Bifunctional recombinant protein GIK for regulating glycolipid metabolism and resisting aging and preparation method therefor

Abstract

Provided are a bifunctional recombinant protein GIK for regulating the glycolipid metabolism and resisting aging, and a preparation method therefor. The recombinant protein GIK comprises an N-terminus, a middle section and a C-terminus. The N-terminus is an optimized human GLP-1 polypeptide having an amino acid sequence of SEQ ID NO: 1; the middle section is an Fc region of human IgG4 having an amino acid sequence of SEQ ID NO: 2; and the C-terminus is an s-klotho polypeptide having an amino acid sequence of SEQ ID NO: 3. The N-terminus is connected to the middle section via a connecting peptide 1 having an amino acid sequence of SEQ ID NO: 4; the middle section is connected to the C-terminus via a connecting peptide 2 having an amino acid sequence of SEQ ID NO: 5; and a signal peptide of human serum albumin having an amino acid sequence of SEQ ID NO: 7 is introduced at the N-terminus, such that a bifunctional recombinant protein that can be secreted and expressed in mammalian cells is obtained, and the bifunctional recombinant protein has an amino acid sequence of SEQ ID NO: 8.

Description

A bifunctional recombinant protein GIK for regulating glycolipid metabolism and anti-aging and its preparation method Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a dual-function recombinant protein GIK for regulating glycolipid metabolism and resisting aging, and a preparation method thereof.

[0002] Background Art

[0003] Diabetes is a common chronic disease worldwide, with the adult diabetes prevalence in my country reaching 12.8%. As the disease progresses, diabetic patients develop a variety of complications, such as vascular disease, peripheral neuropathy, fragility fractures, and distal finger erosions. Numerous clinical and basic research studies have revealed a close association between diabetes and aging. Aging can lead to a variety of chronic metabolic diseases, and the onset and progression of diabetes can also accelerate cellular aging. Studies have shown that senescent cells can secrete aging-related factors such as activin A, IL-6 and TNF-α to promote insulin resistance. Senescent adipocytes can induce macrophages to enter visceral adipose tissue and release chemokines, exacerbating the development of type 2 diabetes [Xu M, Tchkonia T, Ding H, Ogrodnik M, Lubbers ER, Pirtskhalava T, White TA, Johnson KO, Stout MB, Mezera V, Giorgadze N, Jensen MD, LeBrasseur NK, Kirkland JL. JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age. Proc Natl Acad Sci US A. 2015 Nov 17; 112(46): E6301-10. doi: 10.1073 / pnas.1515386112.]. The high-sugar, high-fat environment in diabetic patients increases the production of reactive oxygen species (ROS), causing mitochondrial and DNA damage, thereby triggering cell cycle arrest and senescence. This high-sugar microenvironment also increases the production of advanced glycation end products (AGEs), which facilitate the spread of senescent cells and hinder their clearance by the body.What's more, fat accumulation caused by diabetes can induce chronic sterile inflammation and fat toxicity, further accelerating aging [Berlanga-Acosta JA, Guillén-Nieto GE, Rodríguez-Rodríguez N, Mendoza-Mari Y, Bringas-Vega ML, Berlanga-Saez JO, García Del Barco Herrera D, Martinez-Jimenez I, Hernandez-Gutierrez S, Valdés-Sosa PA. Cellular Senescence as the Pathogenic Hub of Diabetes-Related Wound Chronicity. Front Endocrinol(Lausanne).2020 Sep 16;11:573032.doi:10.3389 / fendo.2020.573032.].

[0004] Most of the treatments for diabetes involve controlling blood sugar by improving lifestyle first, followed by the use of hypoglycemic drugs. Most traditional hypoglycemic drugs have adverse reactions. For example, metformin, glinides, and α-glucosidase inhibitors have little effect on lowering blood sugar and can induce gastrointestinal discomfort; insulin and sulfonylureas increase the risk of hypoglycemia and obesity. In recent years, newly developed glucagon-like peptide-1 receptor agonists (GLP-1 RA) and glucagon-like peptide-1 (Glucagon-like peptide-1, GLP-1) analogs act on GLP-1 receptors to activate pancreatic B cells to secrete insulin in a blood sugar-dependent manner. They have the characteristics of reducing the risk of hypoglycemia and having a wide therapeutic window, which significantly improves the safety of medication [Romera I, Cebrián-Cuenca A, -Guisasola F, Gomez-Peralta F, Reviriego JA Review of Practical Issues on the Use of Glucagon-Like Peptide-1 Receptor Agonists for the Management of Type 2 Diabetes.Diabetes Ther. 2019 Feb;10(1):5-19.doi:10.1007 / s13300-018-0535-9.]. Clinical practice has shown that many drugs for treating diabetes are also effective in treating aging-related diseases. For example, metformin is a weak inhibitor of the senescent associated secretory phenotype (SASP) and has a certain therapeutic effect on cardiovascular diseases and cancer development caused by aging [Huffman DM, Justice JN, Stout MB, Kirkland JL, Barzilai N, Austad SN. Evaluating Health Span in Preclinical Models of Aging and Disease: Guidelines, Challenges, and Opportunities for Geroscience. J Gerontol A Biol Sci Med Sci. 2016 Nov; 71(11): 1395-1406. doi: 10.1093 / gerona / glw106.].Ruxolitinib reduces insulin resistance and slows aging-induced stem cell dysfunction in aged mice [Anderson R, Lagnado A, Maggiorani D, Walaszczyk A, Dookun E, Chapman J, Birch J, Salmonowicz H, Ogrodnik M, Jurk D, Proctor C, Correia-Melo C, Victorelli S, Fielder E, Berlinguer-Palmini R, Owens A, Greaves LC, Kolsky KL, Parini A, Douin-Echinard V, LeBrasseur NK, Arthur HM, Tual-Chalot S, Schafer MJ, Roos CM, Miller JD, Robertson N, Mann J, Adams PD, Tchkonia T, Kirkland JL, Mialet-Perez J, Richardson GD, Passos JF. Length-independent telomere damage drives post-mitotic cardiomyocyte senescence. EMBO. J. 2019Mar 1;38(5):e100492.doi:10.15252 / embj.2018100492.].

[0005] At present, although many of the intrinsic association mechanisms between diabetes and aging have not been fully revealed, it can be foreseen that the combined treatment of the two is a good entry point and has great practical significance for the prevention and treatment of chronic diseases.

[0006] Summary of the Invention

[0007] The present invention provides a bifunctional recombinant protein GIK for regulating glucose and lipid metabolism and resisting aging, and a preparation method thereof, in order to achieve combined treatment of diabetes and aging.

[0008] The specific technical solutions of the present invention are as follows:

[0009] The present invention provides a bifunctional recombinant protein GIK for regulating glycolipid metabolism and anti-aging, characterized in that it comprises three structural units, an N-terminus, a middle section and a C-terminus; wherein the N-terminus is an optimized human GLP-1 polypeptide, whose amino acid sequence is SEQ ID NO: 1; the middle section is the Fc region of human IgG4, into which S010P, F016A and L017A mutations are simultaneously introduced, whose amino acid sequence is SEQ ID NO: 2; the C-terminus is an s-klotho polypeptide, whose amino acid sequence is SEQ ID NO: 3; the N-terminus and the middle section are connected by a connecting peptide 1, and the middle section and the C-terminus are connected by a connecting peptide 2.

[0010] The bifunctional recombinant protein GIK for regulating glycolipid metabolism and anti-aging provided by the present invention also has the following technical features: the amino acid sequence of connecting peptide 1 is SEQ ID NO: 4, and the amino acid sequence of connecting peptide 2 is SEQ ID NO: 5.

[0011] The bifunctional recombinant protein GIK for regulating glycolipid metabolism and anti-aging provided by the present invention also has the following technical features: in order to ensure the normal secretion of the bifunctional recombinant protein GIK in mammalian cells, a signal peptide of human serum albumin is introduced at the N-terminus of the bifunctional recombinant protein GIK, and the amino acid sequence of the signal peptide is SEQ ID NO: 7.

[0012] The bifunctional recombinant protein GIK for regulating glycolipid metabolism and anti-aging provided by the present invention also has the following technical features: the amino acid sequence of the bifunctional recombinant protein GIK that can be secreted and expressed in mammalian cells is SEQ ID NO: 8; the amino acid sequence of the bifunctional recombinant protein GIK that cannot be secreted and expressed in mammalian cells without introducing a signal peptide of human serum albumin at the N-terminus of the bifunctional recombinant protein GIK is SEQ ID NO: 6.

[0013] The present invention also provides a DNA encoding a bifunctional recombinant protein GIK that can be secreted and expressed in mammalian cells, characterized in that the sequence of the encoding DNA is SEQ ID NO: 9 or its codon-optimized sequence (codon-optimized for host cells).

[0014] The present invention also provides a GIK expression vector, characterized in that the GIK expression vector contains the above-mentioned encoding DNA.

[0015] The present invention also provides a GIK host cell, characterized in that the GIK host cell contains the above-mentioned GIK expression vector, or the above-mentioned encoding DNA is integrated into the genome of the GIK host cell, and the GIK host cell is a eukaryotic cell.

[0016] The GIK host cell provided by the present invention also has the following technical characteristics: the eukaryotic cell is selected from mammalian cells, yeast and filamentous fungi, the eukaryotic cell is preferably a mammalian cell or yeast, and the eukaryotic cell is more preferably a mammalian cell.

[0017] The present invention also provides a method for preparing the above-mentioned bifunctional recombinant protein GIK for regulating glycolipid metabolism and anti-aging, which is characterized by comprising the following steps: step S1, constructing a GIK expression vector: placing the coding DNA sequence within the eukaryotic gene expression regulatory sequence to obtain a GIK expression vector that can be expressed in eukaryotic cells; step S2, transfecting the GIK expression vector into eukaryotic cells: introducing the GIK expression vector into the eukaryotic cells through gene transfection technology to achieve stable integration of the GIK expression framework on the chromosomes of the eukaryotic cells for stable expression or transient expression in the form of extracellular genetic material to obtain GIK host cells (i.e., eukaryotic cells containing the GIK expression framework); step S3, high-density suspension culture of the GIK host cells to express the bifunctional recombinant protein GIK at high density and for a long time to obtain a culture containing the bifunctional recombinant protein GIK; step S4, purifying the bifunctional recombinant protein GIK from the culture containing the bifunctional recombinant protein GIK.

[0018] The preparation method of the bifunctional recombinant protein GIK for regulating glycolipid metabolism and anti-aging provided by the present invention also has the following technical features, wherein the specific process of purifying the bifunctional recombinant protein GIK in step S4 is: step S4-1, concentrating and filtering the culture containing the bifunctional recombinant protein GIK to obtain a pretreatment liquid; step S4-2, treating the pretreatment liquid by affinity chromatography to obtain an affinity chromatography purified product; step S4-3, treating the affinity chromatography purified product by ion exchange chromatography to obtain an ion exchange chromatography purified product; step S4-4, using a molecular sieve to remove monomers in the ion exchange chromatography purified product to obtain the bifunctional recombinant protein GIK in the form of a dimer.

[0019] The present invention also provides a recombinant protein, characterized in that the amino acid sequence of the recombinant protein has a homology of ≥85% with the amino acid sequence of the bifunctional recombinant protein GIK that can be secreted and expressed in mammalian cells, preferably a homology of ≥90%, and more preferably a homology of ≥95%.

[0020] The present invention also provides a DNA sequence, characterized in that the DNA sequence encodes the above-mentioned recombinant protein and has a homology of ≥85% with the above-mentioned encoding DNA sequence (SEQ ID NO: 9 or its codon-optimized sequence), preferably a homology of ≥90%, and more preferably a homology of ≥95%.

[0021] The present invention also provides a pharmaceutical preparation, characterized in that the pharmaceutical preparation contains the above-mentioned dual-functional recombinant protein GIK for regulating glycolipid metabolism and anti-aging and / or the above-mentioned recombinant protein.

[0022] Functions and effects of the invention

[0023] Compared with the existing technology, the dual-functional recombinant protein GIK for regulating glucose and lipid metabolism and anti-aging and its preparation method provided by the present invention have the following technical advantages:

[0024] (1) The bifunctional recombinant protein GIK of the present invention contains two functional domains, namely the optimized human GLP-1 polypeptide at the N-terminus and the s-Klotho polypeptide at the C-terminus, which has multiple functions such as hypoglycemic, weight loss and anti-aging. Natural GLP-1 is a hormone produced by intestinal cells, which stimulates and enhances the secretion of insulin, inhibits the secretion of glucagon, and slows the rate of gastric emptying. Based on the amino acid sequence of GLP-1, a number of drugs for treating diabetes and weight loss have been developed, such as liraglutide, exenatide, lixisenatide, dulaglutide, semaglutide, etc. The optimized human GLP-1 polypeptide of the present invention replaces the Ala at the second position of natural GLP-1 with Gly, thereby resisting the enzymatic hydrolysis of dipeptidyl peptidase 4 (DPP-4) in the body and increasing the half-life in the body. s-Klotho polypeptide is an anti-aging protein, and Klotho overexpression can extend the lifespan of mice by 30%. s-Klotho regulates not only phosphate metabolism but also calcium metabolism; it inhibits the insulin / IGF-1 signaling pathway, activates FoxO transcription factors, produces antioxidant enzymes, reduces oxidative stress by removing ROS, and thus downregulates cell apoptosis; it inhibits TNF-α-induced oxidative damage and prevents the translocation of NF-κB to the nucleus in activated B cells; and it inhibits the TGF-β1 and Wnt / β-catenin signaling pathways.

[0025] (2) In order to further improve the in vivo half-life of the optimized human GLP-1 polypeptide and s-Klotho polypeptide, the present invention fuses the Fc fragment of human IgG4 into the recombinant protein. The hinge region of IgG4 is relatively short and has weak binding to FcγRs other than FcγRI (CD64), which cannot cause CDC and ADCC, and can more effectively reduce the immune response of repeated medication. At the same time, the present invention simultaneously introduces S010P, F016A and L017A mutations into the Fc region of IgG4, further removing the effector function of the Fc region. The S010P mutation can also stabilize the dimer state of the bifunctional recombinant protein GIK molecule and prevent the formation of half molecules. The introduction of this mutated Fc region can simultaneously improve the in vivo half-life of both GLP-1 and s-Klotho, and put the two proteins in a bimolecular state, increasing the local ligand density, effectively improving their binding efficiency with the receptor, and improving in vivo function. At the same time, the introduction of the IgG4 Fc region allows the bifunctional recombinant protein GIK to be easily purified through Protein A or Protein G affinity chromatography, making it easy to scale up.

[0026] (3) To ensure the efficacy of each functional domain in the bifunctional recombinant protein GIK, the present invention connects the three structural units together through two linker peptides. To adapt to the characteristics of the short GLP-1 peptide, the present invention uses a flexible linker peptide 1 to connect the optimized GLP-1 to the N-terminus of the Fc region, ensuring that the two functional domains do not interfere with each other. To accommodate the relatively large s-Klotho and Fc regions, the present invention extends the linker peptide 2 to 23 amino acids.

[0027] (4) The present invention uses mammalian cells to prepare the bifunctional recombinant protein GIK. This is because, on the one hand, mammalian cells can complete complex post-translational modifications, ensuring the functionality of each region of the bifunctional recombinant protein GIK; on the other hand, mammalian cell culture systems have been widely used in the pharmaceutical-grade production of therapeutic antibodies, bispecific antibodies, and complex proteins, and the production system is mature. In the present invention, the secretory peptide of human serum albumin is used for the secretory expression of the bifunctional recombinant protein GIK because, compared with the signal peptide of natural immunoglobulin, the albumin signal peptide can mediate better antibody protein expression.

[0028] (5) The preparation method of the bifunctional recombinant protein GIK of the present invention is carried out using eukaryotic cells, which not only ensures that the bifunctional recombinant protein GIK obtains sufficient post-translational modification, so that it has more complete biological functions; at the same time, eukaryotic cells, especially mammalian cells (such as CHO, etc.) have mature production processes that can easily expand the production scale.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a schematic diagram of the structure of the GIK precursor protein according to an embodiment of the present invention.

[0031] FIG2 is a plasmid map of the eukaryotic expression vector pCMV-GIK of the GIK gene according to an embodiment of the present invention.

[0032] Figure 3 is an electrophoresis pattern of restriction enzyme digestion of the pCMV-GIK plasmid of the embodiment of the present invention. In the figure, M is DNA Ladder (0.1-10kb); 1 is pCMV-GIK linearized with KpnI; and 2 is the result of NotI+EcoR double enzyme digestion of pCMV-GIK.

[0033] FIG4 is a GIK-integrated CHO-K1 cell (GIK host cell) clone according to an embodiment of the present invention.

[0034] FIG5 is a Dot-bloting test of clones expressing GIK with relatively high efficiency according to an embodiment of the present invention.

[0035] Figure 6 shows the Protein A-sehparose affinity chromatography of the bifunctional recombinant protein GIK according to an embodiment of the present invention. In the figure, A is the Protein A affinity chromatography chromatogram; B is the SDS-PAGE results of each affinity chromatography peak.

[0036] Figure 7 is a DEAE-sepharose ion exchange chromatography of the bifunctional recombinant protein GIK according to an embodiment of the present invention. In the figure, A is a DEAE ion exchange chromatography chromatogram; B is the SDS-PAGE results of each peak of the ion exchange chromatography.

[0037] FIG8 shows the hypoglycemic effect of the bifunctional recombinant protein GIK of Test Example 1 of the present invention.

[0038] FIG9 shows the anti-aging effect of the bifunctional recombinant protein GIK of Test Example 2 of the present invention.

[0039] DETAILED DESCRIPTION

[0040] The terms used in the present invention generally have the meanings commonly understood by those skilled in the art unless otherwise specified.

[0041] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0042] The reagents used in the following examples were purchased from common commercial channels. The experimental operations and experimental conditions not specified were based on conventional operations and conditions in the art.

[0043] The specific implementation of the present invention is described below with reference to the embodiments and drawings.

[0044] <Example>

[0045] This embodiment provides a bifunctional recombinant protein GIK that regulates glycolipid metabolism and anti-aging, and a method for preparing the same. The bifunctional recombinant protein GIK can be secreted and expressed in mammalian cells, and the steps for preparing the bifunctional recombinant protein GIK are as follows:

[0046] Step S1, constructing a GIK expression vector: placing the coding DNA sequence within a eukaryotic gene expression regulatory sequence to obtain a GIK expression vector capable of expression in eukaryotic cells. The specific process is as follows:

[0047] In order to achieve the dual functions of regulating glycolipid metabolism and anti-aging, the two functional domains of optimized human GLP-1 polypeptide and s-Klotho polypeptide were designed to be placed at the N-terminus and C-terminus of the Fc region of human IgG4, respectively. The N-terminal optimized human GLP-1 polypeptide and the middle human IgG4 Fc region were connected by connecting peptide 1 (amino acid sequence of SEQ ID NO: 4), and the middle human IgG4 Fc region and the C-terminal s-Klotho polypeptide were connected by connecting peptide 2 (amino acid sequence of SEQ ID NO: 5), forming a bifunctional recombinant protein GIK that cannot be secreted and expressed in mammalian cells, whose amino acid sequence is SEQ ID NO: 6.

[0048] To ensure the normal and effective secretory expression of the bifunctional recombinant protein GIK in mammalian cells, a secretory peptide (signal peptide) of human serum albumin was introduced at its N-terminus. The amino acid sequence of the signal peptide is SEQ ID NO: 7, forming a bifunctional recombinant protein GIK (GIK precursor protein) that can be secreted and expressed in mammalian cells. The amino acid sequence of the signal peptide is SEQ ID NO: 8, and its structure is shown in Figure 1.

[0049] The coding DNA sequence of the GIK precursor protein was designed based on its amino acid sequence, and NotI and EcoRI sites were added at both ends to facilitate subcloning into an expression vector. The final coding DNA sequence of the GIK precursor protein was SEQ ID NO:9.

[0050] Finally, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to complete the whole gene synthesis and obtain the pUC-GIK plasmid containing the DNA sequence encoding the GIK precursor protein. The pUC-GIK plasmid was double-digested with NotI and EcoRI, and the GIK gene was recovered by agarose gel electrophoresis. It was then directionally cloned into the expression vector pCMV-Express, which had been double-digested with NotI and EcoRI, to form the eukaryotic expression vector pCMV-GIK plasmid (i.e., the GIK expression vector) for the GIK gene. The map of the eukaryotic expression vector pCMV-GIK plasmid for the GIK gene is shown in Figure 2. The electrophoresis map of the pCMV-GIK plasmid is shown in Figure 3.

[0051] Step S2, transfecting the GIK expression vector into eukaryotic cells: The GIK expression vector is introduced into eukaryotic cells using gene transfection technology to achieve stable integration of the GIK expression framework into the eukaryotic cell chromosomes for stable expression or transient expression in the form of extracellular genetic material, thereby obtaining GIK host cells (i.e., eukaryotic cells containing the GIK expression framework). Screening for high-yielding, stably integrated monoclonal cell lines is then performed. The specific process is as follows:

[0052] Cell transfection:

[0053] pCMV-GIK was linearized with restriction endonuclease KpnI, and the linearized fragment was recovered by 1% agarose gel electrophoresis. The concentration of DNA (i.e., KpnI-linearized pCMV-GIK) was adjusted to 0.5 μg / μl, and the DNA was filtered through 0.22 μm before use for cell transfection.

[0054] CHO-K1 cells stored in liquid nitrogen were revived and subcultured normally in complete medium (DEME containing 10% fetal bovine serum) in an incubator set at 37°C and 5% CO2. DNA transfection was performed using the PEI method, which involved digesting and passage cells into 12-well culture dishes one day before transfection, with a cell number of 2.5×10 5 / ml. On the day of transfection, transfection should begin when the confluence of CHO-K1 cells reaches 70-80%. The DNA dosage for transfection is: DNA / total culture volume = 1 μg / ml. Prepare the DNA-PEI complex: equilibrate the DNA, PEI 40000, and DMEM basal medium to room temperature. Dilute the DNA to 20 μg / ml and the PEI 40000 to 60 μg / ml using DMEM basal medium. Mix 100 μl DNA + 100 μl PEI 40000 at a mass ratio of DNA:PEI = 1:3, pipette up and down 2-3 times to thoroughly mix the DNA and PEI, and let it stand at room temperature for 30 minutes to form a DNA-PEI complex; add 200 μl of DNA-PEI complex dropwise to a CHO cell culture plate, replace with fresh culture medium after 6 hours, and transfer to a 10 cm culture dish at a ratio of 1:20 after 24 hours, and add selection medium (complete medium containing 10 μg / ml puromycin).

[0055] Screening of high-yield stable integrated monoclonal cell lines:

[0056] After pCMV-GIK transfection, the cells (i.e., GIK host cells) were cultured with a medium change every 3 days for 12 days in complete medium containing 10 μg / ml puromycin. Clear single colonies emerged, as shown in Figure 4.

[0057] Single clones were picked: cells were washed with PBS buffer, placed in a cloning ring, trypsinized, and transferred to a 96-well plate. A total of 111 clones were picked. After the 96-well plate became confluent (3-5 days), the clones were sequentially passaged to 24-well, 12-well, 6-well, and 10 cm dishes. The supernatant from each passage was retained for expression analysis of the bifunctional recombinant protein GIK.

[0058] The expression of the bifunctional recombinant GIK protein was screened by dot-blotting. The method involved spotting 2 μl of cell culture supernatant onto a nitrocellulose membrane, allowing it to air dry and blocking it with 5% skim milk powder overnight at 4°C. Hybridization was then performed with a 1:2000 dilution of rabbit anti-human IgG-HRP antibody at room temperature for 3 hours. The membrane was washed with PBS-T and developed using an ECL kit. Human IgG was used as a positive control. Among the 111 cell clones, 12 clones expressing the bifunctional recombinant GIK protein at relatively high efficiency were identified, as shown in Figure 5: 9#, 17#, 23#, 32#, 67#, 53#, 66#, 69#, 76#, 91#, 95#, and 110#. These 12 clones expressing GIK at high efficiency were frozen in liquid nitrogen for further serum-free acclimation.

[0059] Step S3: High-density suspension culture of GIK host cells to express the bifunctional recombinant protein GIK at high density and for a long time to obtain a culture containing the bifunctional recombinant protein GIK. The specific process is as follows:

[0060] Before high-density suspension culture of GIK-expressing clones, they must be serum-free acclimated. Recover each GIK-expressing clone from liquid nitrogen and resuspend in a 10 cm culture plate. Culture medium is complete culture medium supplemented with 10% serum, and the cells are passaged at a 1:3 ratio. At the third passage, the culture medium is replaced with 75% complete culture medium plus 25% serum-free culture medium (Vega CHO Medium, P106390, Aupuma). Once the cells are confluent, the fourth passage is performed, and the culture medium is replaced with 50% complete culture medium plus 50% serum-free culture medium. For the fifth and sixth passages, the culture medium is replaced with 25% complete culture medium plus 75% serum-free culture medium. For the seventh passage, cells from three 10 cm dishes are combined, 25 ml of 100% serum-free culture medium is added, and the cells are placed in a 125 ml shake flask for suspension culture at 120 rpm, 37°C, and 5% CO2. The cell density and viability of the suspension cultured cells were tracked and measured every day. When the cell density reached (2-4)×10 6 / ml for 1:3 passage. After 3 consecutive passages, the cell density can reach (2-3)×10 6 / ml, the activity is ≥90%, and the doubling time is stable, which means that the serum-free acclimation is successful. Take each cell line that has been successfully acclimated, and calculate (0.4-0.6)×10 6 / ml initial cell density was cultured until the cell density reached 2×10 6 / ml, and the supernatant was used for Dot-blotting to detect the expression level of GIK, and the 69# cell line with the highest expression level was selected. The successfully acclimated cells were taken and the cell density was adjusted to 20×10 6 / ml, stored in liquid nitrogen for a long time.

[0061] The serum-free acclimated 69# cell line was cultured in the order of 125ml shake flask → 250ml shake flask → 1L shake flask → 5L shake flask. The culture conditions were serum-free medium, 120rpm, 37℃, 5% CO2. When the cell density reached (2-3)×10 6 The cells were transferred to the flask when the cells were 10 / ml. The initial cell density after the transfer was controlled at (0.4-0.6)×10 6 / ml, and the culture medium volume of each level of shake flask is 25ml, 60ml, 250ml, and 1600ml respectively. Continue to culture for 10-15 days, and wait until the cell density in the 5L shake flask reaches (4-6)×10 6 / ml, feed solution (Vega CHO Feed, P120826, Aupuma) at 5% of the volume of culture medium was added every 2 days until the cell density reached 10×10 6 When the cell viability reaches ≤ 80%, harvest approximately 2 L of culture per bottle containing the bifunctional recombinant GIK protein. This is done by centrifuging the culture at 3000 g for 20 minutes, removing the cell pellet, and storing the supernatant at -20°C until purification of the bifunctional recombinant GIK protein is initiated.

[0062] Step S4: Purify the bifunctional recombinant protein GIK from the culture containing the bifunctional recombinant protein GIK. The steps are as follows: Step S4-1: Concentrate and filter the culture containing the bifunctional recombinant protein GIK to obtain a pretreatment solution. The specific process is as follows: The supernatant obtained in step S3, which was frozen at -20°C, was removed and slowly thawed at 15°C; the thawed supernatant was filtered through a 0.22μm filter membrane, and the filtrate was then ultrafiltered and concentrated through an ultrafiltration membrane with a 10kDa molecular weight cutoff. The supernatant was finally concentrated 5-fold to a final volume of approximately 1.2L, obtaining the pretreatment solution.

[0063] Step S4-2: Affinity chromatography is performed on the pretreatment solution to obtain an affinity chromatography-purified product. The specific process is as follows: the pretreatment solution is purified by affinity chromatography on Protein A-Sepharose 6FF using a mobile phase consisting of 20 mmol / L sodium phosphate buffer, pH 7.4; the elution solution is 20 mmol / L sodium phosphate buffer, pH 2.5; the flow rate is 3 ml / min, and the elution peak is collected, as shown in Figure 6. The eluent pH is adjusted to 7.0 using 1 M Tris-HCl, pH 8.0, to obtain the affinity chromatography-purified product.

[0064] In step S4-3, the affinity chromatography purified product is treated with ion exchange chromatography to obtain an ion exchange chromatography purified product. The specific process is as follows: the affinity chromatography purified product is subjected to DEAE-sepharose ion exchange chromatography with a mobile phase of 20 mmol / L sodium phosphate buffer solution, pH 7.0. The elution peak containing GIK is collected by linear elution with 0-1M NaCl, as shown in Figure 7. As shown in the SDS-PAGE results in Figure 7, GIK is a clear single band. The eluate is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to a protein concentration of 5.0 mg / ml to obtain an ion exchange chromatography purified product.

[0065] Step S4-4: Using molecular sieves to remove monomers from the purified product by ion exchange chromatography, a dimeric bifunctional recombinant protein GIK is obtained. The specific process is as follows: The purified product by ion exchange chromatography is subjected to molecular sieve chromatography using S-200 as a filler and PBS as the mobile phase. The target protein peak (i.e., the dimeric bifunctional recombinant protein GIK) is collected and concentrated by ultrafiltration using a 50 kDa molecular weight cutoff ultrafiltration membrane to a protein concentration of 10 mg / ml. The product is then stored at 4°C for short-term storage of <3 weeks and at -20°C for long-term storage.

[0066] <Test Example 1>

[0067] This test example measures the hypoglycemic effect of the bifunctional recombinant protein GIK in the dimer form obtained in the above example. The measurement process is as follows:

[0068] Healthy 8-week-old C57BL / 6 mice were purchased from Shanghai Model Organisms Science Co., Ltd. under license number SYXK(Shanghai)2023-0005. Mice were housed at 20 ± 2°C and 55 ± 5% relative humidity and provided with a normal diet and water.

[0069] After 7 days of adaptive feeding, C57BL / 6 mice were divided into three groups: a blank control group, a positive control group, and an experimental group, with 6 mice in each group. Mice were fasted overnight before intraperitoneal injection. Mice in the blank control group were injected with 200 μl of 40% glucose solution, mice in the experimental group were injected with 200 μl of 40% glucose solution plus 50 μg of the bifunctional recombinant protein GIK, and mice in the positive control group were injected with 200 μl of 40% glucose solution plus 10 μg of GLP-1(7-36)NH2. GLP-1(7-36)NH2 was purchased from Sigma.

[0070] Immediately after injection, 20 μl of blood was collected from the canthus of each mouse using a heparin-soaked glass pipette. The blood was then mixed with 300 μl of normal saline and centrifuged at 3000 rpm to remove red blood cells. The supernatant was used to measure blood glucose. Subsequently, blood was collected using the same procedure at 30, 60, and 120 minutes after injection, and the supernatant was retained for blood glucose measurement. The results are shown in Figure 8.

[0071] As shown in Figure 8, the blood glucose concentrations of mice in the blank control group increased significantly, then gradually returned to normal levels. The blood glucose concentrations of mice in the experimental and positive control groups remained near normal levels, without any significant increases. This indicates that the injection of GLP-1(7-36)NH2 and the bifunctional recombinant protein GIK has a hypoglycemic effect, which is consistent with the theoretical expectations of the hypoglycemic function of the bifunctional recombinant protein GIK.

[0072] <Test Example 2>

[0073] This test example measures the anti-aging effect of the bifunctional recombinant protein GIK in the dimer form obtained in the above example. The test process is as follows:

[0074] The anti-aging effects of the bifunctional recombinant protein GIK were evaluated in a D-galactose-senescence mouse model. Eight-week-old C57BL / 6J mice were randomly divided into three groups: a normal control group, a D-galactose-senescence group, and a GIK-treated group, with six mice in each group. The medications are listed in Table 1.

[0075] Table 1

[0076] During the experiment, mice were weighed weekly, and changes in food intake, water intake, and behavior were observed. After the final dose, mice were fasted for 12 hours. Blood was collected from the ocular plexus vein and serum was separated. Brain tissue, liver, kidney, thymus, and spleen were dissected and removed. After rinsing with saline, the samples were stored at -80°C for subsequent experiments.

[0077] Blood was collected from the ocular plexus vein, placed in a centrifuge tube, and centrifuged at 2500 rpm for 10 min at 4°C to obtain serum. According to the kit instructions, the superoxide dismutase activity, glutathione peroxidase activity, total antioxidant capacity, and malondialdehyde content in the mouse serum were determined. The measurement results are shown in Figure 9.

[0078] As shown in Figure 9, compared with the normal control group, the D-galactose aging group showed significantly decreased serum superoxide dismutase activity, glutathione peroxidase activity, and total antioxidant capacity, while significantly increased malondialdehyde content. Compared with the D-galactose aging group, the GIK intervention group showed significantly increased serum superoxide dismutase activity, glutathione peroxidase activity, and total antioxidant capacity, while decreased malondialdehyde content. These results indicate that subcutaneous injection of the bifunctional recombinant protein GIK can enhance antioxidant enzyme activity in mouse serum and has a protective effect against D-galactose-induced aging in mice.

[0079] The above is a detailed description of the embodiments to facilitate proper understanding and application of the present invention by those skilled in the art. Any improvements or modifications to the technical solutions derived by those skilled in the art based on the present invention, without inventive effort, solely through analysis, analogy, or limited enumeration, should be within the scope of protection defined by the claims.

Claims

1. A bifunctional recombinant protein GIK for regulating glucose and lipid metabolism and anti-aging, characterized in that: It contains three structural units, the N-terminus, the middle segment and the C-terminus. The N-terminus is an optimized human GLP-1 polypeptide, whose amino acid sequence is SEQ ID NO: 1, the middle section is the Fc region of human IgG4, and S010P, F016A and L017A mutations are simultaneously introduced into the Fc region of human IgG4, whose amino acid sequence is SEQ ID NO: 2, The C-terminal is s-klotho polypeptide, whose amino acid sequence is SEQ ID NO: 3, The N-terminus is connected to the middle segment via a linker peptide 1, The middle section is connected to the C-terminus via a linker peptide 2, Wherein, the amino acid sequence of the connecting peptide 1 is SEQ ID NO: 4, The amino acid sequence of the connecting peptide 2 is SEQ ID NO:

5.

2. The bifunctional recombinant protein GIK for regulating glucose and lipid metabolism and anti-aging according to claim 1, characterized in that: in, In order to allow the bifunctional recombinant protein GIK to be normally secreted in mammalian cells, a signal peptide of human serum albumin is introduced into the N-terminus of the bifunctional recombinant protein GIK, and the amino acid sequence of the signal peptide is SEQ ID NO:

7.

3. The bifunctional recombinant protein GIK for regulating glucose and lipid metabolism and anti-aging according to claim 1 or 2, characterized in that: The bifunctional recombinant protein GIK can be secreted and expressed in mammalian cells, and its amino acid sequence is SEQ ID NO:

8.

4. A DNA encoding the bifunctional recombinant protein GIK according to claim 3, characterized in that: The coding DNA sequence is SEQ ID NO: 9 or its codon optimized sequence.

5. A GIK expression vector, characterized in that: The GIK expression vector contains the coding DNA as claimed in claim 4.

6. A GIK host cell, characterized in that The GIK host cell contains the GIK expression vector according to claim 5, or the coding DNA according to claim 4 is integrated into the genome of the GIK host cell, and the GIK host cell is a eukaryotic cell.

7. The GIK host cell according to claim 6, characterized in that The eukaryotic cell is selected from the group consisting of a mammalian cell, a yeast, and a filamentous fungus.

8. A method for preparing the bifunctional recombinant protein GIK for regulating glucose and lipid metabolism and anti-aging according to any one of claims 1 to 3, characterized in that: The steps include: Step S1, constructing the GIK expression vector as claimed in claim 5: placing the coding DNA sequence as claimed in claim 4 into a eukaryotic gene expression regulatory sequence to obtain a GIK expression vector that can be expressed in eukaryotic cells; Step S2, transfecting the GIK expression vector into eukaryotic cells: introducing the GIK expression vector into eukaryotic cells by gene transfection technology to obtain the GIK host cell as claimed in claim 6 or 7; Step S3, culturing the GIK host cells in high-density suspension to express the bifunctional recombinant protein GIK, to obtain a culture containing the bifunctional recombinant protein GIK; Step S4, purifying the bifunctional recombinant protein GIK from the culture containing the bifunctional recombinant protein GIK.

9. The method for preparing the bifunctional recombinant protein GIK for regulating sugar and lipid metabolism and anti-aging according to claim 8, characterized in that: in, The specific process of purifying the bifunctional recombinant protein GIK in step S4 is as follows: Step S4-1, concentrating and filtering the culture containing the bifunctional recombinant protein GIK to obtain a pretreatment solution; Step S4-2, treating the pre-treatment solution by affinity chromatography to obtain an affinity chromatography purified product; Step S4-3, treating the affinity chromatography purified product with ion exchange chromatography to obtain an ion exchange chromatography purified product; Step S4-4, using molecular sieves to remove monomers in the ion exchange chromatography purification product to obtain a dimeric bifunctional recombinant protein GIK.

10. A pharmaceutical preparation, characterized in that The pharmaceutical preparation contains the bifunctional recombinant protein GIK for regulating glucose and lipid metabolism and resisting aging as described in any one of claims 1 to 3.

Images