Novel oral hypoglycemic polypeptide for treating diabetes and preparation method thereof

Abstract

The invention provides a novel oral blood sugar reducing polypeptide. The polypeptide is an improved derivative based on Exendin-4. According to the present invention, the Exendin-4 is analyzed so as to obtain the core sequence of 28 amino acids, wherein the core sequence is HGEGTFTSDLSKQMEEEAVRLFIEWLKN; in order to improve the stability and bioavailability, four key sites are subjected to amino acid substitution (K12H, R20H, F22A and K27H), AF and FK are respectively added at the N end and the C end, and finally a complete sequence with 32 amino acids is formed. The structural modifications significantly improve the oral absorption efficiency of the polypeptide. Compared with an Exendin-4 prototype, the antihyperglycemic polypeptide Ex32 disclosed by the invention has the advantages that the enzymolysis resistance is obviously improved, and the obtained Ex32-containing polypeptide has good physical and chemical stability and bioavailability and can be directly used for preparing various dosage forms, for example, the Ex32-containing polypeptide can be used for treating type II diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of peptide pharmaceuticals, and specifically relates to a novel oral hypoglycemic polypeptide for treating diabetes and a preparation method thereof. By performing specific amino acid modifications on the Exendin-4 peptide sequence, the present invention improves the stability and oral absorption rate of the polypeptide in the gastrointestinal tract, making it more suitable for oral administration and providing a new treatment option for diabetic patients. Background Art

[0002] The present invention relates to an oral hypoglycemic polypeptide for treating type 2 diabetes (T2DM). As is well known, one of the main pathogenesis of T2DM lies in the insufficient secretion of glucagon-like peptide 1 (GLP-1), which in turn leads to insulin secretion dysfunction. In the prior art, Exendin-4, as a GLP-1 receptor agonist (consisting of 39 amino acid residues), has proven its therapeutic effect in clinical applications. Exendin-4 has a longer half-life than endogenous GLP-1 and maintains multiple biological functions, including but not limited to: promoting glucose-dependent insulin secretion, inhibiting glucagon release, delaying gastric emptying, and suppressing appetite, etc.

[0003] In the prior art, although exenatide (a synthetic analogue of Exendin-4) has been approved for marketing, its administration route is limited to subcutaneous injection. This administration route has many deficiencies: (1) poor patient compliance; (2) high treatment cost; (3) adverse reactions such as infections may occur at the injection site. Although there are already long-acting injectable GLP-1 receptor agonists on the market, the development of oral preparations is still an important technical approach to improve patient compliance and treatment effect.

[0004] However, the oral administration of protein and polypeptide drugs such as Exendin-4 faces significant technical obstacles, mainly manifested in their extremely low oral bioavailability. This is due to the following factors: (1) the physical barrier formed by the mucus layer and tightly connected epithelial cells in the gastrointestinal tract; (2) the chemical barrier formed by the strong acidic environment in the stomach; (3) the degradation effect of various proteases in the digestive system. Therefore, improving the absorption efficiency of oral polypeptide drugs is an urgent technical problem in this field.

[0005] The inventor of the present invention found through a large number of experimental studies that by modifying the amino acids at specific positions in the molecular structure of Exendin-4 (K12H, R20H, F22A, K27H), the recognition and degradation by trypsin can be effectively avoided. To further improve its stability, the present invention adopts an innovative technical solution to co-express the modified Exendin-4 with a peptide segment of bovine lactoferrin (LfcinB). Among them, LfcinB is a cationic peptide containing 25 amino acid residues (sequence: FKCRRWQWRMKKLGAPSITCVRRAF), and its characteristic amphiphilic structure and antiparallel β-sheet conformation endow it with significant anti-enzyme digestion characteristics.

[0006] Systematic studies have shown that in the digestive system, trypsin (generated by the activation of zymogen secreted by the pancreas by enterokinase) mainly exerts proteolytic effects in the small intestine region, especially in the duodenum. Through in-depth research, the present invention determined the key sequence fragment Ex32 with significant hypoglycemic activity: AFHGEGTFTSDLSHQMEEEAVHLAIEWLHNFK. This sequence fragment has excellent intestinal absorption characteristics and significant hypoglycemic effects. Summary of the Invention

[0007] Through molecular dynamics simulations in this study, it was found that the truncated modified Exendin-4 has a high affinity for the GLP-1 receptor ( Figure 1 , 2, 3), and its biological activity may not be lower than that of the prototype Exendin-4. Given that the currently commercially available Exendin-4 preparations mainly use subcutaneous injection for administration, this to a certain extent affects the compliance of patients. To overcome this limitation and achieve independent intellectual property rights, this study is committed to developing an orally administered Exendin-4 analogue with independent intellectual property rights, with the expectation of providing a more cost-effective treatment option for domestic type 2 diabetes patients. Through systematic research, we optimized the structure of truncated Exendin-4, focusing on the targeted modification of amino acid sites that are easily recognized by digestive enzymes, and successfully screened and obtained candidate molecules with significant anti-enzyme digestion characteristics.

[0008] According to the patent application documents, the present invention mainly involves the following innovative points.

[0009] Invented a new orally administered hypoglycemic polypeptide Ex32, which significantly improves the stability of the polypeptide in the gastrointestinal tract by modifying the amino acid sequence of Exendin-4 at specific positions (K12H, R20H, F22A, K27H);

[0010] Innovatively co-expressed the modified Exendin-4 with bovine lactoferrin (LfcinB) at the molecular level to further enhance the anti-enzyme digestion characteristics of the drug;

[0011] Through systematic research, the key sequence fragment Ex32 with significant hypoglycemic activity was identified. This sequence fragment has excellent intestinal absorption characteristics and hypoglycemic efficacy;

[0012] The present invention overcomes the limitation that Exendin-4 in the prior art needs to be administered subcutaneously, provides a new oral administration option for patients, and is expected to improve treatment compliance and reduce adverse reactions;

[0013] The technical solution of the present invention solves the key technical problem of low oral bioavailability of polypeptide drugs and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Schematic diagram of the molecular structural interaction between the hypoglycemic polypeptide Ex32 of the present invention and the GLP-1 receptor. This figure shows the molecular docking mode of the Ex32 polypeptide (presented in the form of a protein backbone) and the GLP-1 receptor (shown in gray). The crystal structure data of the GLP-1 receptor is from the PDB database (accession number: 7LLL). The structural model of the Ex32 polypeptide was obtained by homology modeling based on the structure of Exendin-4 in 7LLL, and the interaction interface between the two molecules is highlighted in the dark region.

[0015] Figure 2 : Molecular dynamics energy conformational map of the binding of Ex32 polypeptide to the GLP-1 receptor. The black region in the upper right corner of the figure characterizes the lowest energy conformational state when Ex32 binds to the receptor.

[0016] Figure 3 : Hydrogen bond network analysis diagram of the binding interface between Ex32 polypeptide and GLP-1 receptor. Figure A shows the hydrogen bond interaction formed between the N-terminal AF residues of Ex32 and the receptor, and K shows the detailed analysis results of the participation of the C-terminal AF residues in the formation of intramolecular hydrogen bonds. DETAILED DESCRIPTION OF THE INVENTION

[0017] The specific implementation scheme of the present invention will be elaborated through the following experimental data and detailed description. All experiments were carried out under standard laboratory conditions. The experimental instruments and reagents used complied with the relevant technical specifications, and the experimental operations were carried out in accordance with the standard operating procedures or the technical guidance provided by the manufacturer.

[0018] Example 1: Construction and expression of the expression vector.

[0019] 1.1 Construction of the expression vector.

[0020] By analyzing the crystal structure of the 7LLL protein, we found HGEGTFTSDLSKQMEEEAVRLFIEWLKNThe sequence shows specific binding to the helical region of the GLP-1 receptor. This 28-amino acid sequence exhibits good receptor binding potential, but faces challenges of being degraded by trypsin and difficult to pass through the gastrointestinal digestive system in practical applications.

[0021] To address these problems, we adopted two key strategies: First, enhance its anti-enzymatic ability through directed amino acid substitution (K12H, R20H, F22A, K27H); Second, add AF and FK at both ends of the sequence to construct a complete 32-amino acid sequence. Furthermore, we co-expressed the modified sequence with the bovine lactoferrin peptide segment (LfcinB) to enhance the anti-enzymatic properties of the overall sequence. To evaluate the impact of these modifications on receptor binding, we conducted molecular dynamics (MD) simulation studies.

[0022] We used the pET28a(+) plasmid to construct the expression system of SQDIDNO: 1. To enable the polypeptide to release the pharmacologically active fragment under the action of trypsin, we added an additional MK amino acid residue at the N-terminus of the SQDIDNO: 2 sequence.

[0023] 1.2 Expression sequence design.

[0024] The complete nucleotide sequence expressed by the vector is: atgaaagcgtttcatggcgaaggcacctttaccagcgatctgagccatcagatggaagaa gaagcggtgcatctggcgattgaatggctgcataactttaaatgccgccgctggcagtgg cgcatgaaaaaactgggcgcgccgagcattacctgcgtgcgccgcgcgtttcatggcgaa ggcacctttaccagcgatctgagccatcagatggaagaagaagcggtgcatctggcgatt gaatggctgcataactttaaatgccgccgctggcagtggcgcatgaaaaaactgggcgcg ccgagcattacctgcgtgcgccgcgcgtttcatggcgaaggcacctttaccagcgatctg agccatcagatggaagaagaagcggtgcatctggcgattgaatggctgcataactttaaa。

[0025] 1.3 Expression characteristics.

[0026] This expression vector contains a histidine tag (His-tag), which is beneficial for subsequent protein purification. After sequence optimization, this construct can achieve high-level expression in the Escherichia coli expression system.

[0027] The experimental process consists of four key stages, and each stage has been systematically optimized to ensure the high-level expression and purification of the target protein. In the molecular cloning stage, the target gene is first chemically synthesized, and then the target gene sequence is amplified by polymerase chain reaction (PCR) and an expression vector is constructed. The specific experimental steps include: designing primers containing restriction enzyme sites, performing double digestion on the PCR product and the pET28a(+) plasmid, using DNA ligase to directionally insert the target fragment into the vector, and finally transforming the recombinant plasmid into DH5α competent cells for amplification.

[0028] In the expression optimization stage, the recombinant plasmid is transformed into the BL21(DE3) expression strain, and the key expression parameters are systematically optimized. Specifically, this includes optimizing parameters such as the culture temperature (16°C, 25°C, 37°C), the concentration of isopropyl-β-D-thiogalactoside (IPTG) for induction (0.1 - 1.0 mM), and the induction time (4 - 16 hours) to obtain the optimal expression conditions.

[0029] Protein purification adopts a multi-step purification strategy: First, ultrasonic cell disruption technology is used to obtain the cell lysate, and the supernatant is collected after centrifugation. Subsequently, nickel ion affinity chromatography is used to purify the His-tag fusion protein. After removing imidazole by dialysis, gel filtration chromatography (SEC) is used for further purification to obtain the target protein with high purity.

[0030] To ensure the quality of protein expression, multiple analytical methods are used for characterization: The purity of the protein is evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the expression of the target protein is verified by Western blot to ensure the expected target product is obtained.

[0031] Through systematic optimization experiments, this study has determined the following optimal expression conditions: Culture temperature: 25°C (degrees Celsius) Concentration of IPTG inducer: 0.5 mM (millimoles per liter) Induction expression time: 12 hours Under the above optimized conditions, the expression level of the target protein reaches 30 mg / L (milligrams per liter) of the culture medium, and the purity is above 95%. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the target protein presents an obvious band at 20 kDa (kilodaltons), and Western blot analysis further confirms the specific expression of the target protein.

[0032] Example 2: In vitro enzymatic stability test of Ex32 tandem polypeptide.

[0033] 2.1 Experimental method.

[0034] In this study, the enzymatic stability of the target polypeptide was systematically evaluated under simulated gastrointestinal conditions. Specifically, the purified target polypeptide Ex32 tandem polypeptide (final concentration 1.0 mg / mL) was incubated with three major digestive enzymes under strictly controlled experimental conditions: pepsin (100 U / mL), trypsin (50 U / mL), and α-chymotrypsin (50 U / mL). To simulate the human gastrointestinal environment, the experiments were carried out at pH 2.0 (simulating the gastric environment) and pH 7.4 (simulating the intestinal environment). Samples were taken at preset time points (0, 30, 60, 120, and 240 minutes), and the residual polypeptide content in the samples was quantitatively analyzed using high performance liquid chromatography (HPLC) technology.

[0035] 2.2 Experimental results.

[0036] Based on the systematic evaluation of enzymatic stability, significant experimental data were obtained in this study. The target polypeptide with optimized structure showed excellent characteristics in terms of enzymatic stability, and the experimental results are detailed as follows: These data show that this product has significantly better stability than the control exenatide under the action of various digestive enzymes, fully confirming the effectiveness of our molecular design strategy. Especially under the simulated gastric environment (pH 2.0), this product still maintained a residual rate of 85% after 4 hours, and under the simulated intestinal environment (pH 7.4), it also showed good resistance to trypsin and α-chymotrypsin.

[0037] Example 3: In vitro cell activity evaluation of hypoglycemic polypeptide Ex32.

[0038] 3.1 Experimental method.

[0039] The preparation of Ex32 protein adopted a complete purification process. First, the expression products of the protein were collected: the Ex32 tandem protein expressing His-tag was collected by centrifugation at 8000 rpm, the bacterial cells were resuspended with PBS buffer containing protease inhibitors, and the target protein was released by ultrasonic disruption or high-pressure homogenization. The disrupted sample was centrifuged at 12000 - 15000 rpm at 4°C, and the supernatant was collected to obtain the crude protein extract.

[0040] Subsequent affinity chromatography purification was carried out: Using a Ni-NTA affinity chromatography column, after equilibration with a detergent-free equilibration buffer 2-3 times, the crude protein extract was slowly loaded at a flow rate of 10 bed volumes per hour. The crude protein was washed 3-5 times with a 10-20 mM low-concentration imidazole buffer to remove impurities, and then the target protein was gradually eluted with a 150-300 mM high-concentration imidazole elution buffer.

[0041] Finally, there was a post-treatment step: The obtained Ex32 tandem protein was degraded into Ex32 short peptides by trypsin treatment and further purified by dialysis and ultrafiltration concentration. To ensure protein stability, 10-20% glycerol was added as a stabilizer to the final product and stored at -80 °C after aliquoting.

[0042] In this study, the INS-1 rat insulinoma cell line was used for in vitro activity evaluation. This cell line has functional characteristics similar to pancreatic islet β cells and can synthesize and secrete insulin. In the experiment, the cells were seeded in 24-well culture plates at a density of 5×10^5 cells / well and cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO2 for 24 hours to allow the cells to adhere well and reach the logarithmic growth phase.

[0043] In the pre-experiment treatment stage, the cells were pre-incubated with sugar-free Krebs-Ringer buffer (KRBH) for 1 hour to remove glucose and other stimulating factors in the residual medium and keep the cells in the basal secretion state. Subsequently, KRBH buffer containing 3 mM glucose was added to the culture wells, and the modified polypeptide Ex32 at different concentrations (0.1-100 nM) was added and incubated for 2 hours. A low-glucose condition was set to facilitate the observation of the promoting effect of the polypeptide on insulin secretion.

[0044] The experimental design included three biological replicates, and a positive control group (the prototype GLP-1 receptor agonist Exendin-4, exenatide) and a negative control group (medium containing only 3 mM glucose) were set up. The insulin content in the culture supernatant was quantitatively detected by enzyme-linked immunosorbent assay (ELISA), and the cell viability was evaluated by the MTT method.

[0045] 3.2 Experimental results.

[0046] The analysis results of insulin secretion and cell survival rate after incubation of the pancreatic islet β cell line INS-1 with the polypeptide for 2 hours are as follows: The research results showed that the modified polypeptide exhibited insulinotropic activity comparable to that of the prototype Exendin-4 in the concentration range of 1 - 10 nM. Dose-effect analysis revealed that the half-maximal effective concentration (EC50) of the modified polypeptide was 3.2 nM, which was similar to the EC50 value (3.5 nM) of the prototype Exendin-4, indicating that the structural modification did not significantly affect its receptor agonist activity.

[0047] At the highest tested concentration (100 nM), the modified polypeptide increased insulin secretion by approximately 4.2-fold compared to the basal level. Time kinetics studies showed that the insulinotropic effect started to appear 30 minutes after administration, reached its peak at 2 hours, and lasted for at least 4 hours, demonstrating that the polypeptide had a rapid and sustained insulinotropic ability.

[0048] The results of cell viability assays showed that the modified polypeptide did not exhibit significant cytotoxicity to INS-1 cells within the tested concentration range (0.1 - 100 nM), and the cell survival rate was comparable to that of the control group. This indicated that the observed changes in insulin secretion during the experiment were indeed due to the biological activity of the polypeptide, rather than changes in cell number or status.

[0049] Example 4: Evaluation of the hypoglycemic effect of the polypeptide Ex32 by in vivo injection.

[0050] 4.1 Experimental method.

[0051] Sixty 10 - 12-week-old db / db diabetic mice were randomly divided into 4 groups (15 mice in each group): Blank control group (subcutaneous injection of normal saline) Low-dose Ex32 group (5 μg / kg / day, subcutaneous injection) High-dose Ex32 group (10 μg / kg / day, subcutaneous injection) Exenatide control group (10 μg / kg / day, subcutaneous injection) Subcutaneous injection was administered once every morning at 8:00 for 4 consecutive weeks. The following indicators were observed: Blood glucose level: Fasting and 2-hour postprandial blood glucose were measured weekly. Body weight change: Body weight was measured every 3 days. Glycated hemoglobin (HbA1c): Measured at the start and end of the experiment. Insulin and GLP-1 levels: Measured at the start and end of the experiment.

[0052] 4.2 Experimental results.

[0053] The hypoglycemic effect of the polypeptide Ex32 by in vivo injection was as follows: The results showed that the high-dose group of Ex32 (10 μg / kg / day) had a comparable hypoglycemic effect to exenatide (10 μg / kg / day), and there was no significant difference in various indicators between the two groups (p>0.05). This indicates that Ex32 has comparable hypoglycemic activity to exenatide when administered subcutaneously.

[0054] Example 5: In vivo pharmacodynamics study of Ex32 tandem polypeptide.

[0055] 5.1 Experimental method.

[0056] Eight-week-old male db / db diabetic mice (n = 40) were selected and randomly divided into 4 groups: blank control group (normal saline), low-, medium-, and high-dose oral administration groups (0.1, 0.5, and 2.5 mg / kg). All animals were fasted for 12 hours before the experiment but were allowed free access to water. Tail vein blood samples were collected at 0, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration, and blood glucose levels were measured using a blood glucose meter. At the same time, the general status, body weight changes, and food intake of the animals were monitored.

[0057] 5.2 Experimental results.

[0058] The blood glucose test results of orally administered Ex32 tandem polypeptide are as follows: Based on the hyperglycemic characteristics of db / db mice, the above data examples showed that the orally modified polypeptide showed an obvious hypoglycemic effect in the high-dose group. The blood glucose level reached the lowest point at 2-4 hours, with a decrease of about 40%, and then gradually recovered to the baseline level.

[0059] The blood glucose level in the blank control group remained at a high level, reflecting the stability of the diabetic model.

[0060] The hypoglycemic effect of the low-dose group was weak, and the blood glucose change was not significant.

[0061] In summary, the modified polypeptide Ex32 showed a good dose-dependent hypoglycemic effect, and the hypoglycemic effect was most significant in the high-dose group (2.5 mg / kg), with a duration of about 12 hours. These results indicate that the polypeptide has the potential to become an oral hypoglycemic drug.

[0062] The results of the analysis of the in vivo pharmacokinetic parameters of Ex32 polypeptide (derived from orally administered Ex32 tandem polypeptide) are as follows: Tmax (h): Time to reach the maximum blood drug concentration, which refers to the time required for the drug to reach the maximum plasma concentration, in hours. This parameter reflects the drug absorption rate.

[0063] Cmax (ng / mL): Maximum blood drug concentration, which refers to the highest concentration that the drug can reach in the plasma, in ng / mL.

[0064] AUC0-24h (ng·h / mL): Area under the plasma concentration-time curve, which refers to the area under the plasma concentration-time curve of the drug within 24 hours after administration, expressed in ng·h / mL.

[0065] t1 / 2 (h): Elimination half-life, which refers to the time required for the plasma drug concentration to decline to half of the maximum concentration, expressed in hours.

[0066] Bioavailability (%): It refers to the ratio of the amount of drug entering the systemic circulation after non-intravenous administration to the total administered dose. In this study, the measurement object in the blood was Ex32 peptide, and the oral administration was Ex32 tandem polypeptide.

[0067] The above table summarizes the main pharmacokinetic parameters under different administration routes and doses. The results show that this product exhibits good absorption characteristics and high bioavailability after oral administration, which is consistent with its enhanced enzymatic stability.

[0068] Example 6: Evaluation of the hypoglycemic effect of long-term administration of Ex32 tandem polypeptide.

[0069] 6.1 Experimental method.

[0070] Sixty 8-week-old db / db diabetic mice were randomly divided into 4 groups (15 mice in each group): blank control group (normal saline), low-dose group (0.5 mg / kg / day), high-dose group (2.5 mg / kg / day), and positive control group (subcutaneous injection of Exendin-4 at 10 μg / kg / day). The drug (Ex32 tandem polypeptide) was administered once every morning at 8:00 for 8 consecutive weeks. Fasting blood glucose (after 12 hours of fasting) and 2-hour postprandial blood glucose were measured twice a week, and at the same time, changes in body weight, water intake, and food intake were monitored. At the end of the experiment, blood samples were collected for glycated hemoglobin (HbA1c) detection, and ELISA method was used to determine plasma insulin and GLP-1 levels.

[0071] 6.2 Experimental results.

[0072] Analysis results of blood glucose levels and treatment indicators of mice after long-term administration of Ex32 tandem polypeptide are as follows: Analysis results of biochemical index detection and analysis of mice after long-term administration of Ex32 tandem polypeptide are as follows: The hypoglycemic effect of the high-dose oral administration group (2.5 mg / kg / day) is comparable to that of subcutaneous injection of Exendin-4 (10 μg / kg / day). Both groups showed significant improvement in various indicators, and the difference was not statistically significant (p>0.05), indicating that this modified polypeptide has good oral bioavailability and therapeutic effect.

[0073] Example 7: Safety assessment of long-term administration of Ex32 tandem polypeptide.

[0074] 7.1 Experimental method.

[0075] Ninety healthy SD rats were selected and randomly divided into 3 groups (30 rats in each group, with an equal number of males and females): control group (normal saline), low-dose group (5 mg / kg / day), and high-dose group (25 mg / kg / day). The Ex32 tandem polypeptide was orally administered once every morning at 8:00 for 12 consecutive weeks. During this period, the breeding environment was strictly controlled: temperature 23±2°C, relative humidity 50±10%, and a 12-hour light-dark cycle. The following indicators were observed: **Body weight change**: Monitor the body weight gain of animals during the 12-week drug administration period. **Hematological indices**: including red blood cell count (RBC), white blood cell count (WBC), platelet count (PLT), etc. **Liver function**: Monitor the changes in liver function indices such as ALT and AST. **Renal function**: Observe the fluctuations in renal function indices such as creatinine and urea nitrogen. **Immunogenicity**: Detect the IgG antibody titer to evaluate whether an immune response occurs.

[0076] 7.2 Experimental results.

[0077] The detailed summary of the safety assessment results of long-term administration of Ex32 tandem polypeptide is as follows: The results of the long-term drug safety study showed that no obvious toxic reactions were observed in the high- and low-dose groups of Ex32 tandem polypeptide. Although there was a slight increase in liver function indices in the high-dose group, they were all within the acceptable range (<1.5 times the upper limit of the normal value), and no corresponding pathological changes were observed. The complement system was not activated during the 12-week drug administration period, the cytokine levels were stable, and no obvious immunogenicity problems were found. All the test animals survived until the end of the experiment, and no serious adverse reactions related to drug administration occurred, indicating that this polypeptide has good safety characteristics.

Claims

1. A hypoglycemic polypeptide, characterized in that: The amino acid sequence of the polypeptide is AFHGEGTFTSDLSHQMEEEAVHLAIEWLHNFK (Sequence Listing SEQ ID NO: 1). Compared with the Exendin-4 prototype, the amino acids at positions 12, 20, 22 and 27 are histidine (H), histidine (H), alanine (A) and histidine (H) respectively, and the N-terminus and C-terminus are modified with alanine-phenylalanine (AF) and phenylalanine-lysine (FK) respectively.

2. A tandem polypeptide of an oral hypoglycemic polypeptide, characterized in that: The tandem polypeptide consists of the following structure (Sequence Listing SEQ ID NO: 2): (1) Three Ex32 core sequence fragments (amino acid sequence: HGEGTFTSDLSHQMEEEAVHLAIEWLHN); (2) Two linker peptides (amino acid sequence: FKCRRWQWRMKKLGAPSITCVRRAF); (3) N-terminal modification group alanine-phenylalanine (AF); (4) To achieve site-specific enzymatic cleavage by trypsin and selective release of the target fragment, the MKAF sequence and the FK sequence are introduced at the amino terminus and carboxyl terminus of the tandem fragment respectively.

3. Use of the hypoglycemic polypeptide according to claim 1 or 2 in the preparation of an oral pharmaceutical composition for the treatment of type 2 diabetes.

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