Fusion protein with GIP and GLP-1 dual receptor agonist function and its application

Abstract

The present application relates to the field of medicine, and specifically discloses a fusion protein having the function of a dual receptor agonist of GIP and GLP-1 and its application. It is obtained by fusing a specifically designed GLP-1 / GIP targeting polypeptide with the N-terminus of the IgG4 heavy chain constant region and the light chain constant region or the N-terminus of Fc. The results of the study showed that the fusion protein of the present invention having the function of a dual receptor agonist of GIP and GLP-1 had a significant effect on reducing the weight of mice, the weight of the mouse liver also decreased, the blood sugar of the mice also decreased significantly, and the total cholesterol in the serum decreased to a certain extent. Therefore, it can be used in drugs or products for weight loss, lowering blood sugar, and treating fatty liver.

Description

Technical Field

[0001] The present application relates to the field of medicine, and in particular to a fusion protein having GIP and GLP-1 dual receptor agonist functions and its application. Background Art

[0002] Obesity and type 2 diabetes are representative metabolic diseases. These diseases are common factors that threaten health. Due to their high incidence, they have become an economic burden on public health.

[0003] Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin-releasing polypeptide (GIP) are representative gastrointestinal hormones and neuronal hormones. GLP-1 receptor agonist drugs are a class of drugs used to treat type 2 diabetes. These drugs work by stimulating the glucagon-like peptide-1 receptor to act as incretins, thereby producing a hypoglycemic effect. They can not only lower blood sugar, but also reduce body weight and have a protective effect on cardiovascular health. Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal regulatory peptide that has physiological effects such as inhibiting gastric acid secretion, inhibiting pepsinogen secretion, and stimulating insulin release by stimulating pancreatic beta cells to secrete insulin and protecting pancreatic beta cells.

[0004] Therefore, it is necessary to develop a fusion protein of GIP and GLP-1 receptor agonist functions with significant weight loss and blood sugar lowering effects to achieve the purpose of treating obesity and hyperglycemia. Summary of the Invention

[0005] Based on the needs of the existing technology, the present application provides a novel fusion protein with dual activities of GLP-1 and GIP and its application.

[0006] First, the present invention provides a protein having the function of a dual receptor agonist of GIP and GLP-1, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0007] The present invention provides a fusion protein having the function of a dual receptor agonist of GIP and GLP-1, which is obtained by fusing the protein having the function of a dual receptor agonist of GIP and GLP-1 with an IgG4 protein or an Fc protein;

[0008] More specifically, the protein having GIP and GLP-1 dual receptor agonist functions as described in claim 1 is fused to the N-terminus of the Fc fragment; and the protein having GIP and GLP-1 dual receptor agonist functions is fused to the N-terminus of the IgG4 heavy chain constant region and the light chain constant region, respectively. Specifically, the N-terminal fusions are connected via a connecting peptide.

[0009] More specifically, the amino acid sequence of Fc is shown in SEQ ID NO.3; the amino acid sequence of the IgG4 heavy chain constant region is shown in SEQ ID NO.4; and the amino acid sequence of the IgG4 light chain constant region is shown in SEQ ID NO.5.

[0010] Specifically, the N-termini of the Fc or IgG4 heavy chain constant region and the light chain constant region are fused to GLP and GIP dual-active proteins via connecting peptides, respectively. Preferably, the amino acid sequence of the connecting peptide is GGGGSGGGGSGGGGS;

[0011] The amino acid sequence of the protein having the dual receptor agonist functions of GIP and GLP-1 is fused to the N-terminus of Fc, as shown in SEQ ID NO.15 or SEQ ID NO.16;

[0012] The amino acid sequences obtained by fusing a protein having GIP and GLP-1 dual receptor agonist functions to the N-termini of the IgG4 heavy chain constant region and the light chain constant region are shown in SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 11, SEQ ID NO. 12, respectively;

[0013] Specifically, the N-terminus of the protein having the function of a dual GIP and GLP-1 receptor agonist is fused with a signal peptide; preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.6.

[0014] The present invention also provides a nucleic acid encoding the protein having the functions of a dual GIP and GLP-1 receptor agonist;

[0015] The nucleotide sequence encoding the protein having the functions of a GIP and GLP-1 dual receptor agonist fused to the N-terminus of Fc is shown in SEQ ID NO. 17 or SEQ ID NO. 18;

[0016] The nucleotide sequence encoding a protein with dual GLP and GIP activity fused to the N-terminus of the IgG4 heavy chain constant region is shown in SEQ ID NO.9 or SEQ ID NO.10, and the nucleotide sequence encoding a protein with dual GIP and GLP-1 receptor agonist function fused to the N-terminus of the IgG4 light chain constant region is shown in SEQ ID NO.13 or SEQ ID NO.14.

[0017] The present invention also provides a recombinant expression vector of the encoding nucleic acid.

[0018] The present invention also provides a method for preparing a cell line of a protein having the function of a dual receptor agonist of GIP and GLP-1, characterized in that the cell line is obtained by transfecting the encoding nucleic acid into mammalian cells, preferably, the mammal is a CHO cell; preferably, the cell line is obtained by constructing a recombinant expression vector containing the encoding nucleic acid and transfecting the mammalian cells.

[0019] The present invention also provides a cell line having a protein having GIP and GLP-1 dual receptor agonist function obtained by the method.

[0020] The present invention particularly provides the use of the protein having GIP and GLP-1 dual receptor agonist function and the cell line in preparing weight loss products, blood sugar lowering products or drugs for treating fatty liver.

[0021] Beneficial effects of the present invention:

[0022] Based on sequence analysis of GIP, GIP-1, and GLP-1 / GIP dual-activity analogs, the present invention comprehensively considers the characteristics of GLP-1 and GIP sequences, selects amino acids suitable for cellular expression, and, based on the design of multiple sequences, screens two novel dual-target proteins with GIP and GLP-1 receptor agonist functions. Furthermore, these proteins form fusion proteins with Fc or IgG4. Research results show that compared with controls, the present fusion proteins with GIP and GLP-1 receptor agonist functions significantly reduce mouse body weight, liver weight, blood sugar levels, and serum total cholesterol levels. Therefore, these proteins can be used in the development of drugs or products for weight loss, blood sugar reduction, and the treatment of fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 , schematic diagram of the results of the pharmacokinetic experiment. DETAILED DESCRIPTION

[0024] The present invention is described below through specific embodiments in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0025] 1. Design or screening of proteins with dual GIP and GLP-1 receptor agonist functions

[0026] Based on the sequence analysis of GIP, GIP-1, and GLP-1 / GIP dual-activity analogs, and considering the characteristics of GLP-1 and GIP sequences, we selected amino acids suitable for cell expression. After designing multiple sequences and screening and verification, we obtained two new dual-target proteins, PY001 and PY002, which have the function of dual GIP and GLP-1 receptor agonists. Their amino acid sequences are as follows:

[0027] PY001 (SEQ ID No: 1): HGEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPPPS;

[0028] PY002 (SEQ ID No: 2): HGEGTFTSDYSIALDKIAQKAFIEWLIAGGPSSGAPPPS.

[0029] The other sequences used are as follows:

[0030] The amino acid sequence of Fc is shown in SEQ ID NO.3: AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0031] The amino acid sequence of the constant region of IgG4 heavy chain is shown in SEQ ID NO.4: PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0032] The amino acid sequence of the IgG4 light chain constant region is shown in SEQ ID NO. 5: APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0033] The amino acid sequence of the signal peptide is shown in SEQ ID NO.6: METDTLLLWVLLLWVPGSTG.

[0034] The amino acid sequence of the connecting peptide is as follows: GGGGSGGGGSGGGGS.

[0035] 2. Construction of expression vector for GLP-1 / GIP-IgG4 fusion protein

[0036] (1) Fusion protein with IgG4: Proteins PY001 and PY002, which have dual receptor agonist functions of GIP and GLP-1, are fused to the N-terminus of the IgG4 heavy chain constant region. The two proteins are connected by a connecting peptide and a signal peptide is added. The amino acid sequences are as follows:

[0037] Heavy chain sequence of PY001-Hc (SEQ ID NO:7,395aa):METDTLLLWVLLLWVPGSTGHGEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0038] PY002-Hc of the heavy chain sequence (SEQ ID NO: 8, 395 aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSIALDKIAQKAFIEWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0039]

[0040]

[0041] The proteins PY001 and PY002, which have dual GIP and GLP-1 receptor agonist functions, were fused to the N-terminus of the IgG4 light chain constant region, respectively. The two proteins were connected by a linker peptide and a signal peptide was added. Their amino acid sequences are as follows:

[0042] Light chain sequence of PY001-Lc (SEQ ID NO: 11, 177aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAPSVFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0043] Light chain sequence of PY002-Lc (SEQ ID NO: 12, 177aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSIALDKIAQKAFIEWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAPSVFIFPPSDEQSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0044] Correspondingly, the PY001-Lc coding nucleotide sequence ((SEQ ID NO: 13, 531 bp): ATGGAAACCGATACCCTCTTGCTTTGGGTACTGCTCCTGTGGGTACCTGGCAGCACCGGTCACGGCGAGGGCACCTTTACCTCTGACTATTCAATACTGTTGGATAAGATCGCTCAAAAGGCTTTTGTCCAGTGGCTTATCGCAGGTGGGCCTTCTTCTGGAGCACCACCACCATCAGGGGGCGGTGGATCTGGGGGTGGAGGTTCTGGCGGTGGGGGGTCCGCACCCTCTGTCTTTATCTTTCCCCCTAGCGATGAGCAGCTTAAATCCGGGACCGCAAGCGTCGTCTGTCTGCTGAACAATTTTTATCCCAGAGAGGCCAAGGTGCAGTGGAAAGTGGACAACGCTCTCCAGAGTGGCAACAGTCAGGAGAGCGTAACAGAGCAGGATAGCAAGGACTCTACTTACTCCCTTAGCTCCACTCTGACCCTGTCAAAAGCTGACTATGAGAAGCACAAAGTGTACGCATGCGAGGTGACACATCAGGGTTTGAGCTCCCCAGTCACAAAGTCTTTTAACCGAGGAGAGTGT。

[0045] Correspondingly, the PY002-Lc encoding nucleotide sequence (SEQ ID NO: 14, 531 bp):.

[0046] (2) Fusion protein with Fc:

[0047] The proteins PY001 and PY002, which have dual GIP and GLP-1 receptor agonist functions, were fused to the N-terminus of the Fc, respectively. The two proteins were connected by a linker peptide and a signal peptide was added. Their amino acid sequences are as follows:

[0048] Amino acid sequence of PY001-Fc (SEQ ID NO: 15, 303 aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0049] Amino acid sequence of PY002-Fc (SEQ ID NO: 16, 303 aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSIALDKIAQKAFIEWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0050] Correspondingly, the PY001-Fc coding nucleotide sequence (SEQ ID NO: 17, 909 bp): ATGGAAACCGATACCCTCTTGCTTTGGGTACTGCTCCTGTGGGTACCTGGCAGCACCGGTCACGGCGAGGGCACCTTTACCTCTGACTATTCAATACTGTTGGATAAGATCGCTCAAAAGGCTTTTGTCCAGTGGCTTATCGCAGGTGGGCCTTCTTCTGGAGCACCACCACCATCAGGGGGCGGTGGATCTGGGGGTGGAGGTTCTGGCGGTGGGGGGTCCGCTGAAAGCAAGTATGGACCTCCTTGCCCACCCTGTCCTGCACCTGAGGCTGCCGGAGGGCCCAGTGTGTTCTTGTTCCCACCAAAACCCAAGGATACCCTCATGATCAGCCGGACCCCAGAGGTAACCTGCGTGGTCGTGGACGTGAGCCAAGAGGACCCAGAAGTACAATTCAATTGGTATGTGGATGGAGTGGAGGTTCATAACGCAAAAACAAAGCCCCGCGAGGAACAGTTCAACTCCACATACCGCGTGGTCAGCGTTCTGACTGTACTGCACCAGGACTGGCTCAACGGAAAGGAGTACAAATGCAAAGTTTCTAACAAGGGTCTGCCATCCAGCATCGAAAAAACTATCTCCAAAGCAAAGGGGCAGCCTCGTGAACCTCAGGTATACACTCTCCCACCCAGCCAGGAGGAAATGACCAAAAATCAGGTATCTCTCACATGTCTGGTCAAGGGATTCTACCCATCCGACATTGCCGTGGAGTGGGAGTCAAATGGCCAGCCCGAAAATAACTATAAGACCACCCCACCCGTCTTGGACAGCGATGGCAGCTTTTTCCTGTACTCACGCCTGACAGTGGATAAGAGCCGTTGGCAGGAGGGTAACGTATTTTCTTGCAGCGTCATGCATGAAGCCCTGCATAATCATTACACCCAGAAGTCTCTGAGCCTGTCCCTGGGC。

[0051] Correspondingly, the PY002-Fc coding nucleotide sequence (SEQ ID NO: 18, 909bp): ATGGAAACCGATACCCTCTTGCTTTGGGTACTGCTCCTGTGGGTACCTGGCAGCACCGGTCATGGAGAGGGGACATTTACATCTGACTACTCCATCGCCCTTGACAAGATTGCTCAGAAGGCCTTTATTGAGTGGCTGATTGCTGGAGGACCATCTTCTGGAGCACCACCTCCAAGTGGGGGCGGTGGATCTGGGGGTGGAGGTTCTGGCGGTGGGGGGTCCGCTGAAAGCAAGTATGGACCTCCTTGCCCACCCTGTCCTGCACCTGAGGCTGCCGGAGGGCCCAGTGTGTTCTTGTTCCCACCAAAACCCAAGGATACCCTCATGATCAGCCGGACCCCAGAGGTAACCTGCGTGGTCGTGGACGTGAGCCAAGAGGACCCAGAAGTACAATTCAATTGGTATGTGGATGGAGTGGAGGTTCATAACGCAAAAACAAAGCCCCGCGAGGAACAGTTCAACTCCACATACCGCGTGGTCAGCGTTCTGACTGTACTGCACCAGGACTGGCTCAACGGAAAGGAGTACAAATGCAAAGTTTCTAACAAGGGTCTGCCATCCAGCATCGAAAAAACTATCTCCAAAGCAAAGGGGCAGCCTCGTGAACCTCAGGTATACACTCTCCCACCCAGCCAGGAGGAAATGACCAAAAATCAGGTATCTCTCACATGTCTGGTCAAGGGATTCTACCCATCCGACATTGCCGTGGAGTGGGAGTCAAATGGCCAGCCCGAAAATAACTATAAGACCACCCCACCCGTCTTGGACAGCGATGGCAGCTTTTTCCTGTACTCACGCCTGACAGTGGATAAGAGCCGTTGGCAGGAGGGTAACGTATTTTCTTGCAGCGTCATGCATGAAGCCCTGCATAATCATTACACCCAGAAGTCTCTGAGCCTGTCCCTGGGC。

[0052] The genes encoding the aforementioned fusion proteins were synthesized using artificially optimized codons preferred by CHO cells. The full-length sequences were commissioned to a gene synthesis company and then constructed into a vector suitable for CHO cell expression. Gene synthesis and vector construction were performed using conventional molecular biology methods.

[0053] 3. Construction of a cell line stably expressing the above fusion protein

[0054] The recombinant expression plasmid was transfected into CHO-K1 cells via electroporation using a voltage of 1100 V, a pulse intensity of 30 ms, and a single electroporation cycle. Forty-eight hours after transfection, cells were cultured in a selection medium containing methionine sulfoximine (MSX). When cell viability reached above 70%, the transfected cells were plated in 96-well plates at a density of 0.5 cells per well for single clone selection using limiting dilution. After 14-20 days of static culture in an incubator, the supernatants of the clones were analyzed by ELISA. Based on the results, the top 10-20 expressing clones were selected and gradually expanded. After 3-7 days of culture in 6-well plates, the cells were counted and protein expression was measured. The top 3-5 expressing clones were then expanded in T125 shake flasks. After 3-7 days of culture, the cells were counted and protein expression was measured. The cells with the highest expression were selected as stable cell lines.

[0055] 4. Expression and purification of the above fusion protein

[0056] High-expressing cells were cultured in serum-free CD OptiCHO medium and the culture supernatant was collected after a certain period of time. The fusion protein was purified by affinity chromatography as follows:

[0057] 1) Centrifuge the cell culture product, obtain the supernatant, filter it through a 0.45 μm filter, and mix it for later use.

[0058] 2) After cleaning and disinfecting the EZ Fast NoVo A affinity chromatography column, equilibrate it with equilibration buffer (20 mM PB, 150 mM NaCl, pH 8.0) before use.

[0059] 3) Load the cell culture supernatant onto an affinity chromatography column and then elute to baseline with wash buffer (0.1 M glycine-HCl, 50 mM Arg, pH 5.5). The elution buffer is 0.1 M glycine-HCl, 50 mM Arg, pH 2.2. A linear elution from pH 5.5 to 2.2 is performed for 20 CV. Collect the elution peaks in separate tubes and combine the samples containing the target protein based on the electrophoresis results.

[0060] 4) The affinity chromatography eluted sample was neutralized to pH 8.0 with 1 M Tris buffer, diluted 1 / 2 with purified water, filtered through a 0.45 μm filter, and mixed for later use.

[0061] 5) After cleaning and disinfecting the POROS 50 HQ anion exchange chromatography column, equilibrate it with equilibration buffer (20 mM Tris-HCl, pH 8.0) and set aside.

[0062] 6) Load the neutralized and diluted sample onto an anion exchange chromatography column and then elute to baseline with equilibration buffer (20 mM Tris-HCl, 1 M NaCl, pH 8.0). Use a linear elution from 0 to 500 mM NaCl for 10 CV. Collect the elution peaks separately and combine the samples containing the target protein based on the electrophoresis results.

[0063] 7) After cleaning and disinfecting the Chromdex 200 gel filtration chromatography column, equilibrate it with buffer (20 mM Pb, 150 mM NaCl, 0.02% Tween 80, pH 6.5) and set aside.

[0064] 8) Load the anion exchange chromatography elution sample onto a gel filtration column, controlling the loading volume within 30% CV, and collect the elution peak.

[0065] 5. Therapeutic efficacy test of the fusion protein of the present invention

[0066] 2-3 week old C57BL / 6JNifdc-DIO male mice were purchased and fed a high-fat diet for 10 weeks before being used for efficacy testing. The drug was administered subcutaneously twice weekly (Tuesday and Friday) for 5 weeks.

[0067] The fusion proteins used in this experiment included the present invention's fusion proteins PY001-IgG4, PY001-Fc, PY002-IgG4, and PY002-Fc, and Eli Lilly's dulaglutide (Dlu-Fc). Each fusion protein was diluted with physiological saline to the desired concentration to obtain a fusion protein solution. Thirty-three mice were randomly divided into 10 experimental groups and one blank control group. The five experimental groups were injected with 75 μg of PY001-IgG4, PY001-Fc, PY002-IgG4, PY002-Fc, and Eli Lilly's dulaglutide, respectively. The five experimental groups were injected with 225 μg of PY001-IgG4, PY001-Fc, PY002-IgG4, PY002-Fc, and Eli Lilly's dulaglutide, respectively. The fusion protein solution for each of the ten experimental groups was 100 μl, while the blank control group was injected with 100 μl of physiological saline.

[0068] The group numbers are shown in Table 1:

[0069] Table 1. Group design

[0070]

[0071] Experimental procedure: The mice were injected once every Tuesday and Friday for 5 consecutive weeks. The mice were weighed before each administration. The results are shown in Table 2.

[0072] Table 2. Average weight change rate (%)

[0073]

[0074] As shown in Table 2, compared with the blank control group, the average weight change rate of the mice in the experimental groups was significantly reduced, especially in Groups 3 and 4, and Groups 7 and 8.

[0075] After the last weighing, the mice were fasted and killed 16 hours later. The livers were removed and weighed, and the serum was taken to measure biochemical indicators.

[0076] The liver test results are shown in Table 3.

[0077] Table 3. Liver weight (unit: g)

[0078]

[0079] From the results in Table 3, it can be seen that compared with the blank control group, the liver weights of the mice in the experimental group were significantly reduced.

[0080] Biochemical markers were measured using a total cholesterol (TC) assay kit (oxidase method) and a triglyceride (TG) assay kit (oxidase method), respectively, to measure the total cholesterol and triglyceride levels in the serum of mice in the experimental and blank control groups. The results of the total cholesterol and triglyceride assays are shown in Table 4.

[0081] Table 4. Biochemical index test results (unit: mmol / L)

[0082]

[0083] The results in Table 4 show that compared with the blank control group, the glucose and total cholesterol levels of the experimental groups were significantly reduced, especially in Groups 3 and 4, and Groups 7 and 8. However, the triglyceride levels in all experimental groups did not change significantly.

[0084] 6. Pharmacokinetics experiments

[0085] Balb / C mice were subcutaneously administered 1 mg of protein, and blood was collected from the eyeball every 24 hours for 7 consecutive days. After 7 days, a conventional ELISA experiment was performed using an anti-GLP-1 monoclonal antibody as the coating antibody and an anti-Fc antibody as the detection antibody to measure the drug concentration in the serum collected daily. The results are shown in Table 5 and Figure 1 shown.

[0086] Table 5. Biochemical assay results (unit: μg / ml)

[0087]

[0088] As can be seen from the figure, peak blood concentrations in experimental groups 3, 4, 7, and 8 all occurred 24 hours after administration, followed by a gradual decline. In the remaining experimental groups, peak blood concentrations occurred approximately 72 hours after administration, followed by a gradual decline. Since the natural GLP-1 polypeptide has a half-life of only a few minutes, the novel fusion proteins of the present invention significantly extend their half-life, demonstrating their potential for development as pharmaceuticals.

Claims

1. A fusion protein having the function of a dual receptor agonist of GIP and GLP-1, characterized in that: It consists of a signal peptide, a protein with GIP and GLP-1 dual receptor agonist functions, a connecting peptide, and an Fc protein, wherein the protein with GIP and GLP-1 dual receptor agonist functions is fused to the N-terminus of the Fc protein via the connecting peptide, and the signal peptide is at the N-terminus of the fusion protein; The amino acid sequence of the protein having the function of a dual GIP and GLP-1 receptor agonist is shown in SEQ ID NO: 1 or SEQ ID NO: 2; The amino acid sequence of Fc is shown in SEQ ID NO.

3.

2. The fusion protein according to claim 1, wherein The amino acid sequence of the connecting peptide is GGGGSGGGGSGGGGS.

3. The fusion protein according to claim 2, wherein The amino acid sequence of the signal peptide is shown in SEQ ID NO.

6.

4. The dual-active fusion protein of GLP and GIP according to claim 3, characterized in that: Its amino acid sequence is shown in SEQ ID NO.15 or SEQ ID NO.

16. The nucleic acid encoding the fusion protein according to claim 1 .

6. The encoding nucleic acid according to claim 5, wherein Its nucleotide sequence is shown in SEQ ID NO.17 or SEQ ID NO.

18.

7. A recombinant expression vector comprising the encoding nucleic acid according to claim 6.

8. A method for preparing a cell line having a fusion protein having GIP and GLP-1 dual receptor agonist function, characterized in that: The encoding nucleic acid according to claim 5 or 6 is transfected into mammalian cells, which is obtained by constructing a recombinant expression vector containing the encoding nucleic acid according to claim 7, linearizing it, and then transfecting it into mammalian cells.

9. The method according to claim 8, wherein The mammalian cell is a CHO cell.

10. A cell line having a fusion protein having GIP and GLP-1 dual receptor agonist functions obtained by the method according to claim 9.

11. Use of the fusion protein according to any one of claims 1 to 4 or the cell line according to claim 10 in the preparation of a weight-loss drug, a blood sugar-lowering drug or a drug for treating fatty liver.

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