Method for producing intestinal insulinotropic receptor activating peptide by using animal mammary gland

Abstract

The invention discloses a method for producing an insulinotropic receptor activating peptide by using animal mammary glands. The invention provides a method for constructing an animal model for producing an insulinotropic receptor activating peptide by using mammary gland, which comprises the following steps: specifically expressing a fusion protein in the mammary gland of an animal, the fusion protein comprising a fusion fragment of the insulinotropic receptor activating peptide and an antibody Fc fragment. The fusion protein can be efficiently expressed in the mammary gland of a transgenic mouse; moreover, the fusion protein secreted into the milk contains complete GIP / GLP-1 functional oligopeptides and Fc fragments, the fusion protein in the milk can lower the weight and the blood sugar level of lactating cubs, and the result proves that the fusion protein has the biological activity of the intestinal insulinotropic receptor activating peptide; that is to say, synthesis of the intestinal insulinotropic receptor activating peptide by using the animal mammary gland is feasible and efficient.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a method for producing an intestinal glucagon-like peptide-1 receptor agonist using the mammary gland of an animal. Background Art

[0002] After designing a vector for specific expression in mammary gland epithelial cells, the exogenous gene is integrated into the animal genome through techniques such as transgenic technology / gene editing to achieve specific expression of the exogenous protein in the mammary gland tissue, which is a method for producing pharmaceutical proteins. This technical system is also known as the "mammary gland bioreactor". As of 2018, there are two drugs produced by the mammary gland bioreactor that have been launched on the market, namely human antihemophilic factor III and human esterase C1 inhibitor; there are more than 20 drugs produced by the mammary gland bioreactor under research, including blood factor VIIa and IX, lactoferrin, fibrinogen, growth hormone, collagen, CD20 monoclonal antibody, and CD137 monoclonal antibody.

[0003] Incretin hormones are a class of gut-derived hormones released by the digestive system, including glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), etc. For GLP-1, under the condition of food stimulation, it is secreted by intestinal L cells and then absorbed into the blood to perform its physiological functions. Its receptors are mainly distributed in the cells of two types of tissues: (1) One is pancreatic β cells. After these receptors are activated by GLP-1 or its receptor activation peptide, they not only stimulate insulin secretion but also inhibit pancreatic α cells from secreting glucagon, thereby regulating postprandial blood glucose and can be used to develop therapeutic drugs for diabetes. Moreover, the secretion of GLP-1 is glucose-dependent and can also avoid the risk of severe hypoglycemia; (2) The other is nerve cells in the brain's appetite control area. After these receptors are activated, they reduce appetite and slow down the gastric emptying process, so they can reduce body weight, can be used to develop weight loss drugs, and have cardio-renal protective effects. The GLP-1 protein is very conservative. In its amino acid sequence, the functional region that binds to and activates the receptor is the 7-37 polypeptide (His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg). Therefore, the research and development of GLP-1 receptor activation peptides targeting 7-37 polypeptide analogs have become a new blue ocean for diabetes treatment and weight loss drug development. Currently, the main production methods of GLP-1 receptor activation peptides sold or under research in the market are total chemical synthesis, semi-biosynthesis, or eukaryotic cell synthesis. In the above methods, the amino acid sequence is often optimized to extend the drug half-life. For example, the Ala at the 8th position in the above amino acid sequence can be modified to Gly (such as Tanzeum developed by GlaxoSmithKline) or modified to α-amino butyric acid (a-Aib, such as Semaglutide developed by Novo Nordisk) to prevent degradation by dipeptidyl peptidase-4 in the body; a long-chain fatty acid chain with 18-20 carbons can also be coupled to the Lys at the 26th position (such as Semaglutide) to increase the affinity of the functional peptide segment for plasma albumin and extend the drug half-life. Currently, the market prospect of GLP-1 receptor activation peptides is broad and the development potential is huge. The financial report of Novo Nordisk shows that the total sales of the Semaglutide series of products in 2024 are close to $30 billion.

[0004] Glucose-dependent insulinotropic polypeptide (GIP) is the first discovered and purified incretin. GIP has dual functions: it has glucagon inhibitory properties in hyperglycemic states and glucagon-like effects in euglycemic and hypoglycemic states. Therefore, the potential additional benefit of GIP is to prevent hypoglycemia. With the research on intestinal polypeptide hormones such as glucagon-like peptide-1 (GLP-1) and GIP, researchers found that GIP and GLP-1 can synergistically promote insulin secretion from pancreatic β-cells, improve insulin sensitivity, and exert glucose concentration-dependent hypoglycemic effects. Therefore, researchers recombined two amino acid sequences to develop GLP-1 / GIP dual receptor agonists. In May 2022, a product called Tirzepatide was successfully developed by Eli Lilly and Company in the United States and obtained approval from the US FDA for the treatment of type II diabetes; in November 2023, it was also approved for chronic weight management. Tirzepatide is modified based on the natural GIP sequence and GLP-1 sequence and consists of a peptide segment composed of 39 amino acids: Tyr-(α-Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(α-Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser. The 2nd and 13th positions are non-natural α-aminoisobutyric acid (α-Aib), which can prevent degradation by endogenous enzymes; a 20-carbon fatty acid branch chain is linked to the lysine at the 20th position, which can assist the functional peptide to better bind to albumin. After these two improvements, the plasma half-life of Tirzepatide exceeds 5 days. Clinical trials have shown that Tirzepatide can effectively control blood glucose and body weight and reduce the risk of cardiovascular diseases, such as improving blood pressure, reducing triglycerides and low-density lipoproteins, etc. It has become one of the star drugs for weight loss and diabetes treatment, and it is estimated that the total sales in 2025 will reach more than $30 billion.

[0005] Fc fusion proteins are fusion proteins formed by recombinant of the Fc fragment of immunoglobulins (such as IgG, IgA, etc.) and the target protein sequence. The Fc segment of this fusion protein can not only extend the half-life and stability of the target protein in plasma, but also facilitate the purification process of the fusion protein, which is of great significance for the development of biological products. For example, the half-life of the Fc-fused interleukin-2 molecule in vivo can be extended from 6.9 minutes to more than 3 days; the half-life of the Fc-fused GLP-1 receptor (Dulaglutide) in vivo is also extended from several minutes to more than 5 days. As of August 2023, at least 13 Fc fusion protein drugs have been approved for marketing globally, such as Etanercept, which is a TNF-alpha receptor of Fc-fused tumor necrosis factor for the treatment of rheumatoid arthritis and was approved in 1998; Dulaglutide, which was approved in 2015; Luspatercept, which is a ligand of the Fc-fused transforming growth factor beta superfamily for the treatment of thalassemia and was approved in 2019, etc. In addition, more than 40 Fc fusion protein drugs are in the clinical research and development stage.

[0006] It can be seen that the key objective of the research and development of glucagon-like peptide-1 receptor agonists is to extend the half-life in vivo, and the specific technical means include the following: (1) modifying the key amino acids at the enzyme degradation site to α-aminobutyric acid by biosemi-synthesis, (2) adding fatty acid branches (such as semaglutide and tirzepatide), (3) fusing immunoglobulin Fc fragments or albumin fragments and expressing the fusion protein in cells (such as dulaglutide), etc. At present, the technical goal of extending the half-life has been basically achieved. Currently, the key restricting the development of glucagon-like peptide-1 receptor agonists is the problem of insufficient production. In the United States, tirzepatide was removed from the FDA's shortage list until December 19, 2024; semaglutide was even later, removed from the shortage list on February 21, 2025. In China, because they are all imported from abroad, there are greater shortages and price problems. Currently, the most commonly used CHO cells can only produce milligrams to grams of recombinant proteins per liter under large-scale culture conditions, and also face high production costs. Summary of the Invention

[0007] The technical problems to be solved by the present invention are how to efficiently produce glucagon-like peptide-1 receptor agonists using animal mammary glands, and / or how to construct an animal model for producing glucagon-like peptide-1 receptor agonists using mammary glands, and / or provide related biological materials for constructing an animal model for producing glucagon-like peptide-1 receptor agonists using mammary glands.

[0008] In the first aspect, the present invention claims to protect a method for constructing an animal model for producing glucagon-like peptide-1 receptor agonists using mammary glands.

[0009] The method claimed in the present invention may include the following steps: specifically expressing a fusion protein in the mammary gland of an animal, wherein the fusion protein comprises a fusion fragment of an incretin receptor agonist and an antibody Fc segment.

[0010] Among them, the incretin hormones are polypeptides secreted by the gastrointestinal tract under food stimulation, which can stimulate insulin secretion, and may include but are not limited to intestinal cell secretion factors such as "glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP)", etc. The incretin receptor agonists (IRAs) may include but are not limited to one, two, and / or multiple incretin receptor agonist components. The same meaning applies hereinafter.

[0011] In one embodiment of the present invention, the incretin receptor agonist is a GIP / GLP-1 dual receptor agonist.

[0012] Furthermore, the amino acid sequence of the GIP / GLP-1 dual receptor agonist is as shown in positions 16-54 of SEQ ID No.1 or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% with the sequence shown in positions 16-54 of SEQ ID No.1.

[0013] In one embodiment of the present invention, the antibody Fc segment is the Fc segment of an IgG4 antibody.

[0014] Furthermore, the amino acid sequence of the Fc segment of the IgG4 antibody is as shown in positions 71-299 of SEQ ID No.1.

[0015] According to needs, the fusion protein may further contain a secretory signal peptide.

[0016] In one embodiment of the present invention, the amino acid sequence of the secretory signal peptide is as shown in positions 1-15 of SEQ ID No.1.

[0017] In the fusion protein, the incretin receptor agonist and the antibody Fc segment may be connected by a linker peptide.

[0018] In one embodiment of the present invention, the amino acid sequence of the linker peptide is as shown in positions 56-70 of SEQ ID No.1.

[0019] Specifically, in one embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID No.1 or has an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with SEQ ID No.1.

[0020] The identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using identity search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0021] The identity of more than 80% can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The identity of more than 85% can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The identity of more than 90% can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The identity of more than 95% can be at least 95%, 96%, 97%, 98% or 99% identity.

[0022] Exemplarily, the method may specifically include the following steps: introducing the coding gene of the fusion protein into the fertilized egg or somatic cell of the animal, and then transplanting the fertilized egg or the embryo cloned from the somatic cell into the fallopian tube or uterus of a pseudopregnant animal, and selecting the F0 generation transgenic animal from the offspring born from pregnancy; establishing a family using the F0 generation transgenic animal, and the homozygous or heterozygous transgenic offspring in the family are the animal models capable of producing the intestinal glucagon-like peptide receptor activating peptide using the mammary gland.

[0023] Among them, when the animal is a mouse, the fertilized egg injection method is mainly used; when the animal is cattle, sheep, pigs, etc., the method of first transferring the expression construct into somatic cells and then cloning the positive cells into animals can be used.

[0024] In one embodiment of the present invention, the coding gene of the fusion protein is injected into the fertilized egg of the animal in the form of an expression cassette.

[0025] The expression cassette contains a mammary gland-specific expression promoter and the coding gene of the fusion protein expressed under the initiation of the mammary gland-specific expression promoter.

[0026] Furthermore, the 5' end of the expression cassette consists of 2X chicken β-globin insulator, the promoter of goat β-casein, the first exon of goat β-casein, the first intron of goat β-casein, and a partial second exon of goat β-casein, and the 3' end consists of the seventh exon of goat β-casein and the subsequent partial genome, and the coding gene of the fusion protein is inserted in the middle of the 5' end and the 3' end.

[0027] Exemplarily, the mammary gland-specific expression promoter can be a β-casein promoter.

[0028] Furthermore, the nucleotide sequence of the β-casein promoter can be as shown in SEQ ID No.3.

[0029] Furthermore, the nucleotide sequence of the coding gene of the fusion protein can be as shown in SEQ ID No.2. SEQ ID No.2 is a sequence optimized for codons.

[0030] In one embodiment of the present invention, the expression cassette is prepared by the following method: inserting the DNA fragment shown in SEQ ID No.2 into the Xho I digestion site of the pBC1 plasmid to obtain a recombinant plasmid; then double-digesting the recombinant plasmid with Sal I and Not I, and recovering the 16.6 kb fragment, which is the expression cassette.

[0031] Exemplarily, the animal can be a mammal, such as a mouse, a cow, a sheep, a pig, or a rabbit.

[0032] In one embodiment of the present invention, the animal is a Kunming mouse.

[0033] In the second aspect, the present invention claims the application of the animal model constructed by the method described in the first aspect above in the production of an incretin receptor activating peptide.

[0034] In one embodiment of the present invention, the incretin receptor activating peptide is a GIP / GLP-1 dual receptor activating peptide.

[0035] Furthermore, the amino acid sequence of the GIP / GLP-1 dual receptor activating peptide is as shown in positions 16-54 of SEQ ID No.1.

[0036] In a third aspect, the present invention claims protection for a method of producing an intestinal glucagon-like peptide-1 (GLP-1) receptor activating peptide using an animal mammary gland.

[0037] The method of producing an intestinal glucagon-like peptide-1 (GLP-1) receptor activating peptide using an animal mammary gland, for which the present invention claims protection, may include the following steps: causing the animal model constructed by the method described in the first aspect above to secrete milk, and obtaining the intestinal glucagon-like peptide-1 (GLP-1) receptor activating peptide from the milk.

[0038] In an embodiment of the present invention, the intestinal glucagon-like peptide-1 (GLP-1) receptor activating peptide is a GIP / GLP-1 dual receptor activating peptide.

[0039] Furthermore, the amino acid sequence of the GIP / GLP-1 dual receptor activating peptide is as shown in positions 16-54 of SEQ ID No.1.

[0040] In a fourth aspect, the present invention claims protection for any of the following biological materials:

[0041] (A1) The fusion protein described in the first aspect above;

[0042] (A2) A nucleic acid molecule encoding the fusion protein;

[0043] (A3) An expression cassette or recombinant plasmid or recombinant bacterium or transfected cell containing the nucleic acid molecule.

[0044] Among them, the nucleic acid molecule described in (A2) may be the encoding gene of the fusion protein described in the first aspect above. The expression cassette described in (A3) may be the expression cassette described in the first aspect above; the recombinant plasmid may be the recombinant plasmid obtained by inserting the DNA molecule shown in SEQ ID No.2 into the restriction enzyme cleavage site XhoI of the pBC1 plasmid.

[0045] In a fifth aspect, the present invention claims protection for the use of the biological materials described in the fourth aspect above in constructing an animal model for producing an intestinal glucagon-like peptide-1 (GLP-1) receptor activating peptide using a mammary gland.

[0046] Exemplarily, the animal may be a mammal, such as a mouse or a cow or a sheep or a pig or a rabbit.

[0047] In an embodiment of the present invention, the animal is a Kunming mouse.

[0048] In a sixth aspect, the present invention claims protection for the milk secreted by the animal model constructed by the method described in the first aspect above or the intestinal glucagon-like peptide-1 (GLP-1) receptor activating peptide obtained from the milk.

[0049] In a seventh aspect, the present invention claims the use of the milk or the incretin receptor activating peptide described in the sixth aspect above in the preparation of a product; the product has any one of the following functions: down-regulating blood glucose and / or body weight, improving cardiovascular or chronic kidney disease symptoms, and assisting in the treatment of diabetic complications.

[0050] Based on the amino acid sequence of tirzepatide, the present invention optimizes the codons of the GIP / GLP-1 short peptide, replaces the unnatural α-amino butyric acid (a-Aib) at positions 2 and 13 with codons of alanine that can be translated by cells, and then couples its codons with the Fc fragment codons of IgG4. This new coding sequence can efficiently express a fusion protein in the mammary glands of transgenic mice under the drive of the β-casein promoter; moreover, the fusion protein secreted into the milk contains the complete GIP / GLP-1 functional short peptide and the Fc fragment. These fusion proteins in the milk can down-regulate the body weight and blood glucose levels of suckling pups, and the results prove that the fusion protein has the biological activity of an incretin receptor activating peptide; that is to say, it is feasible and efficient to synthesize an incretin receptor activating peptide using the animal mammary gland. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is a schematic diagram of the structure of the β-casein promoter-GIP / GLP-1 fragment (expression cassette) in Example 1 and the PCR and Southern blot identification diagrams of transgenic mice. Among them, (A) is a schematic diagram of the structure of the β-casein promoter-GIP / GLP-1 expression cassette; (B) is the PCR identification result of transgenic mice; (C) is the Southern blot identification result of transgenic mice. In (A), after the GIP / GLP-1 synthetic sequence (orange) fused with the Fc fragment is inserted into the Xho I digestion site of the pBC1 plasmid, a mammary gland cell-specific expression structure driven by the β-casein promoter is formed, and the corresponding positions of 5 pairs of PCR primers are listed in the figure. In (B), the amplification results of 5 pairs of PCR primers are shown, and the target fragments can be amplified in both female mice numbered tg-1 and tg-5. In (C), it includes the Southern blot results using probe-311 and probe-746 respectively, showing that a 3954bp positive DNA fragment containing the target fragment can be detected in the genomes of tg-1 and tg-5 mice, indicating that the genomes of these 2 mice have integrated the exogenous expression cassette sequence. The positive sample is the recombinant plasmid containing the β-casein promoter-GIP / GLP-1 fragment + the genomic DNA of wild-type mice; the negative samples are from the genomic DNAs of multiple wild-type mice in the same litter as tg-1 and tg-5.

[0052] Figure 2Immunoblot analysis of the fusion protein in the tissues of transgenic mice in Example 1. Among them, (A) shows the results using the anti-tirzepatide antibody. (B) shows the results using the anti-Fc fragment antibody, with human IgG as the positive control for the Fc fragment. Both (A) and (B) show that in the mouse milk collected on the 8th day of lactation (loading volume 5 μl), only in the milk of tg-1 and tg-5 mice, there is a GIP / GLP-1 receptor activating peptide with a molecular weight of approximately 37 kDa. (C) The results using the anti-tirzepatide antibody show that the fusion protein is only present in the milk of transgenic mice (from tg-1), and the fusion protein is not detected in the brown adipose tissue, heart, liver, spleen, lung, and kidney tissues of the positive offspring of tg-1 mice and negative mice; anti-actin antibody is used as the tissue internal reference.

[0053] Figure 3 Graph showing the changes in blood glucose and body weight of the offspring nursed by transgenic female mice tg-1 and tg-5 respectively in Example 2.

[0054] Among them, (A) is the graph showing the changes in blood glucose and body weight of the offspring nursed by transgenic female mouse tg-1; (B) is the graph showing the changes in blood glucose and body weight of the offspring nursed by transgenic female mouse tg-5. The results show that the fusion protein secreted by the mammary glands of transgenic mice significantly downregulates the blood glucose (left) and body weight (right) of the offspring. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001. Detailed implementation manners

[0055] The present invention will be further described in detail below in combination with the detailed implementation manners. The provided examples are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0056] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0057] Example 1. Production of transgenic mice

[0058] I. Preparation of β-casein promoter-GIP / GLP-1 fragment

[0059] Design concept: Based on the amino acid sequence of Tirzepatide, the codons of GIP / GLP-1 short peptide were optimized, and the unnatural α-amino butyric acid (a-Aib) at the 2nd and 13th positions was replaced with codons of alanine that can be translated by cells. Then it was coupled with the codons of the Fc fragment of IgG4, and this new coding sequence was expressed under the drive of the β-casein promoter.

[0060] First, a DNA optimized sequence containing "GIP / GLP-1 dual receptor activating peptide-(GGGGS)*3 linker-Fc gene fragment" in sequence was designed and synthesized, and the specific sequence is shown in SEQ ID No.2. SEQ ID No.2 encodes the amino acid sequence shown in SEQ ID No.1. The 1st to 15th positions of SEQ ID No.1 are the amino acid sequence of the secretory signal peptide, the 16th to 54th positions are the amino acid sequence of the GIP / GLP-1 dual receptor activating peptide, the 56th to 70th positions are the amino acid sequence of the linker peptide, and the 71st to 299th positions are the amino acid sequence of the Fc segment of the IgG4 antibody.

[0061] Next, the above synthetic sequence (SEQ ID No.2) was inserted into the pBC1 plasmid (Invitrogen, catalog number K270-01) through the Xho I restriction site to construct a new recombinant plasmid. In this recombinant plasmid, the 16.6 kb DNA fragment between the Sal I and Not I restriction enzymes was named β-casein promoter-GIP / GLP-1 (as Figure 1 shown in (A) below). In the β-casein promoter-GIP / GLP-1 fragment, the fusion gene shown in SEQ ID No.2 is expressed under the drive of the β-casein promoter (a mammary gland-specific expression promoter, whose nucleotide sequence is shown in SEQ ID No.3).

[0062] The structure of the β-casein promoter-GIP / GLP-1 fragment from the 5' to 3' end is described in sequence as follows: 2× chicken β-globin insulator, goat β-casein promoter, goat β-casein first exon, goat β-casein first intron, partial second exon of goat β-casein, coding gene of the fusion protein, goat β-casein seventh exon and the genomic sequence behind it. The specific sequence of the β-casein promoter-GIP / GLP-1 fragment is as follows:

[0063]

[0064] Subsequently, using conventional genetic engineering operation methods, the above recombinant plasmid was transformed into Escherichia coli for amplification, extraction, purification. After correct identification by enzyme digestion and sequencing, it was used for the next experiment.

[0065] II. Microinjection

[0066] Referring to the standard method and the kit instruction manual, the large-scale extracted recombinant plasmid was digested with Sal I and Not I double enzymes, and the 16.6 kb β-casein promoter-GIP / GLP-1 fragment (sequence as above) was recovered and purified, and its concentration was adjusted to 10 ng / μl for preparation of microinjection into fertilized eggs. Referring to the standard embryo operation and transgenic methods, Kunming mouse fertilized eggs were obtained, the DNA fragment was microinjected into the pronucleus of the fertilized eggs, and then these fertilized eggs were transplanted into the oviducts of pseudopregnant Kunming female mice to wait for pregnancy and the birth of offspring.

[0067] III. Molecular Identification of Transgenic Mice

[0068] After the offspring were born and weaned, a small amount of tail tip tissue was cut and lysed with the standard SNET + proteinase K solution, and then DNA was extracted with phenol / chloroform / isopentanol (volume ratio 25:24:1) for PCR and Southern blot identification.

[0069] First, PCR screening was carried out. Five pairs of primers were designed (the specific positions are shown in Figure 1 (A) below), and the amplified lengths were 792 bp (F: 5-AGACCCTCCTCCTGTATGGG-3; R: GGATGCACGGAAGTTTTGGC-3), 557 bp (F: 5-AATGGCTGGCAGTGAAAC-3; R: 5-CGTACTTGCTCTCGGCTGAT-3), 311 bp (5-TTCAACTGGTACGTGGACGG; R: 5-GCGATGTCGCTGGGATAGAA-3), 746 bp (F: 5-AGGGGAACGTCTTCTCATGC-3; R: 5-AAAAGTGAGGAGGGGGCATTC-3) and 616 bp (F: 5-CCCTTTCGCAGTTTGGAACC-3; R: 5-TGCAGGTCAACGGATCACTC-3) fragments of the foreign expression vector. A total of 2 PCR-positive female mice were screened out from 95 offspring, and the mouse numbers were tg-1 and tg-5 ( Figure 1(B)); Sequencing analysis showed that the full sequence of the expression vector was correct; then, 311bp and 746bp labeled primers were used as probes to perform Southern blot hybridization on the EcoR I digested products of genomic DNA, respectively, to confirm that both mice had a 3954bp digested product containing the target band, and they were transgenic mice( Figure 1 (C)).

[0070] IV. Identification of the fusion protein

[0071] After mating tg-1 and tg-5 female mice with wild-type Kunming male mice, they could all be normally pregnant and give birth (the day of giving birth was recorded as D0). Subsequently, at D5, D8, D10, D14, and D21 days, according to the conventional method, the milk of the two transgenic female mice and wild-type female mice (negative control) with the same lactation time was collected for standby. Referring to the standard SDS-PAGE protein electrophoresis, protein blotting, and western blot hybridization methods, immunoblot analysis was performed on the milk proteins. During immunoblotting, the GIP / GLP-1 activating peptide in the fusion protein was identified with an anti-tirzepatide antibody (PHK13902, Wuhan Bolebo Biotechnology Co., Ltd.), and after hybridization with an HPR-labeled secondary antibody (Santa Cruz Biotechnology, sc-2004), development was carried out to confirm that there was a positive band of 37 kDa size in the milk samples of tg-1 and tg-5 mice, while there was no target band in the negative milk from wild-type female mice( Figure 2 (A)); Then, the Fc fragment in the fusion protein was directly identified with an HRP-labeled anti-human IgG Fc antibody (A18817, Thermo Fisher, USA), and it was confirmed that there was still a positive band of 37 kDa size in the milk samples of tg-1 and tg-5 mice, and there was no band in the negative milk of wild-type female mice( Figure 2 (B)), and the results showed that intact fusion protein existed in the milk secreted by tg-1 and tg-5 mice.

[0072] Meanwhile, one positive and one negative 10-day-old cub were randomly selected from the first-generation offspring of tg-1. They were dissected, and various tissues and organs were collected and stored at -80 °C for future use. Referring to the standard SDS-PAGE protein electrophoresis, protein blotting, and western blot hybridization methods, immunoblot analysis was performed on each tissue sample to detect whether the foreign gene would be expressed in tissues outside the mammary gland. The results of the anti-actin antibody (Wuhan Sanying Biotechnology Co., Ltd., 20536-1-AP) combined with the HRP-labeled secondary antibody (Santa Cruz Biotechnology, sc-2004) showed that there were positive actin bands in both transgenic and wild-type tissues, indicating that the test samples were reasonable and the experiment could continue. Then, identification was performed using the anti-tirzepatide antibody. The results showed that not only were there no positive bands of 37 kDa in the negative tissues of wild-type mice, but also no positive bands were detected in the brown adipose tissue, heart, liver, spleen, lung, and kidney tissues of transgenic mice, further indicating that the fusion protein was only expressed in the mammary gland tissue and not in other tissues ( Figure 2 in (C) of the above figure). The above results prove that the use of the mammary gland of transgenic animals can produce glucagon-like peptide-1 receptor agonist (GIP / GLP-1 receptor agonist).

[0073] Furthermore, the concentration of the fusion protein in the milk was detected by ELISA. A 96-well plate coated by the conventional method was used, and the operations were as follows: 100 μL of the diluted emulsion (4 μL of milk was diluted with 96 μL of coating buffer) was added to each well, incubated at 37 °C for 2 - 3 hours, washed 3 times with PBST, blocked with 5% fetal bovine serum at room temperature for 1 hour, incubated with 100 μL of anti-human IgG Fc antibody (Thermo Fisher Scientific Inc, A18817) diluted with PBS at 37 °C for 1 hour, washed 5 times with PBST, added with TMB solution and developed color at 37 °C for 10 minutes, the reaction was terminated with the termination solution, and the absorbance value was read by an enzyme-linked immunosorbent assay at a wavelength of 450 nm. The standard product used was human IgG (Beyotime Biotechnology Co., Ltd., A7001). For tg-1 mice, the concentrations of the fusion protein in the D5 and D10 samples were 0.8 and 1.33 g / L, respectively; for tg-5 female mice, the concentrations of the fusion protein in the D8, D10, and D14 milk samples were 1.11, 1.15, and 1.42 g / L, respectively. This shows that the concentration of the fusion protein in the milk samples of both transgenic mice exceeded 1.0 g / L. That is, the method of producing GIP / GLP-1 receptor agonist using the mammary gland of transgenic animals can obtain a high concentration of receptor agonist and has commercial development value.

[0074] Example 2: Biological Activity Analysis of the Fusion Protein Secreted by Transgenic Mammary Glands

[0075] To detect the biological activity of the fusion protein in milk, the present invention observed the pregnancy and postpartum lactation of transgenic female mice. The results showed that both transgenic female mice could reproduce and lactate normally. Further, the blood glucose and body weight parameters of the tail tips of the pups at 8 days old, 10 days old, 14 days old, and 21 days old were detected.

[0076] Compared with the same-age pups born to negative female mice during the same period, the blood glucose values of the pups born to tg-1 female mice at 8 days old, 10 days old, 14 days old, and 21 days old were significantly decreased, with the decreases being 10.37%, 20.92%, 10.88%, and 16.97% respectively. Except for the 14-day-old group, the differences at other ages were significant (p < 0.05). At the same time, the body weights of the transgenic pups at the four monitoring points were also extremely significantly (p < 0.001) decreased, with the body weight decreases reaching 14.86%, 14.36%, 21.88%, and 31.25% respectively ( Figure 3 in (A)), and the results indicate that the fusion protein in the milk of tg-1 female mice can down-regulate the blood glucose level and body weight of the suckling pups.

[0077] For tg-5 female mice, due to the higher yield of their fusion protein, the experimental design was adjusted. The tg-5 female mice in the same pregnancy were caged with a wild-type female mouse. They gave birth to a total of 22 pups. After nursing together for 8 days, the tg-5 and wild-type female mice were separated into different cages, and the 22 pups were randomly and evenly divided between them for continued nursing. Before separation, the 8-day-old blood glucose values of the two groups of pups were not different (p > 0.05). After separation, compared with the wild-type group, the blood glucose values of the pups nursed by tg-5 female mice were significantly (p < 0.05) decreased, and the blood glucose values at 10 days old, 14 days old, and 21 days old decreased by 16.87%, 19.93%, and 39.39% respectively. At the same time, the body weight of the pups in the tg-5 group at 14 days old decreased by 26.42% (p < 0.001), and a large number of them died after 16 days old. 8 out of 11 pups died successively, and only 3 developed to 21 days old, and the body weight decrease of these 3 reached 48.61% ( Figure 3 in (B)), and the results indicate that the fusion protein in the milk of tg-5 female mice efficiently down-regulates the blood glucose level and body weight of the suckling pups, and the excessive concentration of the fusion protein leads to a high mortality rate of the pups.

[0078] The experimental results of the two transgenic mice both indicate that the fusion protein specifically synthesized in the mammary gland in the present invention has obvious incretin-like insulin activity, that is, the dual effects of lowering blood glucose and body weight.

[0079] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art.

Claims

1. A method for constructing an animal model for producing incretin receptor activating peptide using mammary gland, comprising the following steps: specifically expressing a fusion protein in the mammary gland of an animal, wherein the fusion protein comprises a fusion fragment of incretin receptor activating peptide and an antibody Fc segment.

2. The method according to claim 1, characterized in that: The incretin receptor activating peptide is a GIP / GLP-1 dual receptor activating peptide; Further, the amino acid sequence of the GIP / GLP-1 dual receptor activating peptide is as shown in positions 16-54 of SEQ ID No.1 or has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the sequence shown in positions 16-54 of SEQ ID No.1; and / or The antibody Fc segment is the Fc segment of an IgG4 antibody; Furthermore, the amino acid sequence of the Fc segment of the IgG4 antibody is shown in positions 71-299 of SEQ ID No. 1; and / or The fusion protein also contains a secretory signal peptide; Furthermore, the amino acid sequence of the secretory signal peptide is shown in positions 1 to 15 of SEQ ID No. 1; and / or In the fusion protein, the incretin receptor activating peptide and the antibody Fc segment are connected via a connecting peptide; Furthermore, the amino acid sequence of the connecting peptide is shown in positions 56-70 of SEQ ID No.1; Furthermore, the amino acid sequence of the fusion protein is as shown in SEQ ID No.1 or has more than 99%, more than 95%, more than 90%, more than 85% or more identity with SEQ ID No.

1.

3. The method according to claim 1 or 2, characterized in that: The method comprises the following steps: introducing the coding gene of the fusion protein into the fertilized egg or somatic cell of the animal, then transplanting the fertilized egg or the embryo cloned from the somatic cell into the oviduct or uterus of a pseudo-pregnant animal, and selecting F0 generation transgenic animals from the offspring born from pregnancy; establishing a family using the F0 generation transgenic animals, and the homozygous or heterozygous transgenic offspring in the family are animal models that can produce incretin receptor activating peptide using mammary glands.

4. The method according to claim 3, characterized in that: The coding gene of the fusion protein is integrated into the fertilized egg or somatic cell of the animal in the form of an expression cassette; The expression cassette contains a mammary gland-specific expression promoter and a gene encoding the fusion protein whose expression is driven by the mammary gland-specific expression promoter.

5. The method according to claim 4, characterized in that: The mammary gland-specific expression promoter is a β-casein promoter; Furthermore, the nucleotide sequence of the β-casein promoter is shown in SEQ ID No. 3; and / or The nucleotide sequence of the gene encoding the fusion protein is shown in SEQ ID No.

2.

6. The method according to any one of claims 1 to 5, characterized in that: The animal is a mammal; Furthermore, the mammal is a mouse, a cow, a sheep, a pig or a rabbit.

7. Use of the animal model constructed by the method of any one of claims 1 to 6 in the production of incretin receptor activating peptides.

8. A method for producing incretin receptor activating peptides using animal mammary glands, comprising the following steps: causing an animal model constructed using the method described in any one of claims 1 to 6 to secrete milk, and obtaining incretin receptor activating peptides from the milk.

9. Any of the following biological materials or their applications: (A1) the fusion protein according to claim 1 or 2; (A2) a nucleic acid molecule encoding the fusion protein; (A3) an expression cassette or a recombinant plasmid or a recombinant bacterium or a transfected cell containing the nucleic acid molecule; The application is the application of the biomaterial in constructing an animal model for producing incretin receptor activating peptides using mammary glands.

10. Milk secreted by the animal model constructed by the method of any one of claims 1 to 6 or an incretin receptor activating peptide obtained from the milk; or The use of the milk or the incretin receptor activating peptide in the preparation of products; the product has any of the following functions: lowering blood sugar and / or body weight, improving cardiovascular or chronic kidney disease symptoms, and assisting in the treatment of diabetic complications.

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