GLP-1 analogue fusion protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of genetic engineering biology, and particularly discloses a GLP-1 analogue fusion protein as well as a preparation method and application thereof. According to the application, fGLP-1 and a mutant thereof are respectively fused with FSA, IgG4-Fc and a mutant thereof through connecting peptides to form a GLP-1 analogue fusion protein with the effects of high biological activity, long half-life period, high stability and the like, and meanwhile, after pichia pastoris codon is optimized and modified, an efficient pichia pastoris methanol-induced secretory expression system is utilized for expression; the problems of short half-life period, low biological activity, poor thermal stability, potential immunogenicity, complex production process, high purification and preparation cost and the like of the GLP-1 analogue prepared by the existing expression system and related biotechnology are solved.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering biotechnology, and in particular discloses a GLP-1 analog fusion protein and a preparation method and application thereof. Background Art

[0002] Glucagon-like peptide-1 (GLP-1) is a short peptide hormone composed of 37 amino acids synthesized and secreted by intestinal endocrine cells-L cells. It is distributed in organs and tissues throughout the body. 80% of it exists in the form of GLP-1(7-36)NH2 in the body, and GLP-1(7-37) forms about 20%. The whole GLP-1 peptide (1-37) is inactive. The sequence of GLP-1(7-37) is conserved in humans, dogs and cats. GLP-1 mainly regulates blood sugar levels through multiple pathways such as promoting insulin secretion, inhibiting glucose production, slowing down gastrointestinal glucose absorption, inhibiting gastric emptying and reducing appetite. GLP-1 has a glucose concentration-dependent hypoglycemic effect. When blood sugar levels are elevated, GLP-1 exerts a hypoglycemic effect. When blood sugar levels are normal, GLP-1 will not further reduce blood sugar levels. Therefore, GLP-1 reduces the risk of hypoglycemia commonly seen when administering hypoglycemic pharmaceutical compositions and has a high safety profile. Therefore, GLP-1 is a potential ideal drug for the treatment of type II diabetes. In addition, GLP-1 analogs work through multiple mechanisms to reduce appetite, lower blood sugar levels, and promote weight loss, making GLP-1 analogs one of the effective drugs for treating obesity and diabetes.

[0003] The exogenous expression of GLP-1 mainly uses three expression systems: Escherichia coli, Pichia pastoris, and animal cells. Many studies have shown that GLP-1 has good biological activity in Escherichia coli, Pichia pastoris, and CHO cell expression systems. However, due to the lack of an effective release and secretion mechanism for proteins in the Escherichia coli expression system, it faces problems such as low yield and inclusion body expression, and there are cumbersome protein purification processes such as renaturation, removal of endotoxins and heat sources; while the use of animal cells for expression is industrially complex, costly, and difficult for large-scale industrial production; therefore, the Pichia pastoris system, which is genetically stable, low-cost, simple to operate, has the function of post-translational modification of eukaryotic cells, and can achieve high-level secretory expression of exogenous proteins, is conducive to purification and is used for the expression of GLP-1.

[0004] Natural feline GLP-1 (fGLP-1) is a short peptide of only 3.3 kda, and it is difficult to express and purify it directly using the Pichia pastoris expression system or the prokaryotic Escherichia coli expression system. In addition, the low molecular weight natural fGLP-1 is quickly cleared through the kidneys, and because natural fGLP-1 is rapidly inactivated by the protease dipeptidyl peptidase IV (DPP-IV) in the body, the half-life of natural fGLP-1 in plasma is very short, only 2 to 6 minutes, making it difficult to be widely used in clinical practice.

[0005] It is an economical and efficient method to express and produce fGLP-1 analogs through genetic engineering and related molecular biology techniques. However, natural fGLP-1 is susceptible to protease degradation and has a short half-life in the body. Continuous administration can cause side effects such as nausea, headache, and diarrhea, which directly affects the clinical treatment effect, which greatly limits its application in clinical treatment.

[0006] The existing technology usually adopts the following two approaches to solve the problems of low molecular weight and short half-life of natural GLP-1. The first approach is to mutate or modify natural GLP-1 to enhance its ability to resist degradation by DPP-IV and prolong its half-life. The other approach is to increase the molecular weight of GLP-1 analogs to reduce their clearance in the kidneys, and link the two modified GLP-1 peptide chains to serum albumin, thereby prolonging the effective action time and solving the problem of short half-life of GLP-1 analogs.

[0007] However, although the above ideas can be used to modify the fatty acid side chain of GLP-1 or express it by protein fusion, even after fatty acid modification or protein fusion, GLP-1 still has problems such as easy breakage during expression, poor stability, high sensitivity to the environment, easy inactivation in the body, large molecular weight, poor lipid solubility, and difficulty in penetrating biological membranes. For example, the existing GLP-1 / HSA fusion protein has some disadvantages. First, compared with the GLP-1 molecule, the molecular weight of albumin is huge. After the fusion of the two, the biological activity of the GLP-1 / HSA fusion protein is reduced or basically has no biological activity due to steric hindrance. Clinical use requires an increase in dosage, which will increase the risk of antibody formation. In addition, since diabetes requires lifelong treatment, there are general concerns about the immunogenicity of multiple fusion proteins when they are repeatedly administered for a long period of time; due to the problem of product stability, such products are currently sensitive to light and heat, and need to be stored and transported at low temperatures, which is inconvenient for patients to use.

[0008] As the number of dogs and cats in China has exceeded 120 million in 2023, and the number of pet cats is nearly 70 million, pet cats face different health challenges at different stages of life, such as diabetes, obesity, etc. Therefore, it is urgent to develop a GLP-1 analog fusion protein specifically for cats with a long half-life, good stability, and no immunogenicity, for diseases such as diabetes and obesity in cats. Summary of the invention

[0009] The purpose of the present application is to overcome the shortcomings of the prior art natural GLP-1, such as short half-life, poor stability and immunogenicity, and to provide a GLP-1 analog fusion protein and a preparation method and application thereof.

[0010] To achieve the above purpose, the technical solution adopted by this application is:

[0011] The present application provides a GLP-1 analog fusion protein, wherein the GLP-1 analog fusion protein is any one of the following fusion proteins;

[0012] 1) a sequence formed by mutating the amino acid sequence as shown in SEQ ID NO: 1 or by mutating at least one glycine in the amino acid sequence as shown in SEQ ID NO: 1, or / and by mutating at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 1 to glycine, or / and by adding at least one glycine to the amino acid sequence as shown in SEQ ID NO: 1,

[0013] By connecting peptides,

[0014] fusing with the amino acid sequence as shown in SEQ ID NO: 15 or a sequence in which at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 15 is mutated to at least one alanine to obtain a GLP-1 analog fusion protein;

[0015] 2) a sequence formed by mutating the amino acid sequence as shown in SEQ ID NO: 1, or by mutating at least one glycine in the amino acid sequence as shown in SEQ ID NO: 1, or / and by mutating at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 1 to glycine, or / and by adding at least one glycine to the amino acid sequence as shown in SEQ ID NO: 1,

[0016] By connecting peptides,

[0017] The GLP-1 analog fusion protein is obtained by fusing it with the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 33 or a sequence formed by mutation or truncation of the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 33.

[0018] In order to solve the shortcomings of the prior art natural GLP-1, such as short half-life, poor stability and immunogenicity, the present application provides two GLP-1 analog fusion proteins. The first one is fGLP-1 (SEQ ID NO: 1) and its mutants (fGLP-1-mut) fused with feline serum albumin (FSA, SEQ ID NO: 15) and its mutants (FSA-mut), and a linker is added between feline serum albumin and its mutants and fGLP-1 and its mutants, thereby increasing the thermal stability and protease resistance stability of the fusion protein; the other one is fGLP-1 and its mutants fused with IgG-Fc (SEQ ID NO: 29 or 33) and its mutants (Fc-mut), and in the part where fGLP-1 and Fc are fused and expressed, the cat-derived Fc is truncated and replaced, thereby reducing the effector function of the cat Fc protein as much as possible, thereby overcoming the potential immunogenicity problem of heterologous Fc fusion proteins to a large extent and improving the safety and efficacy of the drug.

[0019] At the same time, the GLP-1 analog fusion protein provided in the present application is a sequence formed by mutating different amino acid sites of fGLP-1 (SEQ ID NO: 1) (mutating at least one glycine, or mutating at least one amino acid to glycine, or adding at least one glycine) to form an fGLP-1 mutant. The above mutation formation can reduce the degradation activity of DDP-IV on the GLP-1 analog fusion protein, and can also improve the expression level of the protein and the stability of the drug; when fGLP-1 and its mutants (fGLP-1-mut) are fused with feline serum albumin (FSA) and its mutants (FSA-mut) or with IgG-Fc and its mutants (Fc-mut), the half-life of the protein can be further extended.

[0020] As a preferred embodiment of the GLP-1 analog fusion protein of the present application, in 1) or 2), the glycine at position 22 of the amino acid sequence shown in SEQ ID NO: 1 is arbitrarily mutated;

[0021] Preferably, the glycine at position 22 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to glutamic acid.

[0022] As a preferred embodiment of the GLP-1 analog fusion protein described in the present application, in 1) or 2), at least one amino acid mutation site in the amino acid sequence shown in SEQ ID NO: 1 includes mutation of alanine at position 8 and / or arginine at position 36 to glycine.

[0023] As a preferred embodiment of the GLP-1 analog fusion protein described in the present application, in 1) or 2), at least one glycine is added to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO: 1.

[0024] The present application mutates the amino acids in the amino acid sequence (fGLP-1) shown in SEQ ID NO: 1 at the above sites, which can reduce the degradation activity of DDP-IV on the GLP-1 analog fusion protein. Fusion of fGLP-1 and its mutants with feline serum albumin (FSA) and its mutants (FSA-mut) or with IgG-Fc and its mutants (Fc-mut) can prolong the half-life of the GLP-1 analog fusion protein, and further improve the expression level of the GLP-1 analog fusion protein and the stability of the drug.

[0025] As a preferred embodiment of the GLP-1 analog fusion protein described in the present application, in 1) or 2), the number of glycine added is ≥1, preferably 1 to 3.

[0026] As a preferred embodiment of the GLP-1 analog fusion protein described in the present application, in 1), the mutation site of at least one amino acid in the amino acid sequence shown in SEQ ID NO: 15 includes at least one of phenylalanine at position 330, tyrosine at position 332, tyrosine at position 334, and tyrosine at position 341.

[0027] As a preferred embodiment of the GLP-1 analog fusion protein described in the present application, in the above 2), any mutation is performed at any position among positions 37, 100, 171, 176 and 177 of the amino acid sequence shown in SEQ ID NO: 29.

[0028] As a preferred embodiment of the GLP-1 analog fusion protein described in the present application, in the above 2), in the amino acid sequence shown in SEQ ID NO: 29, the proline at position 37 is mutated to glutamine, the proline at position 100 is mutated to serine, the serine at position 171 is mutated to proline, the phenylalanine at position 176 is mutated to alanine, and the valine at position 177 is mutated to alanine.

[0029] As a preferred embodiment of the GLP-1 analog fusion protein of the present application, the sequence of the connecting peptide is shown as SEQ ID NOs: 9-11.

[0030] The use of the above-mentioned connecting peptide is beneficial to the half-life of the GLP-1 analog fusion protein, thereby improving thermal stability and protease resistance stability.

[0031] As a preferred embodiment of the GLP-1 analog fusion protein of the present application, a histidine tag is also added to the C-terminus of the GLP-1 analog fusion protein.

[0032] As a preferred embodiment of the GLP-1 analog fusion protein described in the present application, the GLP-1 analog fusion protein comprises an amino acid sequence having at least 90% homology with at least one of the sequences SEQ ID NOs: 19-23 and SEQ ID NOs: 35-37.

[0033] As a preferred embodiment of the GLP-1 analog fusion protein described in the present application, the amino acid sequence of the GLP-1 analog fusion protein is shown in SEQ ID NOs: 19 to 23 and SEQ ID NOs: 35 to 37, and the nucleotide sequence of the GLP-1 analog fusion protein is shown in SEQ ID NOs: 24 to 28 and SEQ ID NOs: 38 to 40.

[0034] Preferably, the GLP-1 analog fusion proteins are fGLP-1-L1-FSA-H (SEQ ID NO: 19), fGLP-1-mut1-L1-FSA-H (SEQ ID NO: 20), fGLP-1-mut1-L1-FSA-mut-H (SEQ ID NO: 21), fGLP-1-mut2-L2-FSA-mut-H (SEQ ID NO: 22), fGLP-1-mut3-L3-FSA-mut-H (SEQ ID NO: 23), fGLP-1-mut1-L1-fFc1-H (SEQ ID NO: 35), fGLP-1-mut2-L2-fFc1-mut-H (SEQ ID NO: 36), and fGLP-1-mut3-L3-Fc2-H (SEQ ID NO: 37).

[0035] The GLP-1 analog fusion protein constructed in the present application has a long half-life, which can be up to 28 hours.

[0036] The present application also provides a polynucleotide encoding the above-mentioned GLP-1 analog fusion protein.

[0037] The present application also provides an expression vector, which comprises the above-mentioned polynucleotide.

[0038] As a preferred embodiment of the expression vector described in the present application, the expression vector includes a prokaryotic expression vector, a mammalian cell expression vector, a baculovirus-insect cell expression vector, and a yeast expression vector, and the yeast expression vector includes one of pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pPIC9K, pPIC9, pHIL-S1, pJL-SX, pBLHIS-SX, pYAM75P, pAOX1, pAOX2, pPICZA, pPICZB, pPICZC, pGAPZA, pPIC3K, pPIC3.5K, pHIL-D2, pJL-IX, and pBLHIS-IX, preferably pPICZαA, pPICZαB, and pPICZαC. More preferably, the expression vector is pPICZαA.

[0039] The present application also provides an expression host including a host bacterium or a host cell, wherein the host bacterium includes the above-mentioned polynucleotide or the above-mentioned expression vector.

[0040] As a preferred embodiment of the host bacteria or host cell described in the present application, the host bacteria include Pichia pastoris expression host bacteria, Escherichia coli expression host bacteria, and Bacillus expression host bacteria, preferably Pichia pastoris expression host bacteria, and the Pichia pastoris expression host bacteria include one of X33, GS115, KM71, KM71H, SMD1168, SMD1168 H, SMD1165, SMD1163, MP-36, MC100-3, MS105, and JC100. Preferably, the Pichia pastoris expression host bacteria is X33.

[0041] Pichia pastoris has the characteristics of fast growth and easy operation of prokaryotes, and also has the modification function of eukaryotic cell expression system. The post-translational processing and modification of its expressed proteins are similar to those of higher eukaryotes. Integrating foreign genes into the chromosome of Pichia pastoris has the advantages of high protein expression content, moderate glycosylation, easy formation of disulfide bonds, good biological activity of the expression product, less background protein, simple operation and easy industrial production.

[0042] The above-mentioned GLP-1 analog fusion protein is expressed using an efficient Pichia pastoris methanol-induced secretion expression system, thereby solving the problems of short half-life, low biological activity, poor thermal stability, potential immunogenicity, complex production process, and high purification and preparation cost of GLP-1 analog fusion proteins prepared by existing expression systems and related biotechnologies.

[0043] The present application also provides a method for producing the above-mentioned GLP-1 analog fusion protein, comprising the following steps:

[0044] The host bacteria are cultured to express the GLP-1 analog fusion protein.

[0045] This application selects the highly efficient Pichia pastoris methanol-induced secretion expression system, and utilizes methanol induction technology to efficiently induce protein expression, effectively increasing the expression amount of the target protein to the gram level, which can greatly reduce production costs in large-scale production.

[0046] The present application also provides a pharmaceutical composition, comprising the above-mentioned GLP-1 analog fusion protein, and a pharmaceutically acceptable carrier.

[0047] The present application also provides the use of the above-mentioned GLP-1 analog fusion protein or pharmaceutical composition in the preparation of hypoglycemic drugs, weight loss drugs or long-acting drugs.

[0048] In the present application, fGLP-1 and its mutants are fused with FSA and fFc proteins, and the characteristics of Pichia pastoris methanol-induced secretion expression system, such as moderate glycosylation modification, disulfide bond formation, and efficient induced expression, are utilized to achieve efficient expression of GLP-1 analog fusion protein in Pichia pastoris, so that the GLP-1 analog fusion protein has the characteristics of high biological activity, high stability, and moderate glycosylation modification.

[0049] The fGLP-1-L1-FSA-H, fGLP-1-mut1-L1-FSA-H, fGLP-1-mut1-L1-FSA-mut-H, fGLP-1-mut2-L2-FSA-mut-H, fGLP-1-mut3-L3-FSA-mut-H, fGLP-1-mut1-L1-fFc1-H, fGLP-1-mut2-L2-fFc1-mut-H, and fGLP-1-mut3-L3-Fc2-H in the present application have higher blood sugar and weight loss effects than natural fGLP-1. At the same time, the destruction of the recognition site of DPP-IV and the increase in molecular weight can significantly improve the half-life and stability of the GLP-1 analog fusion protein. The modified GLP-1 analog fusion protein can also reduce potential immunogenicity problems.

[0050] The present application also provides a use of cat serum albumin and its mutants, or IgG-Fc fragments and its mutants in the preparation of long-acting drugs, wherein the sequence of the cat serum albumin is shown in the amino acid sequence SEQ ID NO: 15;

[0051] The mutant of feline serum albumin is a sequence in which at least one amino acid in the amino acid sequence shown in SEQ ID NO: 15 is mutated to at least one alanine;

[0052] The sequence of the IgG-Fc fragment is shown in the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 33;

[0053] The mutant of the IgG-Fc fragment is: a sequence formed by mutation or truncation of the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 33;

[0054] Preferably, in the mutant of feline serum albumin, the mutation site of at least one amino acid in the amino acid sequence shown in SEQ ID NO: 15 includes at least one of phenylalanine at position 330, tyrosine at position 332, tyrosine at position 334, and tyrosine at position 341;

[0055] In the mutant of the IgG-Fc fragment, any mutation is performed at any one of positions 37, 100, 171, 176 and 177 of the amino acid sequence shown in SEQ ID NO: 29.

[0056] The cat serum albumin (FSA, SEQ ID NO: 15) and its mutant (FSA-mut), and IgG-Fc (SEQ ID NO: 29 or 33) and its mutant (Fc-mut) constructed by the present application can be used to prepare long-acting drugs to improve the efficacy of the drugs.

[0057] Compared with the prior art, this application has the following beneficial effects:

[0058] The present application provides a GLP-1 analog fusion protein and a preparation method and application thereof. The present application adopts fGLP-1 and its mutants, and fuses them with FSA, IgG4-Fc and its mutants respectively through connecting peptides to form a GLP-1 analog fusion protein with high biological activity, long half-life, high stability and the like. At the same time, the Pichia pastoris codons are optimized and modified and then expressed using an efficient Pichia pastoris methanol-induced secretion expression system, thereby solving the problems of short half-life, low biological activity, poor thermal stability, potential immunogenicity, complex production process, and high purification and preparation cost of GLP-1 analogs prepared by existing expression systems and related biotechnologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of fGLP-1-Linker-FSA fusion protein;

[0060] Figure 2 Schematic diagram of fGLP-1-Linker-Fc fusion protein;

[0061] Figure 3PCR identification of the recombinant yeast culture medium (M: DL2000; 1-2: pPICZαA-fGLP-1-L1-FSA-H-X33; 3-4: pPICZαA-fGLP-1-mut1-L1-FSA-H-X33;

[0062] 5~6: pPICZαA-fGLP-1-mut1-L1-FSA-mut-H-X33; 7~8: pPICZαA-fGLP-1-mut2-L2-FSA-mut-H-X33

[0063] 9~10: pPICZαA-fGLP-1-mut3-L3-FSA-mut-H-X33; 11~12: pPICZαA-fGLP-1-mut1-L1-fFc1-H-X33;

[0064] 13~14: pPICZαA-fGLP-1-mut2-L2-fFc1-mut-H-X33; 15~16: pPICZαA-fGLP-1-mut3-L3-fFc2-H-X33);

[0065] Figure 4 This is the SDS-PAGE detection diagram of the recombinant yeast induced expression supernatant (M: low molecular weight protein Marker; 1: pPICZαA-fGLP-1-L1-FSA-H-X33; 2: pPICZαA-fGLP-1-mut1-L1-FSA-H-X33; 3: pPICZαA-fGLP-1-mut1-L1-FSA-mut-H-X33; 4: pPICZαA-fGLP-1-mut2-L2-FSA-mut-H-X33;

[0066] 5: pPICZαA-fGLP-1-mut3-L3-FSA-mut-H-X33;

[0067] 6: pPICZαA-fGLP-1-mut1-L1-fFc1-H-X33;

[0068] 7: pPICZαA-fGLP-1-mut2-L2-fFc1-mut-H-X33;

[0069] 8: pPICZαA-fGLP-1-mut3-L3-fFc2-H-X33);

[0070] Figure 5 This is the SDS-PAGE detection diagram of the purified samples (M: protein marker; 1: fGLP-1-L1-FSA-H;

[0071] 2:fGLP-1-mut1-L1-FSA-H; 3:fGLP-1-mut1-L1-FSA-mut-H;

[0072] 4:fGLP-1-mut2-L2-FSA-mut-H;

[0073] 5:fGLP-1-mut3-L3-FSA-mut-H; 6:fGLP-1-mut1-L1-fFc1-H;

[0074] 7:fGLP-1-mut2-L2-fFc1-mut-H; 8:fGLP-1-mut3-L3-fFc2-H);

[0075] Figure 6 This is a graph for measuring the hypoglycemic effect of GLP-1 analog fusion protein;

[0076] Figure 7 This figure is used to evaluate the weight loss effect of GLP-1 analog fusion protein. DETAILED DESCRIPTION

[0077] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0078] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0079] The present application provides a GLP-1 analog fusion protein (including the fGLP-1-Linker-FSA fusion protein and fGLP-1-Linker-Fc fusion protein constructed below), and the amino acid sequence of the GLP-1 analog fusion protein is modified by Pichia pastoris codon.

[0080] Example 1: A GLP-1 analog fusion protein and its construction method

[0081] This embodiment provides a process for constructing a GLP-1 analog fusion protein, comprising the following steps:

[0082] 1. Synthesis of whole gene fragments and construction of recombinant plasmids:

[0083] The feline glucagon-like peptide-1 precursor (pro-glucagon) protein sequence (XP_019693729.1) queried from NCBI consists of 180 amino acids, of which the mature peptide fGLP-1 (1-37) is HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG. The full sequence of fGLP-1 was obtained by whole gene synthesis.

[0084] In order to improve the ability of fGLP-1 to resist DPP-IV degradation and prolong its half-life, the fGLP-1 amino acid sequence (SEQ ID NO: 1) was mutated at different sites to form fGLP-1(7-37)-mut1, fGLP-1(7-37)-mut2 and fGLP-1(7-37)-mut3.

[0085] fGLP-1(7-37) amino acid sequence: HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1).

[0086] fGLP-1(7-37) Pichia pastoris codon-optimized nucleic acid sequence:

[0087] CACGCTGAAGGTACCTTCACTTCCGACGTTTCTTCTTACCTGGAAGGTCAG GCTGCTAAGGAGTTCATTGCATGGTTGGTCAAGGGACGTGGA (SEQ ID NO: 2).

[0088] fGLP-1(7-37)-mut1 amino acid sequence: alanine at position 8 mutated to glycine (A8G), glycine at position 22 mutated to glutamic acid (G22E), and a glycine was added to the end (G38);

[0089] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGG (SEQ ID NO: 3).

[0090] fGLP-1(7-37)-mut1 Pichia pastoris codon-optimized nucleic acid sequence:

[0091] CACGGAGAAGGTACCTTCACTTCCGACGTTTCTTCTTACCTGGAAGAACA GGCTGCTAAGGAGTTCATTGCATGGTTGGTCAAGGGACGTGGA (SEQ ID NO: 4).

[0092] The amino acid sequence of fGLP-1(7-37)-mut2 is as follows: alanine at position 8 is mutated to glycine (A8G), glycine at position 22 is mutated to glutamic acid (G22E), arginine at position 36 is mutated to glycine (R36G), and a glycine is added to the end (G38);

[0093] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGG (SEQ ID NO: 5).

[0094] fGLP-1(7-37)-mut2 Pichia pastoris codon-optimized nucleic acid sequence:

[0095] CACGGAGAAGGTACCTTCACTTCCGACGTTTCTTCTTACCTGGAAGAACA GGCTGCTAAGGAGTTCATTGCATGGTTGGTCAAGGGAGGTGGAGGT (SEQ ID NO: 6).

[0096] The amino acid sequence of fGLP-1(7-37)-mut3 is as follows: alanine at position 8 mutates to glycine (A8G), glycine at position 22 mutates to glutamic acid (G22E), arginine at position 36 mutates to glycine (R36G), and two glycines (G38, G39) are added at the end;

[0097] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGG (SEQ ID NO: 7).

[0098] fGLP-1(7-37)-mut3 Pichia pastoris codon-optimized nucleic acid sequence:

[0099] CACGGAGAAGGTACCTTCACTTCCGACGTTTCTTCTTACCTGGAAGAA CAGGCTGCTAAGGAGTTCATTGCATGGTTGGTCAAGGGAGGTGGAGGTGG A (SEQ ID NO: 8).

[0100] On the basis of the above, linker peptides (Linker1, Linker2, Linker3) were screened, and fGLP-1 and its mutants (fGLP-1(7-37)-mut1, fGLP-1(7-37)-mut2 and fGLP-1(7-37)-mut3) were fused with feline serum albumin FSA and its mutant (FSA-mut) to further improve the half-life of the protein, and the fGLP-1-Linker-FSA fusion protein (fGL P-1-L1-FSA-H, fGLP-1-mut1-L1-FSA-H, fGLP-1-mut1-L1-FSA-mut-H, fGLP-1-mut2-L2-FSA-mut-H, fGLP-1-mut3-L3-FSA-mut-H); for ease of purification, a histidine tag (6×His, HHHHHH, sequence after Pichia codon optimization: CATCATCACCACCATCAT) was added to the C-terminus.

[0101] Linker1 (abbreviated as L1) amino acid sequence: GGGSGGGSGGGS (SEQ ID NO: 9).

[0102] Linker1 Pichia pastoris codon optimized sequence:

[0103] GGAGGAGGATCCGGAGGAGGATCTGGTGGCGGCTCT (SEQ ID NO: 12).

[0104] Linker2 (abbreviated as L2) amino acid sequence: GGSEGSEGSGGGS (SEQ ID NO: 10).

[0105] Linker2 Pichia pastoris codon optimized sequence:

[0106] GGAGGATCTGAAGGTTCTGAAGGATCAGGTGGAGGTTCT (SEQ ID NO: 13).

[0107] Linker3 (abbreviated as L3) amino acid sequence: EGSGGSEGSGGGS (SEQ ID NO: 11).

[0108] Linker3 Pichia pastoris codon optimized sequence:

[0109] GAGGGCTCAGGTGGATCAGAAGGTTCCGGAGGTGGCTCA (SEQ ID NO: 14).

[0110] Feline serum albumin FSA amino acid sequence (NCBI sequence accession number NP_001009961.1) (SEQ ID NO: 15).

[0111] The sequence of feline serum albumin FSA after Pichia pastoris codon optimization:

[0112]

[0113] FSA-mut amino acid sequence: LGTFLYEYSRRHPEY. This region contains chymotrypsin cleavage sites F and YEY. The phenylalanine at position 330 mutates to alanine (F330A), the tyrosine at position 332 mutates to alanine (Y332A), the tyrosine at position 334 mutates to alanine (Y334A), and the tyrosine at position 341 mutates to alanine (Y341A).

[0114] EAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLVNEVTEFAKGCVADQSAAN

[0115] CEKSLHELLGDKLCTVASLRDKYGEMADCCEKKEPERNECFLQHKDDNPGFGQLVTPEAD

[0116] AMCTAFHENEQRFLGKYLYEIARRHPYFYAPELLYYAEEYKGVFTECCEAADKAACLTPKV

[0117] DALREKVLASSAKERLKCASLQKFGERAFKAWSVARLSQKFPKAEFAEISKLVTDLAKIHK

[0118] ECCHGDLLECADDRADLAKYICENQDSISTKLKECCGKPVLEKSHCISEVERDELPADLPPL

[0119] AVDFVEDKEVCKNYQEAKDVFLGTALAEASRRHPEASVSLLLRLAKEYEATLEKCCATDDP

[0120] PACYAHVFDEFKPLVEEPHNLVKTNCELFEKLGEYGFQNALLVRYTKKVPQVSTPTLVEVSR

[0121] SLGKVGSKCCTHPEAERLSCAEDYLSVVLNRLCVLHEKTPVSERVTKCCTESLVNRRPCFS

[0122] ALQVDETYVPKEFSAETFTFHADLCTLPEAEKQIKKQSALVELLKHKPKATEEQLKTVMGDFGSFVDKCCAAEDKEACFAEEGPKLVAAAQAALA (SEQ ID NO: 17).

[0123] FSA-mut Pichia pastoris codon optimized sequence:

[0124]

[0125] The amino acid sequences of fGLP-1-L1-FSA-H, fGLP-1-mut1-L1-FSA-H, fGLP-1-mut1-L1-FSA-mut-H, fGLP-1-mut2-L2-FSA-mut-H and fGLP-1-mut3-L3-FSA-mut-H are shown in SEQ ID NOs: 19 to 23, respectively, and the nucleic acid sequences of fGLP-1-L1-FSA-H, fGLP-1-mut1-L1-FSA-H, fGLP-1-mut1-L1-FSA-mut-H, fGLP-1-mut2-L2-FSA-mut-H and fGLP-1-mut3-L3-FSA-mut-H after Pichia pastoris codon optimization are shown in SEQ ID NOs: 24 to 28, respectively.

[0126] Based on the connecting peptides screened out from the FSA fusion protein, fGLP-1 and its mutants (fGLP-1(7-37)-mut1, fGLP-1(7-37)-mut2 and fGLP-1(7-37)-mut3) were fused with cat-derived IgG4-Fc (fFc1, fFc2) and its mutants (fFc1-mut) for expression, and Fc was truncated or replaced to varying degrees to obtain fGLP-1-Linker-Fc fusion proteins (fGLP-1-mut1-L1-fFc1-H, fGLP-1-mut2-L2-fFc1-mut-H, fGLP-1-mut3-L3-Fc2-H).

[0127] fFc1 amino acid sequence (NCBI sequence accession number ATI97615.1):

[0128] PEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQ PHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK(SEQ ID NO: 29).

[0129] fFc1 Pichia pastoris codon-optimized nucleic acid sequence:

[0130] CCAGAAATGTTAGGTGGACCATCTATTTTCATCTTCCCTCCCAAGCCTAAGGACACGTTG

[0131] TCTATATCCAGAACCCCAGAAGTTACTTGTCTAGTTGTCGACTTGGGCCCAGATGACTCT

[0132] GATGTGCAAATCACTTGGTTTGTCGATAACACTCAGGTCTACACAGCTAAGACTTCTCCA

[0133] CGAGAAGAGCAGTTTAATAGTACTTATAGAGTGGTCTCAGTTCTACCAATTCTTCACCAA

[0134] GACTGGCTTAAGGGCAAGGAGTTTAAGTGCAAAGTCAACTCTAAGAGTTTGCCCAGTCC

[0135] CATCGAGAGAACTATTAGTAAGGCCAAGGGACAACCTCACGAACCCCAAGTTTACGTTT

[0136] TACCCCTGCTCAGGAAGAATTATCCCGAAACAAGGTTCTCCGTTACTTGTTTGATAAAGT

[0137] CATTTCATCCACCTGATATTGCTGTCGAATGGGAAATAACAGGTCAACCTGAACCTGAAA

[0138] ACAACTATAGAACAACTCCCCCTCAGTTGGACAGTGATGGTACTTACTTCGTTTACTCCA

[0139] AACTTTCAGTCGATCGTTCTCATTGGCAACGAGGAAACACCTACACATGCAGGTGTGTCA

[0140] CATGAAGCCTTGCATTCTCACCATACCCAAAAGTCTAACACAGTCTCCTGGTAAA(SEQ ID NO:30)。

[0141] fFc1-mut amino acid sequence: the proline at position 37 mutated to glutamine (P37Q), the proline at position 100 mutated to serine (P99S), the serine at position 171 mutated to proline (S171P), and the phenylalanine and valine at positions 176 and 177 mutated to alanine (F176A, V177A).

[0142] PEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGQDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSSIERTISKAKGQ PHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDPDGTYAAYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK(SEQ ID NO: 31).

[0143] fFc1-mut1 Pichia pastoris codon-optimized nucleic acid sequence:

[0144] CCAGAAATGTTAGGTGGACCATCTATTTTCATCTTCCCTCCCAAGCCTAAGGACACGTTG

[0145] TCTATATCCAGAACCCCAGAAGTTACTTGTCTAGTTGTCGACTTGGGCCAGGATGACTCT

[0146] GATGTGCAAATCACTTGGTTTGTCGATAACACTCAGGTCTACACAGCTAAGACTTCTCCA

[0147] CGAGAAGAGCAGTTTAATAGTACTTATAGAGTGGTCTCAGTTCTACCAATTCTTCACCAA

[0148] GACTGGCTTAAGGGCAAGGAGTTTAAGTGCAAAGTCAACTCTAAGAGTTTGCCCAGTA

[0149] GTATCGAGAGAACTATTAGTAAGGCCAAGGGACAACCTCACGAACCCCAAGTTTACGTT

[0150] TTACCCCCTGCTCAGGAAGAATTATCCCGAAACAAGGTCTCCGTTACTTGTTTGATAAAG

[0151] TCATTTCATCCACCTGATATTGCTGTCGAATGGGAAATAACAGGTCAACCTGAACCTGAA

[0152] AACAACTATAGAACAACTCCCCCTCAGTTGGACCCTGATGGTACTTACGCTGCTTACTCC

[0153] AAACTTTCAGTCGATCGTTCTCATTGGCAACGAGGAAACACCTACACATGCAGTGTGTC

[0154] ACATGAAGCCTTGCATTCTCACCATACCCAAAAAAGTCTAACACAGTCTCCTGGTAAA(SEQ ID NO: 32).

[0155] fFc2 amino acid sequence (GenBank: ATI97438.1):

[0156] PGSLAGSPGQQVTMNCRASQSVSSYLAWYQQKPGQHPKLLIYSASTRASGVPDRFSGSGSG

[0157] TDFTLTISNLQAEDVASYYCQQYYSSPPNFGQGTKLEIKRTDAQPSVFLFQPSLDELHTGSAS

[0158] IVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFNRSECQRE(SEQ ID NO: 33).

[0159] fFc2 nucleic acid sequence after codon optimization in Pichia pastoris:

[0160] CCAGGATCACTTGCAGGTTCCCCTGGCCAACAAGTAACAATGAACTGCAGAGCCAGTCAGTCTGTCTCTTCATATTTGGCTTGGTACCAGCAAAAACCTGGTCAACATCCTAAAT TGTTGATATATTCCGCTTCAACAAGAGCCTCCGGTGTACCTGATAGATTTTCTGGTTCAGGATCTGGTACCGATTTCACTTTGACTATTAGTAACCTGCAAGCCGAAGACGTGGCTTCTTATTACTGTC AACAATACTACTCATCTCCACCAAATTTCGGCCAAGGCACTAAGCTGGAAATCAAACGTACCGATGCACAACCTTCAGTATTTCTTTTCAGCCTTCACTGGACGAGTTGCATACAGGTTCAGCAAGTAT CGGTGTGTATCTTGAACGACTTCTATCCTAAGGAGGTCAACGTTAAGTGGAAGGTAGATGGTGTCGTGCAAAATAAGGGTATTCAGGAGTCAACCACTGAGCAAAACTCAAAAGACTCTACTTACAGTCTG TCCTCAACTTTGACGATGAGTAGTACCGAGTACCAGTCACACGAGAAATTCTCCTGCGAAGTGACCCACAAATCTTTGGCTAGTACTCTAGTTAAGTCTTTTAACAGATCTGAATGCCAAAGAGAG(SEQ ID NO: 34).

[0161] The amino acid sequences of fGLP-1-mut1-L1-fFc1-H, fGLP-1-mut2-L2-fFc1-mut-H, and fGLP-1-mut3-L3-Fc2-H are shown in SEQ ID NOs: 35 to 37, respectively, and the nucleic acid sequences of fGLP-1-mut1-L1-fFc1-H, fGLP-1-mut2-L2-fFc1-mut-H, and fGLP-1-mut3-L3-Fc2-H after Pichia pastoris codon optimization are shown in SEQ ID NOs: 38 to 40, respectively.

[0162] The amino acid sequence of the above fusion protein was modified with Pichia pastoris codons and synthesized by Nanjing GenScript Biotechnology Co., Ltd. in the Pichia pastoris expression vector pPICZαA. The cloning sites were EcoRI and XbaI. The construction diagram is shown in the figure. Figure 1 , Figure 2 As shown, the recombinant plasmids pPICZαA-fGLP-1-L1-FSA-H, pPICZαA-fGLP-1-mut1-L1-FSA-H, pPICZαA-fGLP-1-mut1-L1-FSA-mut-H, and pPICZαA-fGLP-1-mut2-L2-FSA-mut-H were obtained. , pPICZαA-fGLP-1-mut3-L3-FSA-mut-H, pPICZαA-fGLP-1-mut1-L1-fFc1-H, pPICZαA-fGLP-1-mut2-L2-fFc1-mut-H and pPICZαA-fGLP-1-mut3-L3-fFc2-H.

[0163] 2. Enzyme linearization and purification of recombinant plasmid:

[0164] Referring to the enzyme digestion test manual of TAKARA, the recombinant plasmids pPICZαA-fGLP-1-L1-FSA-H, pPICZαA-fGLP-1-mut1-L1-FSA-H, pPICZαA-fGLP-1-mut1-L1-FSA-mut-H, pPICZαA-fGLP-1-mut2-L2-FSA-mut-H, pPICZαA-fGLP-1-mut3-L3-FSA-mut-H, pPICZαA-fGLP-1-mut1-L1-fFc1-H, pPICZαA-fGLP-1-mut2-L2-fFc1-mut-H and pPICZαA-fGLP-1-mut3-L3-fFc2-H were digested with Sac I and the linearization was complete by agarose gel electrophoresis. The linearized product was purified and recovered. The purification and recovery method was referred to the instruction manual of the kit.

[0165] 3. Preparation of Pichia pastoris X33 competent cells:

[0166] 1) Inoculate a single colony of yeast recipient bacteria on a YPD plate and culture at 30°C for 2 days;

[0167] 2) Pick a single colony from the plate and inoculate it into 10 mL YPD liquid culture medium and shake it overnight at 30°C;

[0168] 3) After overnight culture, inoculate about 1% of the inoculum into 100 mL YPD medium and shake culture until the OD value is 1.2-1.5;

[0169] 4) Centrifuge at 5000 rpm for 5 min at 4°C to collect the precipitated cells, and resuspend them in 100 mL of pre-cooled sterile water;

[0170] 5) Centrifuge at 4°C, 5000 rpm for 10 min to collect the precipitated cells, and resuspend the cells in 100 mL of pre-cooled sterile water;

[0171] 6) Centrifuge again at 4°C, 5000 rpm for 10 min to collect the precipitated cells, and resuspend the cells in 100 mL of pre-cooled sterile water;

[0172] 7) 20 ml, 1 mol / L sorbitol, wash once;

[0173] 8) Dissolve the cells in 1 mL of 1 M pre-cooled sorbitol without glycerol and place at -80°C for several hours until transformation;

[0174] 4. Linearized expression plasmid electroporated into Pichia pastoris X33 competent cells:

[0175] 1) Prepare 80 μl of competent yeast and mix with 2 μg of linearized recombinant plasmid (precooled on ice for 15 min), quickly put into a 0.2 cm electroporation cup (precooled and sterilized on ice), and electroporate; the electroporation parameters are Voltage: 1500 V; Capacitance: 25 μF; Resistance: 200 Ω; Cuvette (mm): 2 mm.

[0176] 2) After the electroporation, add 1 mL of sorbitol (1 M) quickly, place on ice for 15 min, and then place in a 30°C incubator for 1 h. Add 1 mL of YPD liquid culture medium, shake and culture at 30°C, 200 r / min for 1 hour, centrifuge at 4 000 r / min at room temperature to collect the cells, apply them to a YPDS plate containing 100 μg / μL, and place at 30°C for 3 days. The following recombinant yeasts were obtained:

[0177] PICZαA-fGLP-1-L1-FSA-H, pPICZαA-fGLP-1-mut1-L1-FSA-H-X33, pPICZαA-fGLP-1-mut1-L1-FSA-mut-H-X33,

[0178] pPICZαA-fGLP-1-mut2-L2-FSA-mut-H-X33,

[0179] pPICZαA-fGLP-1-mut3-L3-FSA-mut-H-X33,

[0180] pPICZαA-fGLP-1-mut1-L1-fFc1-H-X33,

[0181] pPICZαA-fGLP-1-mut2-L2-fFc1-mut-H-X33 and

[0182] pPICZαA-fGLP-1-mut3-L3-fFc2-H-X33.

[0183] 5. Identification of recombinant yeast and screening of high copies:

[0184] Use a sterilized pipette tip to carefully pick up a single colony with Zeocin resistance growing on a YPDS plate, inoculate it into 2 mL of YPD liquid culture medium (containing 150 μg / mL Zeocin), and culture overnight at 30°C and 200 r / min with shaking. Use bacterial liquid PCR to analyze P. pastoris transformants. The PCR identification procedure is shown in Table 1. The PCR product is subjected to 1% agarose gel electrophoresis to identify the clones that can amplify the target band with the primers and define them as positive transformants. The screening of high copies requires the combination of the band brightness in the PCR identification and the test results of the high-resistance YPD plate (200 μg / mL Zeocin). The bacterial liquid PCR identification of recombinant yeast is as follows: Figure 3 shown.

[0185] The results of PCR identification of recombinant yeast showed that all identified strains were positive recombinant yeast strains, indicating that electrotransformation of X33 was successful. The corresponding strains were selected for streaking on YPD plates (containing 100 μg / mL Zeocin) for subsequent induced expression.

[0186] Table 1 PCR identification procedures for recombinant yeast

[0187]

[0188] 6. Induced expression of high-copy recombinant yeast:

[0189] (1) Use a sterilized pipette tip to carefully pick up a single colony with Zeocin resistance growing on the YPD plate and place it in 20 mL of BMGY liquid medium for activation culture. Shake at 30°C, 200 rpm overnight until the OD 600 =2~6, at this time the cells are in the logarithmic growth phase.

[0190] (2) Centrifuge at 3000 rpm for 5 min at room temperature to collect the precipitate, resuspend it in 1 mL of BMMY, wrap it with four layers of clean gauze and two layers of newspaper, and culture it in a 250 mL conical flask with shaking.

[0191] (3) Add 100% methanol every 24 hours to a final concentration of 1-1.5% for induction culture.

[0192] (4) After 72 hours of induction culture, collect the samples, centrifuge them, and take the supernatant for SDS-PAGE immediately or store them at -80°C.

[0193] 7. SDS-PAGE of the supernatant of recombinant yeast induced expression:

[0194] The supernatant of the recombinant yeast induced expression for 3 days was subjected to SDS-PAGE protein electrophoresis, and a corresponding empty plasmid pPICZαA-X33 control group was set up. Figure 4 As shown. The SDS-PAGE protein electrophoresis operation process is as follows: 1. Sample treatment: Add 5× loading buffer to the sample and boil at 100℃ for 10min. 2. Electrophoresis: Load 10μL of sample into the SDS-PAGE gel, add diluted electrophoresis solution inside and outside, cover the electrode and connect the electrophoresis instrument to prepare for running the equipment. After running at 80V for 20min, the sample is compressed into a line. After concentrated gel electrophoresis, increase the voltage to 130V and run for 50-70min. End the electrophoresis after the Marker bands are clearly separated. 3. Staining: After the electrophoresis is completed, remove the gel plate after electrophoresis, disassemble the gel plate with a spatula, remove the concentrated gel, rinse the separation gel with tap water and then transfer it to Coomassie Brilliant Blue dye solution for staining, and accelerate the staining on a decolorizing shaker for 1-2h.

[0195] The results are as follows Figure 4 As shown, fGLP-1-L1-FSA-H, fGLP-1-mut1-L1-FSA-H, and fGLP-1-mut1-L1-fFc1-H were broken during expression, while the fusion proteins fGLP-1-mut1-L1-FSA-mut-H, fGLP-1-mut2-L2-FSA-mut-H, fGLP-1-mut3-L3-FSA-mut-H, fGLP-1-mut1-L1-fFc1-H, fGLP-1-mut2-L2-fFc1-mut-H, and fGLP-1-mut3-L3-fFc2-H were all efficiently expressed in the yeast expression system without breaking.

[0196] 8. Purification and recovery of expression products:

[0197] The supernatant of the GLP-1 analog fusion protein induced by recombinant yeast was purified. The principle of specific binding of His Tag to nickel column was used to select nickel column affinity chromatography method for specific binding, washing and elution of protein to obtain high-purity target protein. The protein collected by chromatography was concentrated and dialyzed into 5-100mM PB, 0-300mM NaCl, pH6.0-9.5 buffer using 3-10kD ultrafiltration membrane package, and finally concentrated to a protein solution with a concentration of 0.5-20mg / ml.

[0198] The results are as follows Figure 5 As shown, the GLP-1 analog fusion protein can be purified by the above purification process to obtain a high-purity protein.

[0199] The protein contents of fGLP-1-mut1-L1-FSA-mut-H, fGLP-1-mut2-L2-FSA-mut-H, fGLP-1-mut3-L3-FSA-mut-H, fGLP-1-mut1-L1-fFc1-H, fGLP-1-mut2-L2-fFc1-mut-H, and fGLP-1-mut3-L3-fFc2-H were much higher than those of the fGLP-1-L1-FSA-H, fGLP-1-mut1-L1-FSA-H, and fGLP-1-mut1-L1-fFc1-H groups.

[0200] Example 2: Determination of the half-life of GLP-1 analog fusion protein

[0201] The half-life of the purified sample of the GLP-1 analog fusion protein obtained in Example 1 was determined by measuring the blood drug concentration of the GLP-1 analog fusion protein in the serum.

[0202] Ob / ob mice, 8 weeks old, were randomly divided into 10 groups according to body weight, 5 mice in each group. The test group was subcutaneously injected with GLP-1 analog fusion protein at a dose of 1.0 mg / kg, and the model group and blank control group were injected with an equal volume of normal saline. The concentration of GLP-1 analog fusion protein in the serum of each group of animals was detected by ELISA method before administration (0h), 1h, 2h, 4h, 6h, 12h, 24h, 48h, and 72h after administration. DAS pharmacokinetic software was used for curve fitting and calculation of relevant parameters. The results are shown in Table 2.

[0203] Table 2 Determination of half-life of purified samples

[0204] Group half life fGLP-1-L1-FSA-H 4h fGLP-1-mut1-L1-FSA-H 12h fGLP-1-mut1-L1-FSA-mut-H 22h fGLP-1-mut2-L2-FSA-mut-H 28h fGLP-1-mut3-L3-FSA-mut-H 24h fGLP-1-mut1-L1-fFc1-H 18h fGLP-1-mut2-L2-fFc1-mut-H 24h fGLP-1-mut3-L3-fFc2-H 28h

[0205] Example 3: Determination of the hypoglycemic effect of GLP-1 analog fusion protein

[0206] Streptozotocin (STZ) is a pancreatic β-cell toxic agent that can specifically destroy β-cells by inducing the breakage of pancreatic β-cell deoxyribonucleic acid. The degree of β-cell damage is dose-dependent. STZ is widely used in the establishment of type 1 diabetes models.

[0207] Fifty-five 12-month-old Chinese rural cats were selected and divided into 11 groups, with 5 cats in each group, namely fGLP-1-L1-FSA-H group, fGLP-1-mut1-L1-FSA-H group, fGLP-1-mut1-L1-FSA-mut-H group, fGLP-1-mut2-L2-FSA-mut-H group, fGLP-1-mut3-L3-FSA-mut-H group, fGLP-1-mut1-L1-fFc1-H group, fGLP-1-mut2-L2-fFc1-mut-H group, fGLP-1-mut3-L3-Fc2-H group, human insulin control group, model control group, and blank control group. Except for the blank control group, the rest of the groups need to establish a diabetic model. Fasting for 12 hours before the start of the experiment, STZ solution (30 mg / kg) was intravenously injected according to the weight of the Chinese rural cats, and the blank control group was injected with an equal amount of sodium chloride injection. After 5 days, the cats' fasting blood glucose and random blood glucose changes were tested. Cats with blood glucose levels above 200 mg / dL were considered to have successfully established models and could be used for subsequent experiments. After the model was successfully established, the drug group was subcutaneously injected with purified, filtered, sterile fGLP-1 and its mutant fusion protein at 40 μg / kg under fasting conditions, and the model control group and blank control group were injected with an equal volume of sodium chloride injection. Blood glucose levels before and after drug injection were measured to evaluate the hypoglycemic effect of each group of samples.

[0208] The results are as follows Figure 6 As shown, the results showed that the hypoglycemic activity of the fGLP-1-mut1-L1-FSA-H group, fGLP-1-mut1-L1-FSA-mut-H group, fGLP-1-mut2-L2-FSA-mut-H group, fGLP-1-mut3-L3-FSA-mut-H group, fGLP-1-mut1-L1-fFc1-H group, fGLP-1-mut2-L2-fFc1-mut-H group, and fGLP-1-mut3-L3-Fc2-H group was better than that of the fGLP-1-L1-FSA-H group and the human insulin group. The modification of the fGLP-1 mutant site and the fusion with FSA and Fc proteins are conducive to improving the hypoglycemic biological activity of the GLP-1 analog fusion protein, and can effectively maintain the blood sugar of hyperglycemic dogs at a normal level for a long time.

[0209] Example 4: Evaluation of the weight loss effect of GLP-1 analog fusion protein

[0210] Establishing an obesity model with a high-fat diet is an effective method for evaluating weight loss drugs. 40 healthy adult Chinese rural cats were selected as experimental animals and divided into a drug group, a model control group, and a blank control group. The obesity model was established by feeding the cats with a 40% fat content feed for 8 consecutive weeks. After the model was successfully established, normal feeding was carried out, and the drug group was subcutaneously injected at 40 μg / kg once a day for 12 consecutive weeks. The performance of GLP-1 analog fusion protein in weight loss was evaluated by measuring the changes in body weight.

[0211] The results are as follows Figure 7 As shown, at the end of the experiment, the body weight of the model control group continued to increase slowly, while the body weight of the blank control group did not change significantly. The GLP-1 analog fusion protein had a good weight loss effect, with a weight loss effect of 8.8-11.3%.

[0212] In the present application, fGLP-1 and its mutants are fused with FSA and fFc proteins, and the characteristics of Pichia pastoris methanol-induced secretion expression system, such as moderate glycosylation modification, disulfide bond formation, and efficient induced expression, are utilized to achieve efficient expression of fGLP-1 mutant fusion protein in Pichia pastoris, so that the fGLP-1 mutant fusion protein has the characteristics of high biological activity, high stability, and moderate glycosylation modification. The fGLP-1-L1-FSA-H, fGLP-1-mut1-L1-FSA-H, fGLP-1-mut1-L1-FSA-mut-H, fGLP-1-mut2-L2-FSA-mut-H, fGLP-1-mut3-L3-FSA-mut-H, fGLP-1-mut1-L1-fFc1-H, fGLP-1-mut2-L2-fFc1-mut-H, and fGLP-1-mut3-L3-Fc2-H in the present application have higher blood sugar and weight loss effects than natural fGLP-1. At the same time, the destruction of the recognition site of DPP-IV and the increase in molecular weight can significantly improve the half-life and stability of the GLP-1 analog fusion protein. The modified GLP-1 analog fusion protein can also reduce potential immunogenicity problems.

[0213] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A GLP-1 analog fusion protein, characterized in that: The GLP-1 analog fusion protein is any one of the following fusion proteins; 1) a sequence formed by mutating the amino acid sequence as shown in SEQ ID NO: 1 or by mutating at least one glycine in the amino acid sequence as shown in SEQ ID NO: 1, or / and by mutating at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 1 to glycine, or / and by adding at least one glycine to the amino acid sequence as shown in SEQ ID NO: 1, By connecting peptides, fusing with the amino acid sequence as shown in SEQ ID NO: 15 or a sequence in which at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 15 is mutated to at least one alanine to obtain a GLP-1 analog fusion protein; 2) a sequence formed by mutating the amino acid sequence as shown in SEQ ID NO: 1, or by mutating at least one glycine in the amino acid sequence as shown in SEQ ID NO: 1, or / and by mutating at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 1 to glycine, or / and by adding at least one glycine to the amino acid sequence as shown in SEQ ID NO: 1, By connecting peptides, The GLP-1 analog fusion protein is obtained by fusing it with the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 33 or a sequence formed by mutation or truncation of the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO:

33.

2. The GLP-1 analog fusion protein according to claim 1, characterized in that: In 1) or 2), any mutation is performed on the glycine at position 22 of the amino acid sequence shown in SEQ ID NO: 1; Preferably, the glycine at position 22 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to glutamic acid.

3. The GLP-1 analog fusion protein according to claim 1, characterized in that: In the above 1) or 2), at least one amino acid mutation site in the amino acid sequence shown in SEQ ID NO: 1 includes mutating the 8th alanine and / or the 36th arginine to glycine.

4. The GLP-1 analog fusion protein according to claim 1, characterized in that: In the above 1) or 2), at least one glycine is added to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO:

1.

5. The GLP-1 analog fusion protein according to claim 1, characterized in that: In the above 1) or 2), the number of glycine added is ≥1, preferably 1 to 3.

6. The GLP-1 analog fusion protein according to claim 1, characterized in that: In the above 1), the mutation site of at least one amino acid in the amino acid sequence shown in SEQ ID NO: 15 includes at least one of phenylalanine at position 330, tyrosine at position 332, tyrosine at position 334, and tyrosine at position 341.

7. The GLP-1 analog fusion protein according to claim 1, characterized in that: In the above 2), any mutation is performed at any of the positions 37, 100, 171, 176 and 177 of the amino acid sequence shown in SEQ ID NO:

29.

8. The GLP-1 analog fusion protein according to claim 7, characterized in that: In the above 2), in the amino acid sequence shown in SEQ ID NO: 29, the proline at position 37 is mutated to glutamine, the proline at position 100 is mutated to serine, the serine at position 171 is mutated to proline, the phenylalanine at position 176 is mutated to alanine, and the valine at position 177 is mutated to alanine.

9. The GLP-1 analog fusion protein according to claim 1, characterized in that: The sequences of the connecting peptides are shown in SEQ ID NOs: 9-11.

10. The GLP-1 analog fusion protein according to claim 1, characterized in that: The GLP-1 analog fusion protein comprises an amino acid sequence having at least 90% homology to at least one of the sequences SEQ ID NOs: 19 to 23 and SEQ ID NOs: 35 to 37; Preferably, the amino acid sequence of the GLP-1 analog fusion protein is shown in SEQ ID NOs: 19-23 and SEQ ID NOs: 35-37, and the nucleotide sequence of the GLP-1 analog fusion protein is shown in SEQ ID NOs: 24-28 and SEQ ID NOs: 38-40.

11. A polynucleotide, characterized in that Encodes the GLP-1 analog fusion protein according to any one of claims 1 to 10.

12. An expression vector, characterized in that: The vector comprises the polynucleotide according to claim 11; The expression vector includes a prokaryotic expression vector, a mammalian cell expression vector, a baculovirus-insect cell expression vector, and a yeast expression vector. The yeast expression vector includes one of pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pPIC9K, pPIC9, pHIL-S1, pJL-SX, pBLHIS-SX, pYAM75P, pAOX1, pAOX2, pPICZA, pPICZB, pPICZC, pGAPZA, pPIC3K, pPIC3.5K, pHIL-D2, pJL-IX, and pBLHIS-IX, preferably pPICZαA, pPICZαB, and pPICZαC.

13. An expression host comprises a host bacterium or a host cell, characterized in that: The host bacteria or host cell comprises the polynucleotide according to claim 11 or the expression vector according to claim 12; The host bacteria include Pichia pastoris expression host bacteria, Escherichia coli expression host bacteria, and Bacillus expression host bacteria, preferably Pichia pastoris expression host bacteria. The Pichia pastoris expression host bacteria include one of X33, GS115, KM71, SMD1168, SMD1168 H, SMD1165, SMD1163, MP-36, MC100-3, MS105, and JC100. Preferably, the Pichia pastoris expression host bacteria is X33.

14. A method for producing the GLP-1 analog fusion protein according to any one of claims 1 to 10, characterized in that: The following steps are involved: Cultivate the host bacteria or host cells as claimed in claim 13 to express the GLP-1 analog fusion protein as claimed in any one of claims 1 to 10.

15. A pharmaceutical composition, characterized in that It comprises the GLP-1 analog fusion protein according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

16. Use of the GLP-1 analog fusion protein according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 15 in the preparation of hypoglycemic drugs and weight loss drugs.

17. Use of cat serum albumin and its mutants, or IgG-Fc fragment and its mutants in the preparation of long-acting drugs, characterized in that: The sequence of the feline serum albumin is shown in the amino acid sequence SEQ ID NO: 15; The mutant of feline serum albumin is a sequence in which at least one amino acid in the amino acid sequence shown in SEQ ID NO: 15 is mutated to at least one alanine; The sequence of the IgG-Fc fragment is shown in the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 33; The mutant of the IgG-Fc fragment is: a sequence formed by mutation or truncation of the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 33; Preferably, in the mutant of feline serum albumin, the mutation site of at least one amino acid in the amino acid sequence shown in SEQ ID NO: 15 includes at least one of phenylalanine at position 330, tyrosine at position 332, tyrosine at position 334, and tyrosine at position 341; In the mutant of the IgG-Fc fragment, any mutation is performed at any one of positions 37, 100, 171, 176 and 177 of the amino acid sequence shown in SEQ ID NO: 29.

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