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

Abstract

The invention relates to the field of medicines, and particularly discloses a fusion protein with a GIP and GLP-1 dual receptor agonist function and application of the fusion protein. A specifically designed GLP-1 / GIP targeting polypeptide is fused with the N end of an IgG4 heavy chain constant region and the N end of a light chain constant region or the N end of Fc to obtain the GLP-1 / GIP targeting polypeptide. A research result shows that the fusion protein with the GIP and GLP-1 dual receptor agonist function has an obvious effect of reducing the weight of a mouse, the weight of the liver of the mouse is also reduced, the blood sugar of the mouse is also remarkably reduced, and the total cholesterol in serum is reduced to a certain extent. Therefore, the compound can be applied to medicines or products for slimming, reducing 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 the functions of a dual receptor agonist of GIP and GLP-1 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 the high incidence of these diseases, they have become an economic burden on public health.

[0003] Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are representative gastrointestinal hormones and neuronal hormones. GLP-1 receptor agonist drugs are a class of drugs for the treatment of type 2 diabetes. This type of drug exerts the effect of incretin by stimulating the glucagon-like peptide-1 receptor, thereby producing a hypoglycemic effect. They can not only lower blood sugar, but also reduce body weight and have a protective effect on cardiovascular system. 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 β cells to secrete insulin and protecting pancreatic β cells.

[0004] Therefore, it is necessary to develop a fusion protein of GIP and GLP-1 receptor agonist function 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 prior art, the present application provides a novel fusion protein having 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, and the amino acid sequence thereof 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; More specifically, the protein having the dual receptor agonist function of GIP and GLP-1 as claimed in claim 1 is fused to the N-terminus of Fc; and the protein having the dual receptor agonist function of GIP and GLP-1 is fused to the N-terminus of the heavy chain constant region and the light chain constant region of IgG4. Specifically, the N-terminal fusion is connected via a connecting peptide.

[0008] 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.

[0009] Specifically, the N-termini of the Fc or IgG4 heavy chain constant region and the light chain constant region are fused to a GLP and GIP dual-active protein via a connecting peptide, respectively. Preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO.7 or SEQ ID NO.8; The amino acid sequence obtained by fusing the protein having the dual receptor agonist function of GIP and GLP-1 as claimed in claim 1 to the N-terminus of Fc is shown in SEQ ID NO.9 or SEQ ID NO.10; The amino acid sequences obtained by fusing a protein having the functions of a dual receptor agonist of GIP and GLP-1 to the N-terminus of the heavy chain constant region and the light chain constant region of IgG4 are shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13, SEQ ID NO.14, respectively; In another specific aspect, a signal peptide is fused to the N-terminus of the protein having the function of a dual receptor agonist of GIP and GLP-1; preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.6.

[0010] The present invention also provides a nucleic acid encoding the protein having the functions of a dual receptor agonist of GIP and GLP-1; The nucleotide sequence encoding the protein having the functions of GIP and GLP-1 dual receptor agonist fused to the N-terminus of Fc is shown in SEQ ID NO.15 or SEQ ID NO.16; The nucleotide sequence encoding a protein with dual activity of GLP and GIP fused to the N-terminus of the IgG4 heavy chain constant region is shown as SEQ ID NO.17 or SEQ ID NO.18, and the nucleotide sequence encoding a protein with dual receptor agonist function of GIP and GLP-1 fused to the N-terminus of the IgG4 light chain constant region is shown as SEQ ID NO.19 or SEQ ID NO.20.

[0011] The present invention also provides a recombinant expression vector of the encoding nucleic acid.

[0012] 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 as described in claim 6 and transfecting the mammalian cells.

[0013] The present invention also provides a cell line of a protein having the function of a dual receptor agonist of GIP and GLP-1 obtained by the method.

[0014] The present invention particularly provides the use of the protein having the function of a dual receptor agonist of GIP and GLP-1, and the cell line in the preparation of weight loss products, blood sugar lowering products or drugs for treating fatty liver.

[0015] Beneficial effects of the present invention: The present invention selects amino acids suitable for cell expression by comprehensively considering the characteristics of GLP-1 and GIP sequences based on the analysis of GIP, GIP-1, and GLP-1 / GIP dual-activity analog sequences, and screens out two new dual-target proteins with GIP and GLP-1 dual receptor agonist functions on the basis of designing multiple sequences. Further, it forms a fusion protein with Fc or IgG4. The research results show that compared with the control, the fusion protein with GIP and GLP-1 dual receptor agonist functions of the present invention has a significant effect of reducing the weight of mice, the weight of the mouse liver also decreases, the blood sugar of the mouse also decreases significantly, and the total cholesterol content in the serum decreases to a certain extent. Therefore, it can be applied to the development of drugs or products for weight loss, blood sugar reduction, and treatment of fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 , schematic diagram of the results of pharmacokinetic experiments. DETAILED DESCRIPTION

[0017] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation to the present invention.

[0018] 1. Design or screening of proteins with dual receptor agonist functions of GIP and GLP-1 Based on the analysis of GIP, GIP-1, and GLP-1 / GIP dual-activity analogue sequences, the characteristics of GLP-1 and GIP sequences were comprehensively considered, and amino acids suitable for cell expression were selected. After designing multiple sequences, two new dual-target proteins PY001 and PY002 with GIP and GLP-1 dual receptor agonist functions were obtained through screening and verification. The amino acid sequences are as follows: PY001 (SEQ ID No: 1):HGEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPPPS; PY002 (SEQ ID No: 2):HGEGTFTSDYSIALDKIAQKAFIEWLIAGGPSSGAPPPS.

[0019] Among them, the other sequences used are as follows: The amino acid sequence of Fc is as SEQ ID Shown in NO.3: AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0020] The amino acid sequence of the IgG4 heavy chain constant region is as SEQ ID Shown in NO.4: PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0021] The amino acid sequence of the IgG4 light chain constant region is shown in SEQ ID NO.5: APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0022] The amino acid sequence of the signal peptide is shown in SEQ ID NO.6: METDTLLLWVLLLWVPGSTG.

[0023] The amino acid sequence of the connecting peptide is as follows: GGGGSGGGGSGGGGS.

[0024] 2. Construction of expression vector for GLP-1 / GIP-IgG4 fusion protein (1) Fusion protein with IgG4: The proteins PY001 and PY002 with dual receptor agonist functions of GIP and GLP-1 are fused to the N-terminus of the IgG4 heavy chain constant region, respectively. The two are connected by a connecting peptide and a signal peptide is added. The amino acid sequences are as follows: Heavy chain sequence of PY001-Hc (SEQ ID NO:7,395aa):METDTLLLWVLLLWVPGSTGHGEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0025] PY002-Hc of the heavy chain sequence (SEQ ID NO: 8, 395 aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSIALDKIAQKAFIEWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0026]

[0027]

[0028] The proteins PY001 and PY002, which have the functions of dual receptor agonists of GIP and GLP-1, were fused to the N-terminus of the IgG4 light chain constant region, respectively. The two were connected by a connecting peptide and a signal peptide was added. The amino acid sequences are as follows: Light chain sequence of PY001-Lc (SEQ ID NO: 9, 177aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAPSVFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0029] Light chain sequence of PY002-Lc (SEQ ID NO: 10, 177aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSIALDKIAQKAFIEWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAPSVFIFPPSDEQSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0030] Correspondingly, the PY001-Lc coding nucleotide sequence ((SEQ ID NO: 11, 531 bp): ATGGAAACCGATACCCTCTTGCTTTGGGTACTGCTCCTGTGGGTACCTGGCAGCACCGGTCACGGCGAGGGCACCTTTACCTCTGACTATTCAATACTGTTGGATAAGATCGCTCAAAAGGCTTTTGTCCAGTGGCTTATCGCAGGTGGGCCTTCTTCTGGAGCACCACCACCATCAGGGGGCGGTGGATCTGGGGGTGGAGGTTCTGGCGGTGGGGGGTCCGCACCCTCTGTCTTTATCTTTCCCCCTAGCGATGAGCAGCTTAAATCCGGGACCGCAAGCGTCGTCTGTCTGCTGAACAATTTTTATCCCAGAGAGGCCAAGGTGCAGTGGAAAGTGGACAACGCTCTCCAGAGTGGCAACAGTCAGGAGAGCGTAACAGAGCAGGATAGCAAGGACTCTACTTACTCCCTTAGCTCCACTCTGACCCTGTCAAAAGCTGACTATGAGAAGCACAAAGTGTACGCATGCGAGGTGACACATCAGGGTTTGAGCTCCCCAGTCACAAAGTCTTTTAACCGAGGAGAGTGT。

[0031] Correspondingly, the PY002-Lc encoding nucleotide sequence (SEQ ID NO: 12, 531 bp):.

[0032] (2) Fusion protein with Fc: The proteins PY001 and PY002, which have dual receptor agonist functions of GIP and GLP-1, were fused to the N-terminus of Fc, respectively. The two were connected by a connecting peptide and a signal peptide was added. The amino acid sequences are as follows: Amino acid sequence of PY001-Fc ((SEQ ID NO: 13, 303 aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0033] Amino acid sequence of PY002-Fc ((SEQ ID NO: 14, 303 aa): METDTLLLWVLLLWVPGSTGHGEGTFTSDYSIALDKIAQKAFIEWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0034] Correspondingly, the PY001-Fc coding nucleotide sequence ((SEQ ID NO: 15, 909bp): ATGGAAACCGATACCCTCTTGCTTTGGGTACTGCTCCTGTGGGTACCTGGCAGCACCGGTCACGGCGAGGGCACCTTTACCTCTGACTATTCAATACTGTTGGATAAGATCGCTCAAAAGGCTTTTGTCCAGTGGCTTATCGCAGGTGGGCCTTCTTCTGGAGCACCACCACCATCAGGGGGCGGTGGATCTGGGGGTGGAGGTTCTGGCGGTGGGGGGTCCGCTGAAAGCAAGTATGGACCTCCTTGCCCACCCTGTCCTGCACCTGAGGCTGCCGGAGGGCCCAGTGTGTTCTTGTTCCCACCAAAACCCAAGGATACCCTCATGATCAGCCGGACCCCAGAGGTAACCTGCGTGGTCGTGGACGTGAGCCAAGAGGACCCAGAAGTACAATTCAATTGGTATGTGGATGGAGTGGAGGTTCATAACGCAAAAACAAAGCCCCGCGAGGAACAGTTCAACTCCACATACCGCGTGGTCAGCGTTCTGACTGTACTGCACCAGGACTGGCTCAACGGAAAGGAGTACAAATGCAAAGTTTCTAACAAGGGTCTGCCATCCAGCATCGAAAAAACTATCTCCAAAGCAAAGGGGCAGCCTCGTGAACCTCAGGTATACACTCTCCCACCCAGCCAGGAGGAAATGACCAAAAATCAGGTATCTCTCACATGTCTGGTCAAGGGATTCTACCCATCCGACATTGCCGTGGAGTGGGAGTCAAATGGCCAGCCCGAAAATAACTATAAGACCACCCCACCCGTCTTGGACAGCGATGGCAGCTTTTTCCTGTACTCACGCCTGACAGTGGATAAGAGCCGTTGGCAGGAGGGTAACGTATTTTCTTGCAGCGTCATGCATGAAGCCCTGCATAATCATTACACCCAGAAGTCTCTGAGCCTGTCCCTGGGC。

[0035] Correspondingly, the PY002-Fc coding nucleotide sequence ((SEQ ID NO: 16, 909bp): ATGGAAACCGATACCCTCTTGCTTTGGGTACTGCTCCTGTGGGTACCTGGCAGCACCGGTCATGGAGAGGGGACATTTACATCTGACTACTCCATCGCCCTTGACAAGATTGCTCAGAAGGCCTTTATTGAGTGGCTGATTGCTGGAGGACCATCTTCTGGAGCACCACCTCCAAGTGGGGGCGGTGGATCTGGGGGTGGAGGTTCTGGCGGTGGGGGGTCCGCTGAAAGCAAGTATGGACCTCCTTGCCCACCCTGTCCTGCACCTGAGGCTGCCGGAGGGCCCAGTGTGTTCTTGTTCCCACCAAAACCCAAGGATACCCTCATGATCAGCCGGACCCCAGAGGTAACCTGCGTGGTCGTGGACGTGAGCCAAGAGGACCCAGAAGTACAATTCAATTGGTATGTGGATGGAGTGGAGGTTCATAACGCAAAAACAAAGCCCCGCGAGGAACAGTTCAACTCCACATACCGCGTGGTCAGCGTTCTGACTGTACTGCACCAGGACTGGCTCAACGGAAAGGAGTACAAATGCAAAGTTTCTAACAAGGGTCTGCCATCCAGCATCGAAAAAACTATCTCCAAAGCAAAGGGGCAGCCTCGTGAACCTCAGGTATACACTCTCCCACCCAGCCAGGAGGAAATGACCAAAAATCAGGTATCTCTCACATGTCTGGTCAAGGGATTCTACCCATCCGACATTGCCGTGGAGTGGGAGTCAAATGGCCAGCCCGAAAATAACTATAAGACCACCCCACCCGTCTTGGACAGCGATGGCAGCTTTTTCCTGTACTCACGCCTGACAGTGGATAAGAGCCGTTGGCAGGAGGGTAACGTATTTTCTTGCAGCGTCATGCATGAAGCCCTGCATAATCATTACACCCAGAAGTCTCTGAGCCTGTCCCTGGGC。

[0036] The genes encoding the above fusion proteins are all artificially optimized with CHO cell preferred codons, and the full-length sequences are synthesized by a gene synthesis company and constructed into a vector suitable for CHO cell expression. Gene synthesis, vector construction and other experimental methods use conventional molecular biology experimental methods.

[0037] 3. Construction of a cell line stably expressing the above fusion protein The recombinant expression plasmid was transfected into CHO-K1 cells by electrofection. The electrofection parameters were voltage of 1100V, pulse intensity of 30ms, and number of electric shocks of 1 time. After 48 hours of transfection, the screening medium containing methionine sulfoximine (MSX) was added for culture. When the cell viability rose to more than 70%, the transfected cells were plated on 96-well plates for monoclonal screening by limiting dilution method. The cell density of the plate was 0.5 per well. After the cells were statically cultured in the incubator for 14-20 days, the monoclonal cell expression supernatant was detected by ELISA. According to the test results, the 10-20 monoclonal cell lines with the highest expression were screened for gradual expansion and culture. After 3-7 days of culture in 6-well plates, the cells were counted and the protein expression was detected. The 3-5 cells with the highest protein expression were selected for expansion culture in T125 shake flasks. After 3-7 days of culture, the cells were counted and the protein expression was detected. The cells with the highest expression were selected as stable cell lines.

[0038] 4. Expression and purification of the above fusion protein The high-expressing cells were cultured in serum-free CD OptiCHO and the culture supernatant was collected after a certain period of time. The fusion protein was purified by the following affinity chromatography method: 1) Centrifuge the cell culture product to obtain the supernatant, filter it through a 0.45 μm filter, and mix it for later use.

[0039] 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) for later use.

[0040] 3) Load the supernatant of the cell culture product onto the affinity chromatography column, and then elute to the baseline with elution 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. Use pH 5.5-2.2 linear elution for 20 CV. Collect the elution peaks in different tubes, and combine the samples containing the target protein according to the electrophoresis detection results for later use.

[0041] 4) The affinity chromatography eluted sample was neutralized to pH 8.0 with 1M Tris buffer, diluted 1 / 2 with purified water, filtered through a 0.45 μm filter, and mixed for later use.

[0042] 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) for later use.

[0043] 6) Load the neutralized and diluted sample onto an anion exchange chromatography column, then elute to the baseline with equilibrium buffer. The elution buffer is 20mM Tris-HCl, 1M NaCl, pH8.0. Use 0-500mM NaCl linear elution for 10CV. Collect the elution peaks in separate tubes. Combine the samples containing the target protein according to the electrophoresis detection results for later use.

[0044] 7) After cleaning and disinfecting the Chromdex 200 gel filtration chromatography column, balance it with buffer (20 mM Pb, 150 mM NaCl, 0.02% Tween 80, pH 6.5) for later use.

[0045] 8) Load the anion exchange chromatography eluted sample onto the gel filtration chromatography column, control the loading amount within 30%CV, and collect the elution peak.

[0046] 5. Therapeutic efficacy test of the fusion protein of the present invention 2-3 week old C57BL / 6JNifdc-DIO male mice were purchased and fed with a high-fat diet for 10 weeks before being used for drug efficacy experiments. The experiment was conducted in this model mouse by subcutaneous injection for 5 weeks with a dosing frequency of 2 times / week (Tuesday and Friday).

[0047] The fusion proteins used in this experiment include the fusion proteins PY001-IgG4, PY001-Fc, PY002-IgG4, PY002-Fc of the present invention and Eli Lilly's dulaglutide (Dlu-Fc) protein. The fusion proteins used were diluted to the required concentration using physiological saline to obtain each fusion protein solution. 33 mice were randomly divided into 10 experimental groups and 1 blank control group. The mice in the 5 experimental groups were injected with 75 μg of PY001-IgG4, PY001-Fc, PY002-IgG4, PY002-Fc and Eli Lilly's dulaglutide, and the mice in the 5 experimental groups were injected with 225 μg of PY001-IgG4, PY001-Fc, PY002-IgG4, PY002-Fc and Eli Lilly's dulaglutide, and the fusion protein solutions of the 10 experimental groups were all 100 μl, and the blank control group was injected with 100 μl of physiological saline.

[0048] The group numbers are shown in Table 1: Table 1. Group design

[0049] 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.

[0050] Table 2. Average weight change rate (%)

[0051] 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, Groups 7 and 8.

[0052] 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.

[0053] The liver test results are shown in Table 3.

[0054] Table 3. Liver weight (unit: g)

[0055] 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.

[0056] Determination of biochemical indices The total cholesterol (TC) assay kit (oxidase method) and the triglyceride (TG) assay kit (oxidase method) were used to detect the total cholesterol and triglyceride content in the serum of mice in the experimental group and the blank control group. The test results of total cholesterol and triglyceride are shown in Table 4.

[0057] Table 4. Biochemical index determination results (unit: mmol / L)

[0058] From the results in Table 4, it can be seen that compared with the blank control group, the glucose and total cholesterol levels of the mice in the experimental groups were significantly reduced, especially in Groups 3 and 4, Groups 7 and 8. However, the triglyceride content in each experimental group did not change significantly.

[0059] 6. Pharmacokinetics experiments Balb / C mice were subcutaneously administered 1 mg of protein, and then blood was collected from the eyeball every 24 hours for 7 consecutive days. After 7 days, a conventional ELISA experiment was performed using anti-GLP-1 monoclonal antibody as the coating antibody and anti-Fc antibody as the detection antibody to detect the drug concentration in the serum collected every day. The results are shown in Table 5 and Figure 1 shown.

[0060] Table 5. Biochemical test results (unit: μg / ml)

[0061] As can be seen from the figure, the peak blood concentration of experimental groups 3, 4, 7, and 8 all appeared 24 hours after administration, and then the blood concentration gradually decreased, while the peak blood concentration of the remaining experimental groups appeared about 72 hours after administration, and then the blood concentration gradually decreased. The half-life of the natural GLP-1 polypeptide is only a few minutes, so the new fusion protein of the present invention greatly prolongs the half-life and has the potential value of being developed into a drug.

Claims

1. A protein having the function of a dual receptor agonist of GIP and GLP-1, characterized in that: Its amino acid sequence is shown as SEQ ID NO.1 or SEQ ID NO.

2.

2. A fusion protein having the function of a dual receptor agonist of GIP and GLP-1, characterized in that: The protein having the dual receptor agonist function of GIP and GLP-1 as claimed in claim 1 is fused with IgG4 protein or Fc protein; More specifically, they are fused to the N-terminus of Fc The protein having the function of dual receptor agonist of GIP and GLP-1 as claimed in claim 1 is obtained; the N-terminus of the IgG4 heavy chain constant region and the light chain constant region are fused respectively The protein having the function of a dual receptor agonist of GIP and GLP-1 as claimed in claim 1 is obtained; Specifically, the N-terminal fusion is connected via a connecting peptide.

3. The dual-active fusion protein of GLP and GIP according to claim 2, characterized in that: 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.

4. The dual-active fusion protein of GLP and GIP according to claim 2, characterized in that: The N-termini of the Fc or IgG4 heavy chain constant region and the light chain constant region are fused to the dual-active proteins of GLP and GIP through connecting peptides, respectively. Preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO.7 or SEQ ID NO.8; The amino acid sequence obtained by fusing the protein having the dual receptor agonist function of GIP and GLP-1 as claimed in claim 1 to the N-terminus of Fc is shown in SEQ ID NO.9 or SEQ ID NO.10; The amino acid sequences obtained by fusing a protein having dual receptor agonist functions of GIP and GLP-1 to the N-terminus of the IgG4 heavy chain constant region and the light chain constant region are shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13, SEQ ID NO.14, respectively.

5. The dual-active fusion protein of GLP and GIP according to claim 2, characterized in that: The protein having the function of a dual receptor agonist of GIP and GLP-1 is fused with a signal peptide at its N-terminus; preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.

6.

6. The nucleic acid encoding the protein having the function of a dual receptor agonist of GIP and GLP-1 according to any one of claims 2 to 5; The nucleotide sequence encoding the protein having the functions of GIP and GLP-1 dual receptor agonist fused to the N-terminus of Fc is shown in SEQ ID NO.15 or SEQ ID NO.16; The nucleotide sequence encoding a protein with dual activity of GLP and GIP fused to the N-terminus of the IgG4 heavy chain constant region is shown as SEQ ID NO.17 or SEQ ID NO.18, and the nucleotide sequence encoding a protein with dual receptor agonist function of GIP and GLP-1 fused to the N-terminus of the IgG4 light chain constant region is shown as SEQ ID NO.19 or SEQ ID NO.

20.

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

8. A method for preparing a cell line having a protein having GIP and GLP-1 dual receptor agonist function, characterized in that: The method is obtained by transfecting a mammalian cell with the encoding nucleic acid as claimed in claim 5 or 6, preferably, the mammal is a CHO cell; preferably, the method is obtained by constructing a recombinant expression vector containing the encoding nucleic acid as claimed in claim 6, and then transfecting the vector into a mammalian cell after linearization.

9. A cell line having a protein having GIP and GLP-1 dual receptor agonist function obtained by the method according to claim 8.

10. Use of the protein having GIP and GLP-1 dual receptor agonist function as claimed in any one of claims 2 to 5, and the cell line as claimed in claim 9 in the preparation of weight loss products, blood sugar lowering products or drugs for treating fatty liver.

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