alternatively
The amino acid sequence of the connecting peptide is (GGGGS)nN is 3 or 4; or
The amino acid sequence of the connecting peptide is (EAAAK)nAnd n is 2, 3, 4 or 5.
3. The fusion protein for improving the oral administration stability of polypeptide drugs according to claim 1, wherein the polypeptide comprises GLP-1, erythropoietin, thymic hormone, cytokine, interferon, calcitonin, tumor necrosis factor or tumor marker molecule.
4. A recombinant expression vector comprising a gene encoding the fusion protein of claim 1 for improving the stability of oral administration of a polypeptide drug.
5. A recombinant strain comprising a gene encoding the fusion protein of claim 1 for improving the oral administration stability of a polypeptide drug.
6. Use of the fusion protein of claim 1 for improving the oral administration stability of polypeptide drugs in the preparation of oral dosage form drugs.
7. The fusion protein MANNase-GLP-1 is characterized in that the amino acid sequence is shown as SEQ ID No. 4.
8. A recombinant expression vector comprising a gene encoding the fusion protein MANNase-GLP-1 according to claim 7.
9. A process for the preparation of the fusion protein MANNase-GLP-1 according to claim 7, characterized in that it comprises the following steps:
(1) transforming host cells by using a recombinant vector containing a fusion protein MANNase-GLP-1 coding gene to obtain a recombinant strain;
(2) culturing the recombinant strain, and inducing the expression of the fusion protein MANNase-GLP-1;
(3) recovering and purifying the expressed fusion protein MANNase-GLP-1.
10. Use of the fusion protein MANNase-GLP-1 according to claim 7 for the preparation of a GLP-1 oral medicament.
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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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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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