CN113150172B

CN113150172B - GLP-1R/GIPR double-target agonist fusion protein and preparation method and application thereof

Info

Publication number: CN113150172B
Application number: CN202110465074.2A
Authority: CN
Inventors: 谭树华; 赵雅嫱; 童守芳; 沈家琪
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2023-09-22
Anticipated expiration: 2041-04-28
Also published as: CN113150172A

Abstract

The application discloses a long-acting fusion protein with dual activation activity on a glucagon-like peptide-1 (GLP-1R) receptor (GLP-1R) and a glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR), and a preparation method and application thereof. The fusion protein is formed by sequentially connecting three structural functional domains of exenatide (exendin-4) or a mutant thereof, human glucose-dependent insulinotropic polypeptide (GIP) or a mutant thereof and Fc fragment of human immunoglobulin IgG from N-terminal to C-terminal through connecting peptide (linker) or directly. The application also provides a genetic engineering preparation method for soluble expression of the fusion protein in escherichia coli, which has simple process and is convenient for directly purifying from cell wall-broken liquid to obtain a protein product with biological activity. The fusion protein has obvious double agonist activity on GLP-1R and GIPR, and can be applied to the preparation of medicaments for treating diabetes, obesity, hyperlipidemia and other related diseases.

Description

GLP-1R/GIPR double-target agonist fusion protein and preparation method and application thereof

Technical Field

The application belongs to the biopharmaceutical technology, and particularly relates to a GLP-1R/GIPR double-target agonist fusion protein, a preparation method thereof and application thereof in preparing medicaments for treating diabetes, obesity, hyperlipidemia and other related diseases.

Background

Diabetes mellitus (Diabetes Mellitus, DM) is an endocrine, metabolic disease characterized by hyperglycemia, mainly caused by insulin secretion defects and/or insulin insufficiency. Diabetes mellitus can be classified into Type 1 (Type 1 diabetes mellitus,T1DM) and Type 2 (Type 2 diabetes mellitus,T2DM). Wherein, T1DM does not produce insulin, belongs to insulin-dependent diseases, and T2DM mainly generates insulin resistance because the organism is insensitive to insulin reaction, and accounts for more than 90 percent of diabetics. Diabetes can lead to a variety of fatal complications, such as obesity, cardiovascular disease, kidney disease, etc., which are serious hazards to human health.

Drugs for the treatment of type 2 diabetes mainly include sulfonylureas, biguanides, glinide, thiazolidinediones, α -glucosidase inhibitors, dipeptidylpeptidase IV (DPP-4) inhibitors, glucagon-like peptide-1 (GLP-1R) receptor (GLP-1R) agonists, sodium-glucose cotransporter 2 (SGLT 2) inhibitors, and the like. Wherein, the DPP-4 inhibitor and the GLP-1 receptor agonist have the advantages of reducing blood sugar, rarely causing hypoglycemia, having better safety and tolerance, and the like, and also have the protection effect on multiple systems such as cardiovascular systems, central nervous systems, digestion and the like.

Exenatide (exendin-4) is an exogenous GLP-1R agonist (GLP-1 RAs) found in North America Exendin saliva, consisting of 39 amino acids, has 53% homology with the human incretin GLP-1 amino acid sequence, and functions physiologically in mammals similarly to GLP-1. Since exenatide is insensitive to DPP-4, its in vivo half-life is remarkably prolonged as compared with GLP-1, up to 3.3-4 hours. Exenatide was developed and marketed by amyin and Eli Lilly company in the united states in month 4 2005 and marketed in china in month 8 2009 as the first marketed GLP-1 receptor agonist drug.

GLP-1 receptor agonist drugs are widely applied to the treatment of type 2 diabetes in clinic, and the specific mechanism is that after the GLP-1 receptor agonist drugs are combined with GLP-1 receptors, insulin secretion of islet beta cells can be stimulated, glucagon secretion of islet alpha cells can be inhibited, and the sensitivity of tissues to insulin is enhanced, so that the effect of reducing blood sugar is achieved. The most obvious advantage of the medicine is that the stimulation of insulin secretion has glucose dependency, namely, the medicine only plays a role in high blood sugar concentration and does not play a role in normal or low blood sugar, thus effectively avoiding the occurrence of hypoglycemia phenomenon and further ensuringSafety of clinical medication. Glucose-dependent insulinotropic polypeptide (GIP) is also an incretin, comprising 42 amino acid residues, secreted by K cells in the duodenum and upper jejunum. GIP can bind to GIP receptor (GIPR) on the surface of islet beta cells, promote insulin synthesis and secretion by glucose-dependent means, i.e., stimulate insulin secretion under hyperglycemic conditions, and lower blood glucose. However, GIP induces glucagon production under hypoglycemic conditions, thereby maintaining blood glucose balance (Christensen et al (2011) Diabetes 60:3103-9).P.K. et al report that GIP analog ZP4165 can enhance the hypoglycemic effect and slimming effect of GLP-1 agonist (/ -A)>et al (2018) Diabetes Obes Metab 20:60-68). Finan, B.et al report a single molecule dual insulinotropic (unimolecular dual incretins) polypeptide with coactivation to GLP-1R and GIPR, which showed good insulin secretion and hypoglycemic effects in db/db mice, ZDF rats, monkeys and humans (Finan et al (2013) Sci Transl Med 5:2090151).

Immunoglobulin IgG is one of the most abundant proteins in the blood and has an in vivo half-life of up to 21 days. Thus, human IgGFc fragments (i.e., the hinge and constant regions CH2-CH3 of human IgG) can be fused to other active proteins or polypeptides to obtain long-acting fusion protein drugs with significantly prolonged in vivo half-lives. For example, the Amgen company in the United states of Amgen in 2008 develops the marketed Romipristim (romistin), which is a fusion protein formed by connecting a thrombopoietin receptor (TPO) binding peptide with human IgG1Fc, and the fusion protein not only maintains the function of the TPO binding peptide for activating the Thrombopoietin (TPO) receptor, but also greatly prolongs the in vivo half-life, and is clinically used for treating chronic Immune Thrombocytopenic Purpura (ITP). The fusion protein forms inclusion bodies when expressed in E.coli, the downstream treatment process is complex, and the fusion protein can form soluble protein with biological activity through denaturation and renaturation treatment.

At present, no report related to GLP-1R/GIPR double-target agonist fusion protein is found.

Disclosure of Invention

The application aims to: aiming at the prior art, the application provides a novel agonist fusion protein with double activation activity on human GLP-1R and GIPR and a preparation method thereof, and also provides application of the fusion protein in preparing medicaments for treating diabetes, obesity, hyperlipidemia and other related diseases.

The technical scheme is as follows: the GLP-1R/GIPR double-target agonist fusion protein is formed by sequentially connecting three structural functional domains of exenatide (exendin-4) or a mutant thereof, human glucose-dependent insulinotropic polypeptide (GIP) or a mutant thereof and Fc fragment of human immunoglobulin IgG from N-terminal to C-terminal through connecting peptide (linker) or directly.

The wild exenatide sequence is shown as SEQ ID NO.1, and the mutant is formed by mutating Leu at 21 st position of the wild exenatide into Lys, arg or His, and has an amino acid sequence shown as SEQ ID NO. 2-4 or a sequence with homology of more than 90% with the sequence.

The natural human GIP (1-30) sequence is shown in SEQ ID NO. 5, and the mutant is formed by mutating Ala at the 2 nd position into Gly or Ser, and has the amino acid sequence shown in SEQ ID NO. 6-7 or homology of more than 90% with the sequence.

The Fc fragment of the human immunoglobulin IgG is any one of human IgG1Fc, igG2Fc, igG3Fc or IgG4 Fc.

The connecting peptide is flexible peptide rich in Gly and/or Ala and/or Ser, the length is between 1 and 100 amino acid residues, and the connecting peptide is preferably (Gly-Gly-Gly-Gly-Ser) n, wherein n is an integer of 2 to 10.

The Fc fragment of the human immunoglobulin IgG comprises a hinge region and a constant region CH2-CH3. The hinge region may be a natural or optimally mutated human IgG hinge region. The constant region CH2-CH3 may be the natural human IgG constant region CH2-CH3 or a sequence having more than 90% homology thereto.

The preferred human IgG1 hinge region has the sequence shown in SEQ ID NO. 8, which is mutated from the native human IgG1 hinge region original sequence-VEPKSCDKTHTCPPCP- (SEQ ID NO. 9).

Preferred human IgG1 constant regions CH2-CH3: has the sequence shown in SEQ ID NO. 10 or the sequence with more than 90 percent of homology with the sequence.

Further preferably, the GLP-1R/GIPR dual-target agonist long-acting fusion proteins such as EX-GIP-Fc, EX-L21K-GIP-Fc, EX-L21R-GIP-Fc and EX-L21H-GIP-Fc respectively have the amino acid sequences shown in SEQ ID NO. 11-14 or have more than 90% homology with the sequences.

The application also discloses a preparation method of the GLP-1R/GIPR double-target agonist long-acting fusion protein, which comprises the following steps:

(1) Designing and synthesizing a coding gene of the fusion protein;

(2) After construction into expression plasmid, colibacillus or yeast is transformed or mammalian cell is transfected for expression;

(3) Collecting cell wall-broken liquid or culture solution supernatant, and separating and purifying to obtain soluble target fusion protein.

The application also discloses application of the GLP-1R/GIPR double-target agonist fusion protein in preparing medicaments for treating diabetes, obesity, hyperlipidemia and other related diseases.

The beneficial effects are that: the GLP-1R/GIPR double-target agonist fusion protein provided by the application can activate human GLP-1R and GIPR simultaneously, has the remarkable advantages of high hypoglycemic activity, quick effect and long duration of hypoglycemic effect, is beneficial to reducing the dosage of medicines for obtaining the therapeutic effect, reduces the administration frequency, improves the compliance of medicine treatment, improves the therapeutic effect, and provides a new medicine for treating diabetes, obesity hyperlipidemia and other related diseases. The GLP-1R/GIPR double-target agonist fusion protein can be subjected to soluble expression in E.coli and exists in a soluble dimer form, so that an expression product with bioactivity can be directly obtained by directly separating and purifying from E.coli wall-broken liquid supernatant, and complex downstream denaturation and renaturation treatment processes caused by inclusion body formation are avoided.

Drawings

FIG. 1 is a schematic diagram of the structure of a GLP-1R/GIPR dual-target agonist fusion protein EX-L21K-GIP-Fc, mainly comprising: the initiator methionine Met, exenatide mutant EX-L21K, the connecting peptide (GGGGS) 3, GIP-A2G, the connecting peptide (GGGGS) 2, the mutated human IgG1 hinge region (-DKTHTCPPCP-), the human IgG1 constant region CH2-CH3. The 5 'end of the coding gene is an initiation codon ATG, and the 3' end of the coding gene is two termination codons TAATAG;

FIG. 2 is a GLP-1R/GIPR dual target agonist fusion protein EX-L21K-GIP-Fc expression plasmid map, wherein T7promoter: a T7 promoter; EX-L21K-GIP-Fc: the fusion protein EX-L21K-GIP-Fc coding gene is cloned between NdeI and HindIII cleavage sites; f1 origin: f1 a replication region sequence of phage genomic DNA; kan: kanamycin resistance gene; ori: a plasmid replication origin; lacI: lactose operon repressor coding gene;

FIG. 3 is a Westernblot for detecting expression of GLP-1R/GIPR dual-target agonist long-acting fusion protein EX-L21K-GIP-Fc, wherein the detection antibody is an HRP-labeled anti-human IgG antibody;

FIG. 4 is an FPLC gel chromatography elution profile;

FIG. 5 is a graph showing the results of detection of purified products EX-L21K-GIP-Fc by 12% (w/v) reduced SDS-PAGE (A) and western blot (B);

FIG. 6 is a graph showing the results of analytical SEC-HPLC detection of standard proteins (BSA, chicken ovalbumin, chymotrypsinogen and lysozyme);

FIG. 7 is a graph showing the results of analytical SEC-HPLC detection of fusion protein EX-L21K-GIP-Fc purity and molecular weight;

FIG. 8 is a graph showing the result of analytical RP-HPLC detection of the purity of fusion protein EX-L21K-GIP-Fc;

FIG. 9 is a graph showing the results of activation of HEK293/GLP-1R/CRE4 cells by fusion protein EX-L21K-GIP-Fc;

FIG. 10 is a graph showing the results of activation of HEK293/GIPR/CRE4 cell GIPR by fusion protein EX-L21K-GIP-Fc;

FIG. 11 is a graph of area under the blood glucose-time curve (AUC) results after oral glucose loading at D36 for each group of animals; "+++". Representation of the phase ratio in the normal control group, P <0.001; ", represents P <0.001 compared to model control group;

FIG. 12 is a graph of pancreatic HE staining results.

Detailed Description

The present application will be described in detail with reference to specific examples.

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

Materials, reagents, devices, instruments, equipment and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 design of GLP-1R/GIPR double-target agonist long-acting fusion proteins

The GLP-1R/GIPR double-target agonist long-acting fusion protein structure is shown in figure 1, and sequentially consists of the following polypeptide domains in series from the N end to the C end: high activity exenatide mutant (EX-L21K), gly-rich connecting peptide (GGGGS) 3, human glucose-dependent insulinotropic polypeptide (GIP) mutant (GIP-A2G), gly-rich connecting peptide (GGGGS) 3, optimized mutated human IgG1 hinge region DKTTCPPCP (underlined), human IgG1 constant region CH2-CH3.

Wherein, the high-activity exenatide mutant EX-L21K is obtained by mutating Leu at the 21 st position of wild exenatide into Lys.

The GIP mutant GIP-A2G is obtained by mutating Ala at the 2 nd position of the natural GIP into Gly.

Optimization of the hinge region of mutated human IgG 1: has an amino acid sequence shown as-DKTHTCPP- (SEQ ID NO: 8).

Human IgG1 constant region CH2-CH3: has the sequence shown in SEQ ID NO. 10.

The amino acid sequence of the GLP-1R/GIPR double-target agonist fusion protein EX-L21K-GIP-Fc is shown as SEQ ID NO.12, and the encoding gene sequence is shown as SEQ ID NO. 15.

EXAMPLE 2 cloning and expression of GLP-1R/GIPR double-target agonist long-acting fusion proteins

(1) Construction of recombinant plasmid pET27b-IgG1Fc

The following forward and reverse primers (underlined to represent BamH I and HindIII cleavage sites, respectively) were designed using the laboratory synthesized plasmid containing the human IgG1Fc encoding gene (SEQ ID NO: 16) as a template:

BamHI-IgG1Fc-F：AATTGGATCCGGTGGTGGTGGTTCTGACAAAACCCACACCTGC(SEQ ID NO:17)；

HindIII-IgG1Fc-R：GGCCGCAAGCTTCTATTATTTACCCGGA(SEQ ID NO:18)。

the above primers were synthesized by Nanjing GenScript Biotechnology.

And (3) amplifying by adopting a PCR technology to obtain an IgG1Fc gene fragment, and inserting corresponding enzyme cutting sites of an expression vector pET27b after enzyme cutting BamH I and HindIII to obtain a recombinant plasmid pET27b-IgG1Fc containing the human IgG1Fc coding gene.

(2) Construction of recombinant plasmid pET27b-EX-L21K-GIP-Fc

The gene (SEQ ID NO: 19) encoding the EX-L21K-GIP fusion protein was synthesized and cloned by Shanghai, digested with Nde I and BamH I, subcloned into the above-mentioned plasmid pET27b-IgG1Fc containing the human IgG1Fc gene, thereby constructing plasmid pET27b-EX-L21K-GIP-Fc (FIG. 2) expressing the GLP-1R/GIPR dual-target agonist long-acting fusion protein EX-L21K-GIP-Fc, and after DNA sequencing was correct, caCl ₂ Transformed E.coli BL21 (DE 3) host bacteria were cultured on LBK (LB medium containing 50. Mu.g/mL kanamycin) plates at 37℃and stored in glycerol tubes at-70 ℃.

(3) Shake flask expression of fusion protein EX-L21K-GIP-Fc and product identification

The pET27b-EX-L21K-GIP-Fc/E.coliBL21 (DE 3) engineering bacteria were streaked overnight on LBK plates at 37℃and single colonies were picked up to 30ml LBK liquid medium and shake cultured at 220rpm at 37℃for 8h. Transferring to 50mL TB culture medium (tryptone 1.2% (w/v), yeast powder 2.4% (w/v), glycerol 0.4% (v/v), 17mM KH2PO4, 72mM K2HPO4.3H2O, 50 μg/mL kanamycin) according to 2% (v/v), culturing at 37deg.C with shaking at 220rpm until OD600 is about 1, adding lactose to 1g/L, and inducing expression at 25deg.C at 220rpm for 12-16 h. Simultaneously, pET27b/E.coliBL21 (DE 3) control was established, and shake flask culture and lactose-induced expression were performed under the same conditions.

After the expression was completed, 2mL of the fermentation broth was centrifuged at 4℃at 600 rpm for 10min, the bacterial sludge was collected, and 1mL of 100mM Tris-HCl solution (pH 8.0) was added for resuspension, and the mixture was subjected to 3s gap ultrasonication for 1min under ice bath conditions. The cell disruption solution was centrifuged (12000 rpm,30 min) at 4 ℃. Taking pET27b-EX-L21K-GIP-Fc/E.coliBL21 (DE 3) wall-broken liquid supernatant, precipitating the heavy suspension by the wall-broken liquid, and carrying out 12% SDS-PAGE electrophoresis on the whole fungus wall-broken liquid of the pET27b/E.coliBL21 (DE 3) control group. Then, the proteins were transferred onto PVDF membrane (Roche, cat# 03010040001) at constant pressure of 100V at 4℃for 100 min. The membranes were placed in 5% blocking solution (TBS with 5% skim milk and 0.1% tween 20) and blocked for 2h at room temperature. At 5% blocking solution according to 1: incubation was carried out overnight at 4deg.C in 5000 dilution of HRP-conjugated Rabbit anti-human IgG antibody (cat#D 110149, shanghai). TBST (TBS with 0.1% Tween 20) was washed 5 times, 5min each, and ECL developed. The results are shown in FIG. 3, lane 1: pET27b-EX-L21K-GIP-Fc/BL21 (DE 3) cell wall-breaking supernatant; lane 2: precipitating pET27b-EX-L21K-GIP-Fc/BL21 (DE 3) thallus wall-breaking liquid; lane 3: pET27b/BL21 (DE 3) empty plasmid control cell wall breaking liquid. As can be seen from the figure, the cell wall-broken supernatant of EX-L21K-GIP-Fc expression had a distinct band at a molecular weight of 35kDa (lane 1), whereas cell wall-broken sediment had only a trace of protein at 35kDa (lane 2). In addition, the empty vector pET27b/BL21 (DE 3) control group had no protein band at 35 kDa. This demonstrates that the target protein EX-L21K-GIP-Fc was expressed in a soluble manner under the above expression conditions.

Example 3 fusion protein EX-L21K-GIP-Fc engineering bacterium fermentation, separation and purification

(1) Seed liquid preparation

The glycerol tubes were picked up and subjected to three-fold streak culture on LBK plates 2 times. Single colonies were picked from LBK plates, inoculated with 30mL LBK liquid medium, incubated at 37℃for about 12h at 220rpm, which was the primary seed solution. Transferring the first seed liquid into two 500mL conical flasks containing 100mL LBK liquid culture medium at 37deg.C and 220rpm for 12 hr at 2% (v/v), fermenting at high density,

(2) Fed-batch fermentation

Inoculating 4% (v/v) of the secondary seed solution into 4.5L fermentation medium (yeast powder 2.4% (w/v), tryptone 1.2% (w/v), glycerol 0.4% (v/v), 17mM KH2PO4, 72mM K2HPO4.3H ₂ O, defoamer 0.1% (v/v), sterilizing at 121 ℃ for 20min, sterilizing and coolingKanamycin at a final concentration of 100. Mu.g/mL) was added to the 7L fermenter. Culturing at 37 deg.c, stirring at 230-400 rpm/min, sampling every one hr to detect OD ₆₀₀ . When the culture was carried out to the logarithmic phase (about 3 hours), the temperature of the fermenter was lowered to 25℃and about 10g of lactose was fed into the fermenter by a peristaltic pump, and the protein expression was continuously induced for 9 hours, and the total fermentation period was about 12 hours. Meanwhile, the pH is controlled to be about 7.0, and the dissolved oxygen is controlled to be 10% by adjusting the rotating speed and the ventilation. When the dissolved oxygen and the pH value rise obviously, the glucose in the culture medium is exhausted, the growth of the thalli is slowed down, and the nitrogenous organic matters are used as carbon sources to carry out feeding in a mode of dissolved oxygen and pH feedback.

(3) Cell disruption

The fermentation broth was centrifuged to collect the bacterial sludge, which was resuspended in a 10% (w/v) ratio with 100mM Tris-HCl solution (pH 8.0) and added with arginine solution (pH 8.0) at a final concentration of 0.2M. Cells were broken with an ATS homogenizer (AH 100B, ATS Engineering inc., canada) until the breaking fluid was clear, maintaining the homogenizer pressure at about 600bar during the breaking process. The cell disruption solution was collected, centrifuged (12000 rpm,30 min) at 4℃and the supernatant was collected, filtered through a 0.45 μm filter and then through a 0.22 μm filter.

(4) Protein A column affinity chromatography

Protein A-Agaros packing (Cat #11134515001, roche) was loaded onto the column, washed with water to remove 20% ethanol stock solution, and the column was then equilibrated thoroughly with 100mM Tris-HCl solution (pH 8.0). Loading the filtered supernatant to a well-balanced chromatographic column, and adjusting the loading flow rate to be 1mL/min. After loading, the column was washed with 10mM Tris-HCl solution (pH 8.0) to remove unbound hetero-protein, and the target protein was eluted with 100mM Glycine (pH 3.0), and the protein elution peak was collected by a separate tube. The elution collection solution was immediately added with 10% (v/v) of a neutralization buffer 1M Tris buffer (pH 8.0) and mixed well, and the pH of the sample was adjusted to 7.5-8.0. To maintain protein activity, affinity chromatography was performed in a chromatography cabinet at 4 ℃.

(5) Ultrafiltration concentration

Protein A affinity chromatography the eluate was centrifuged (5500 rpm,30 min) at 4℃and the supernatant was collected. Then, the mixture was subjected to centrifugal ultrafiltration at 5500rpm at 4℃until the mixture was concentrated to about 1mL using an Amicon Ultra-15 10K ultrafiltration tube (Cat#UFC 901096, millipore). 10mM citrate buffer (pH 5.8) was added to the ultrafiltration tube and centrifuged at 5500rpm at 4℃until the volume of the solution in the tube was about 1mL. The citrate buffer was replaced 3 times with the solution. Collecting protein in the ultrafiltration tube, filtering with 0.22 μm filter membrane, and temporarily storing at 4deg.C.

(6) Gel chromatography

The separation and purification were performed on FPLC system (biological Duo-Flow, BIO-RAD) using gel chromatography, the column was Superdex 200increase 10/300 (Cat#17-5715-01, GE). After equilibration of the column with equilibration buffer (10 mM citrate buffer, pH 5.8), 500. Mu.L of sample was loaded for separation at a flow rate of 0.4mL/min and a detection wavelength of 280nm. The elution peak diagram is shown in FIG. 4, and peak 1 is trace amount of target protein polymer; peak 2 is the target protein dimer. Different protein elution peaks were collected according to peak mapping tube. Collecting target protein eluting peak, performing aseptic push filtration on the sample with 0.22 μm filter membrane in an ultra clean bench, subpackaging, and storing in a refrigerator at-80deg.C for a long time.

Samples collected during separation and purification were analyzed by 12% SDS-PAGE and western blot, and the results are shown in FIGS. 5A and 5B, respectively, wherein M: standard protein marker, lane 1: cell wall-broken supernatant, lanes 2-5: protein A column pass through, 10mM Tris-HCl wash, 100mM Gly eluent (tube 1), 100mM Gly eluent (tube 2), lanes 6-7: superdex 200Increate 10/300 molecular sieve column chromatography eluting peak 1 and peak 2.

Example 4 molecular weight detection and purity analysis of fusion protein EX-L21K-GIP-Fc

(1) Fusion protein EX-L21K-GIP-Fc molecular weight detection

Molecular weight measurements were performed on an HPLC system (LC-2010A HT,SHIMADZU Corp.) using the size exclusion method (Size Exclusion Chromatography, SEC), column Shodex PROTEIN KW-802.5 (cat#F 6989000, SHOWA DENKO K.K., japan), mobile phase 20mM phosphate buffer (pH 7.4), containing 0.1M Na ₂ SO ₄ (added according to the requirements of the chromatographic column specification), the flow rate is 0.7mL/min, the detection wavelength is 280nm, the loading amount is 20 mu L, and the purity of the sample is calculated according to an area normalization method.

Analysis was performed using BSA (MW 66.4 kDa), chicken ovalbumin (MW 45 kDa), chymotrypsinogen (MW 25.6 kDa) and lysozyme (MW 14.3 kDa) as standards, obtained by Shanghai Seisakusho Biotech, with retention times of 10.509min,11.216min,12.434min, 13.650 min (FIG. 6A), corresponding retention volumes of 7.63mL,7.85mL,8.70mL,9.56mL, respectively. A standard curve was made according to the molecular weight and retention volume of each standard protein (fig. 6B), resulting in a molecular weight formula lgmw= -0.299ve+4.019.

The average retention time of three injections of fusion protein EX-L21K-GIP-Fc was 10.489min, corresponding to a retention volume of 7.34mL. The molecular weight of the target protein EX-L21K-GIP-Fc calculated according to the above formula is 66.64kDa, which is 2 times the predicted monomer molecular weight of 34.81kDa of the ExPASy website (http:// web. ExPASy. Org/computer_pi /). This demonstrates that the purified target protein EX-L21K-GIP-Fc was present in the form of a soluble dimer.

(2) Fusion protein EX-L21K-GIP-Fc purity detection

Using the SEC-HPLC system described above (HPLC system LC-2010A HT,SHIMADZU Corp., chromatography column ShodexROTEIN KW-802.5. Eluent 20mM phosphate buffer (pH 7.4) +0.1M Na) ₂ SO ₄ The fusion protein EX-L21K-GIP-Fc was detected at a flow rate of 0.7mL/min, a detection wavelength of 280nm and a loading of 20. Mu.L (FIG. 7A), a solvent peak was observed at a retention time of about 14.5min (FIG. 7B), the solvent peak was subtracted, and the purity of the sample EX-L21K-GIP-Fc was 97.24% as calculated by an area normalization method. The average retention time of three injections is 10.489min, and the corresponding retention volume is 7.34mL, so that the target protein EX-L21K-GIP-Fc molecular weight is 66.64kDa.

EX-L21K-GIP-Fc purity was measured on an HPLC system (LC-2010A HT,SHIMADZU Corp) using reverse phase high performance liquid chromatography (RP-HPLC) with a reverse phase column of Symmetry 300 C4 (4.6 mm. Times.250 mm,5 μm) (cat# 186000289, waters). Phase a (0.05% aqueous trifluoroacetic acid) and phase B (0.05% acetonitrile trifluoroacetic acid). The elution was carried out at room temperature gradient for 14min, and the phase B was increased from 30% to 60%. Flow rate: 0.5mL/min; detection wavelength: 280nm; sample loading amount: 10 mu L. A. Detecting fusion protein EX-L21K-GIP-Fc; B. the solvent was detected. The results are shown in FIG. 8A. The solvent peak appeared at about 6min of the retention time, and the purity of the sample EX-L21K-GIP-Fc was 99.65% as calculated by area normalization method by subtracting the solvent peak as shown in FIG. 8B.

Example 5 activation of glucagon-like peptide-1 receptor (GLP-1R) by fusion protein EX-L21K-GIP-Fc

(1) Construction of HEK293/CRE4 cells

To clone a DNA fragment containing 4 non-palindromic CRE (cAMP response element) sequences (Chepurny et al (2007) J Biomol Screen 12:740-6), the following 2 complementary oligonucleotide strands (CRE sequences shown underlined) were synthesized:

CRE4-F：5-CTAGCAGCCTGACGTCCGAGAGCCTGACGTCCGAGAGCCTGACGTCCGAGAGCCTGACG TCCGAGA-3(SEQ ID NO:20)

CRE4-R：5-GATCTCTCGGACGTCAGGCTCTCGGACGTCAGGCTCTCGGACGTCAGGCTCTCGGACGT CAGGCTG-3(SEQ ID NO:21)

10. Mu.l each of the above-mentioned positive strand (100. Mu.M) and negative strand (5 Xannealing buffer (cat#D0251, beyotime Co.) were mixed with 8. Mu.l of ddH2O 2. Mu.l, denatured at 95℃for 5min, and cooled slowly to room temperature (within 2 h) for annealing to obtain a double-stranded DNA fragment containing 4 CREs. In addition, pGL4.26 plasmid (cat#E8441, promega) was double digested with restriction enzymes NheI and Bgl-II, and vector fragments were recovered by agarose electrophoresis. Then, after ligating the 4 CRE-containing DNA fragment with pGL4.26 vector with T4DNA ligase (cat#M0202S, NEB) at 16℃for 2 hours, E.coli DH 5. Alpha. Host bacteria were transformed and plated on ampicillin-resistant LB plate, positive clones were selected, and DNA sequencing verification was performed by Shanghai worker, and the recombinant plasmid was named pGL4.26-CRE.

Human embryonic kidney HEK293 cells (purchased from Shanghai Life sciences institute cell Bank) were cultured in DMEM (cat# 12800-058, gibco) complete medium. 6 well plate cell plating, 10 ⁶ Cells/well, cultured for 18-24 hours. After washing the cells with PBS, 1mL of serum-free and double antibody-free opti-MEM (cat# 31985-062, gibco) was added to each well and the incubation was continued for 1 hour. Mu.l opti-MEM was used to dilute 2.5. Mu.g of plasmid pGL4.26-CRE, after mixing, 7. Mu.l of the transfection reagent lipo3000 transfection reagent (cat#L 3000001, thermosusher) (plasmid w: transfection reagent v=1:3) was added,mixing for 8s, and standing for 10min. The plasmid-transfection reagent mixture was added to the 6-well plate cells and after 6 hours it was replaced with DMEM complete medium for further cultivation for 48 hours. Selection medium containing 50. Mu.g/ml hygromycin B (cat# 31282-04-9, beyotime Co.) was used for pressure screening, medium was changed every 3 days, and wells without transfected plasmid were used as controls for about 2 weeks. Cells were digested and diluted to 5 cells/ml per ml, plated in 96-well plates, 1 cell/200 μl/well. Culturing for 2-3 weeks, transferring to 24-well plate, and transferring to 6-well plate after full growth. Extracting genome, amplifying luciferase gene (Luc) by PCR method, screening HEK293/CRE4 cell positive clone with pGL4.26-CRE plasmid high copy integrated into genome.

(2) Construction of HEK293/GLP-1R/CRE4 cells

The human GLP-1R encoding gene (NM-002062.2) was synthesized by Jin Weizhi Biotechnology. In-Fusion PCR primers were designed using TAKARA software:

GLP-1R-F：5-ACCCAAGCTGGCTAGCATGGCCGGCGCCCCCGGC-3(SEQ ID NO:22)

GLP-1R-R：5-GCTGATCAGCGGTTTAAACTCAGCTGCAGGAGGCCTGG-3(SEQ ID NO:23)

the synthesized human GLP-1R coding gene is taken as a template, and the human GLP-1R coding gene is amplified by a fidelity DNA polymerase PCR and recovered by a gel recovery kit. In addition, pcDNA3.1 (-) plasmid (cat#V 79520, invitrogen) was double digested with NheI and PmeI endonucleases, and vector fragments were recovered. Then, the human GLP-1R encoding gene was cloned into pcDNA3.1 using In-Fusion HD Cloning kit (cat# 638910, takara), and DNA sequencing verified to give pcDNA3.1-GLP1R recombinant plasmid.

The pcDNA3.1-GLP1R plasmid was transfected into HEK293-pGL4.26-CRE4 cells constructed in example 5 (1) using Lipo3000 transfection reagent. After 300. Mu.g/ml G418 (cat# 108321-42-2, biofrox) pressure screening, the positive monoclonal of pcDNA3.1-GLP1R plasmid integrated into the genome was screened out by paving a monoclonal in a 96-well plate, extracting genomic DNA and using the genomic DNA as a template by adopting a method of PCR amplification of G418 resistance gene. The primer sequences required for the G418 resistance gene PCR are as follows:

G418-F：5-CTATTCGGCTATGACTGGGC-3(SEQ ID NO:24)

G418-R：5-AATATCACGGGTAGCCAACG-3(SEQ ID NO:25)

and (3) extracting proteins from positive monoclonals of the screened pcDNA3.1-GLP1R plasmid integrated into genome, performing western blot detection by using an Anti-GLP-1R antibody (cat# 26196-1-AP, protein), and screening out clones with high expression of the GLP1R protein. Then, the mixture was subjected to pressure screening with 300. Mu.g/ml G418 and 50. Mu.g/ml hygromycin for 4 weeks, and then subjected to monoclonal isolation culture in a 96-well plate by a dilution method to obtain HEK293/GLP-1R/CRE4 monoclonal.

On this basis, EK293/GLP-1R/CRE4 cells obtained by the above screening were plated with Beyogold whole black 96-well cell culture plates (CAT#FCP 966, beyotime Co.) at a cell density of 10000 cells/100. Mu.l/well, and cells were treated with 10-fold gradient-diluted (0.0001-10000 nM) exenatide (cat#052143, HPLC purity 99.41, gill Biochemical) for 6 hours, while HEK293/CRE4 cell negative control group was set. By Bright-Lumi ^TM The firefly luciferase reporter gene detection kit (cat#D7170M, beyotime company) is used for performing chemiluminescence detection on a multifunctional enzyme-labeled instrument, and HEK293/GLP-1R/CRE4 clones with the strongest reactivity to exenatide agonists are screened out for subsequent GLP-1R agonist activity detection.

(3) Detection of agonist Activity of fusion protein EX-L21K-GIP-Fc on GLP-1R

10-fold gradient dilutions (0.0001-10000 nM) of exenatide and EX-L21K-GIP-Fc fusion protein drug solutions (calculated as monomer molecular weight for the EX-L21K-GIP-Fc fusion protein) were prepared in DMEM complete medium with 10% FBS.

HEK293/GLP-1R/CRE4 cells were treated with exenatide and EX-L21K-GIP-Fc fusion protein (purity. Gtoreq.95%) as in the drug treatment method described in example 5 (2), and luciferase activity was detected using a Varioskan flash full-wave fluorescence microplate reader. Chemiluminescent signals were read with a read time of 1500ms per well. Experiments were repeated 3 times. Results are expressed as mean ± SEM. Dose-response relationships were analyzed using GraphPad prism.6 software, and dose response curves were fitted using a four parameter equation and EC50 was calculated. The results are shown in FIG. 9 and Table 1, which demonstrate that fusion protein EX-L21K-GIP-Fc has significant activation effect on GLP-1R and activity superior to that of exenatide as positive control drug.

TABLE 1 agonist activity of fusion protein EX-L21K-GIP-Fc on GLP-1R

Example 6 activation of glucose-dependent insulinotropic polypeptide receptor (GIPR) by fusion protein EX-L21K-GIP-Fc

(1) Construction of HEK293/GIPR/CRE4 cells

The human GIPR coding gene (NM-000164.2) was synthesized by Jin Weizhi Biotechnology. In-Fusion PCR primers were designed using TAKARA software:

GIPR-F：5-ACCCAAGCTGGCTAGCGCCACCATGACTACCTCTCCG-3(SEQ ID NO:26)

GIPR-R：5-GCTGATCAGCGGTTTAAACCTAGCAGTAACTTTCCAACTCCCGG-3(SEQ ID NO:27)

the synthesized human GIPR coding gene is used as a template, and the high-fidelity DNA polymerase PCR is used for amplifying the human GIPR coding gene. In addition, double cleavage of pcDNA3.1 (-) plasmid (cat#V 79520, invitrogen) with NheI and PmeI was performed and the vector fragment was recovered. Then, the human GIPR encoding gene was cloned into pcDNA3.1 using In-Fusion HD Cloning kit (cat# 638910, takara) to obtain pcDNA3.1-GIPR recombinant plasmid.

The pcDNA3.1-GIPR plasmid was transfected into HEK293-pGL4.26-CRE4 cells constructed in example 5 (1) as described for pcDNA3.1-GLP1R plasmid transfection method in example 5 (2), and HEK293/GIPR/CRE4 clone having the strongest reactivity to GIP-A2G polypeptide agonist was obtained by screening for subsequent GIPR agonist activity detection.

(2) Detection of agonist Activity of fusion protein EX-L21K-GIP-Fc on GIPR

HEK293/GLP-1R/CRE4 cells were treated with GIP-A2G and EX-L21K-GIP-Fc fusion proteins (purity. Gtoreq.95%) and luciferase activity was assayed using a Varioskan flash full-wavelength fluoromicroplate reader as described in example 5 (2). Chemiluminescent signals were read with a read time of 1500ms per well. Experiments were repeated 3 times. Results are expressed as mean ± SEM. Dose-response relationships were analyzed using GraphPad prism.6 software, and dose response curves were fitted using a four parameter equation and EC50 was calculated. The results are shown in FIG. 10 and Table 2, which demonstrate that fusion protein EX-L21K-GIP-Fc has significant activation on GIPR and activity superior to GIP-A2G positive control.

TABLE 2 agonist activity of fusion protein EX-L21K-GIP-Fc on GIPR

Example 7 in vivo efficacy test of fusion protein EX-L21K-GIP-Fc

(1) Laboratory animals and groups

Male db mice at 5 weeks of age and C57BL/6 background mice (Jiangsu Jiugang Biotech Co., ltd.), C57BL/6 mice were fed normally, db mice were fed high fat feed. The feed is suitable for 1 week, and the administration time is 6 weeks old. Blood is collected from the dbdb rat tail tip to measure blood glucose. The qualified model mice are obtained without fasting blood sugar being more than or equal to 11.6.

The experimental animals were grouped (n=7) as in table 3. The normal control group is C57BL/6 mice, and normal feed is fed. The other groups are db mice and are fed with high-fat feed.

TABLE 3 group dosing of experimental animals

(2) Animal administration

Model control group: d1, D8, D11, D15, D18, D22, D25, D29 are administered once;

positive drug (Exendin-4) group: d1 single administration; D8-D29 is administrated twice a day;

EX-L21K-GIP-Fc group: d1, D8, D11, D15, D18, D22, D25, D29 are administered once;

(3) Weight of body

All animals were weighed 1 time before grouping; d8, D15, D22, D29, D35 were each weighed 1 time and the results (table 4) showed that EX-L21K-GIP-Fc had significant weight loss effects.

Table 4 statistical data of body weight (g)

In comparison with the control group of the model, ^* P＜0.05， ^** P＜0.01

(4) Blood glucose detection

All animals D1 and D29 were assayed for 1 blood glucose each 30min, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 60h, 72h, 96h, 120h, 144h before and after dosing, and the results are shown in tables 5-6, respectively. D9, D16, D23 were each assayed for 1 non-fasting blood glucose (positive control was assayed 1h after the first administration on the day) and the results are shown in table 7. D12, D19, D26 were each assayed for 1 fasting glucose (16 h fasted; positive control group 1h after the first dose on the day) and the results are shown in table 8. The blood taking method comprises the following steps: blood was taken from the mouse tail tip with a surgical blade, the first drop was discarded and the second drop was collected for blood glucose testing. The results show that the EX-L21K-GIP-Fc has remarkable hypoglycemic effect and remarkable long-acting property.

TABLE 5 D1 blood glucose statistics for single administration

/>

In comparison with the control group of the model, ^* P＜0.05， ^** P＜0.01

in comparison with the control group of the model, ^* P＜0.05， ^** P＜0.01

TABLE 6D 29 statistical data on blood glucose levels

In comparison with the control group of the model, ^* P＜0.05， ^** P＜0.01

in comparison with the control group of the model, ^* P＜0.05， ^** P＜0.01

TABLE 7 non-fasting blood glucose statistics

In comparison with the control group of the model, ^* P＜0.05， ^** P＜0.01

table 8 fasting blood glucose statistics

In comparison with the control group of the model, ^* P＜0.05， ^** P＜0.01

(5) Oral glucose tolerance (OGTT) assay

After all animals D36 had measured fasting blood glucose, 1g/kg of glucose solution was administered by gavage, and 15min, 30min, 60min, 90min, 120min of blood glucose were measured after glucose loading.

Calculate the area under the blood glucose-time curve (AUC): auc=1/2 (0min+15 min) ×0.25h+1/2 (15min+30 min) ×0.25h+1/2 (30min+60 min) ×0.5h … …. The results (FIG. 11) show that the model group had significantly impaired glucose tolerance compared to the normal control group. Compared with the model group, the positive control drug Exendin-4 and the double-target fusion protein EX-L21K-GIP-Fc have obvious improvement on db/db mouse glucose tolerance, but the improvement effect of EX-L21K-GIP-Fc is more obvious.

(6) Glycosylated hemoglobin (HbA 1 c)

After the experiment of all animals D36 (the normal control group is D37), isoflurane is used for anesthetizing the lower abdominal aorta, blood is taken for 0.3-0.5 mL, EDTA-2K is anticoagulated, and a German Rogowski P800 full-automatic biochemical analyzer is used for measuring glycosylated hemoglobin (HbA 1 c). The results show that compared with the model group, the double-target fusion protein EX-L21K-GIP-Fc and the positive drug Exendin-4 have obvious HbA1c reducing effect, and the EX-L21K-GIP-Fc is obviously superior to the positive drug Exendin-4 (Table 9).

(7) Blood lipid measurement

After all animals D36 (normal control group is D37) are subjected to experiments, isoflurane is used for anesthetizing the lower abdominal aorta, 0.3-0.5 mL of blood is taken, a procoagulant tube is placed, the blood is centrifuged at 3000rpm for 10min to separate serum, and a German Rogowski P800 full-automatic biochemical analyzer is used for measuring Total Cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol. The results show that the double-target fusion protein EX-L21K-GIP-Fc and the positive drug Exendin-4 have the effects of obviously reducing LDL-C and TC, and the EX-L21K-GIP-Fc is superior to the positive drug Exendin-4. In addition, EX-L21K-GIP-Fc and the positive drug Exendin-4 both had obvious TG-lowering effect (Table 9).

TABLE 9 glycosylated hemoglobin and blood lipid statistics

In comparison with the control group of the model, ^* P＜0.05， ^** p is less than 0.01; in comparison with the Exendin-4 group, ^# P＜0.05， ^## P＜0.01

(8) Pancreatic HE staining

At the end of the experiment, after the pancreatic tissues of the mice are dyed by HE, the results are shown in figure 12, and the normal group C57BL/6 mice have complete islet structure and orderly cell arrangement. The model group db/db mice had severe islet damage and had atrophy and deformation of the islet edges. Compared with the model group, the EX-L21K-GIP-Fc and Exendin-4 have obvious protection effect on the islet injury of the mice with db/db diabetes mellitus after being dosed, and the islet structure and the cell morphology are obviously improved.

Sequence listing

<110> university of Chinese medical science

<120> GLP-1R/GIPR double-target agonist fusion protein and preparation method and application thereof

<141> 2021-04-28

<160> 27

<170> SIPOSequenceListing 1.0

<210> 1

<211> 39

<212> PRT

<213> Gila monster Heloderma suspectum

<400> 1

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 2

<211> 39

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Lys Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 3

<211> 39

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Arg Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 4

<211> 39

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg His Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 5

<211> 30

<212> PRT

<213> human

<400> 5

Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys

20 25 30

<210> 6

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Tyr Gly Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys

20 25 30

<210> 7

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Tyr Ser Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys

20 25 30

<210> 8

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10

<210> 9

<211> 16

<212> PRT

<213> Human

<400> 9

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 10

<211> 217

<212> PRT

<213> Human

<400> 10

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215

<210> 11

<211> 322

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Met His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu

1 5 10 15

Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Tyr Gly Glu Gly Thr Phe Ile Ser Asp

50 55 60

Tyr Ser Ile Ala Met Asp Lys Ile His Gln Gln Asp Phe Val Asn Trp

65 70 75 80

Leu Leu Ala Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

85 90 95

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

100 105 110

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

115 120 125

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

130 135 140

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

145 150 155 160

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

165 170 175

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

180 185 190

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

195 200 205

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

210 215 220

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

225 230 235 240

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

245 250 255

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

260 265 270

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

275 280 285

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

290 295 300

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

305 310 315 320

Gly Lys

<210> 12

<211> 322

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Met His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu

1 5 10 15

Glu Glu Ala Val Arg Lys Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Tyr Gly Glu Gly Thr Phe Ile Ser Asp

50 55 60

Tyr Ser Ile Ala Met Asp Lys Ile His Gln Gln Asp Phe Val Asn Trp

65 70 75 80

Leu Leu Ala Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

85 90 95

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

100 105 110

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

115 120 125

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

130 135 140

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

145 150 155 160

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

165 170 175

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

180 185 190

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

195 200 205

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

210 215 220

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

225 230 235 240

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

245 250 255

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

260 265 270

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

275 280 285

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

290 295 300

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

305 310 315 320

Gly Lys

<210> 13

<211> 322

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

Met His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu

1 5 10 15

Glu Glu Ala Val Arg Arg Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Tyr Gly Glu Gly Thr Phe Ile Ser Asp

50 55 60

Tyr Ser Ile Ala Met Asp Lys Ile His Gln Gln Asp Phe Val Asn Trp

65 70 75 80

Leu Leu Ala Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

85 90 95

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

100 105 110

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

115 120 125

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

130 135 140

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

145 150 155 160

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

165 170 175

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

180 185 190

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

195 200 205

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

210 215 220

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

225 230 235 240

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

245 250 255

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

260 265 270

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

275 280 285

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

290 295 300

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

305 310 315 320

Gly Lys

<210> 14

<211> 322

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

Met His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu

1 5 10 15

Glu Glu Ala Val Arg His Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Tyr Gly Glu Gly Thr Phe Ile Ser Asp

50 55 60

Tyr Ser Ile Ala Met Asp Lys Ile His Gln Gln Asp Phe Val Asn Trp

65 70 75 80

Leu Leu Ala Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

85 90 95

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

100 105 110

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

115 120 125

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

130 135 140

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

145 150 155 160

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

165 170 175

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

180 185 190

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

195 200 205

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

210 215 220

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

225 230 235 240

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

245 250 255

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

260 265 270

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

275 280 285

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

290 295 300

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

305 310 315 320

Gly Lys

<210> 15

<211> 972

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

atgcacggtg aaggtacctt cacctctgac ctgtctaaac agatggaaga agaagcggtt 60

cgtaaattca tcgaatggct gaaaaacggt ggtccgtctt ctggtgcgcc gccgccgtct 120

ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttcttacgg tgaaggtacc 180

ttcatctctg actactctat cgctatggac aaaatccacc agcaggactt cgttaactgg 240

ctgctggctc agaaaggtgg tggtggatcc ggtggtggtg gttctgacaa aacccacacc 300

tgcccgccgt gcccggcgcc ggaactgctg ggtggtccgt ctgttttcct gttcccgccg 360

aaaccgaaag acaccctgat gatctctcgt accccggaag ttacctgcgt tgttgttgac 420

gtttctcacg aagacccgga agttaaattc aactggtacg ttgacggtgt tgaagttcac 480

aacgcgaaaa ccaaaccgcg tgaagaacag tacaactcta cctaccgtgt tgtttctgtt 540

ctgaccgttc tgcaccagga ctggctgaac ggtaaagaat acaaatgcaa agtttctaac 600

aaagcgctgc cggcgccgat cgaaaaaacc atctctaaag cgaaaggtca gccgcgtgaa 660

ccgcaggttt acaccctgcc gccgtctcgt gacgaactga ccaaaaacca ggtttctctg 720

acctgcctgg ttaaaggttt ctacccgtct gacatcgcgg ttgaatggga atctaacggt 780

cagccggaaa acaactacaa aaccaccccg ccggttctgg actctgacgg ttctttcttc 840

ctgtactcta aactgaccgt tgacaaatct cgttggcagc agggtaacgt tttctcttgc 900

tctgttatgc acgaagcgct gcacaaccac tacacccaga aatctctgtc tctgtctccg 960

ggtaaataat ag 972

<210> 16

<211> 837

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

atgcacggtg aaggtacctt cacctctgac ctgtctaaac agatggaaga agaagcggtt 60

cgtaaattca tcgaatggct gaaaaacggt ggtccgtctt ctggtgcgcc gccgccgtct 120

ggtggtggtg gttctggtgg tggtggttct gacaaaaccc acacctgccc gccgtgcccg 180

gcgccggaac tgctgggtgg tccgtctgtt ttcctgttcc cgccgaaacc gaaagacacc 240

ctgatgatct ctcgtacccc ggaagttacc tgcgttgttg ttgacgtttc tcacgaagac 300

ccggaagtta aattcaactg gtacgttgac ggtgttgaag ttcacaacgc gaaaaccaaa 360

ccgcgtgaag aacagtacaa ctctacctac cgtgttgttt ctgttctgac cgttctgcac 420

caggactggc tgaacggtaa agaatacaaa tgcaaagttt ctaacaaagc gctgccggcg 480

ccgatcgaaa aaaccatctc taaagcgaaa ggtcagccgc gtgaaccgca ggtttacacc 540

ctgccgccgt ctcgtgacga actgaccaaa aaccaggttt ctctgacctg cctggttaaa 600

ggtttctacc cgtctgacat cgcggttgaa tgggaatcta acggtcagcc ggaaaacaac 660

tacaaaacca ccccgccggt tctggactct gacggttctt tcttcctgta ctctaaactg 720

accgttgaca aatctcgttg gcagcagggt aacgttttct cttgctctgt tatgcacgaa 780

gcgctgcaca accactacac ccagaaatct ctgtctctgt ctccgggtaa ataatag 837

<210> 17

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

aattggatcc ggtggtggtg gttctgacaa aacccacacc tgc 43

<210> 18

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ggccgcaagc ttctattatt tacccgga 28

<210> 19

<211> 277

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

tacatatgca cggtgaaggt accttcacct ctgacctgtc taaacagatg gaagaagaag 60

cggttcgtaa attcatcgaa tggctgaaaa acggtggtcc gtcttctggt gcgccgccgc 120

cgtctggtgg tggtggttct ggtggtggtg gttctggtgg tggtggttct tacggtgaag 180

gtaccttcat ctctgactac tctatcgcta tggacaaaat ccaccagcag gacttcgtta 240

actggctgct ggctcagaaa ggtggtggtg gatccag 277

<210> 20

<211> 66

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

ctagcagcct gacgtccgag agcctgacgt ccgagagcct gacgtccgag agcctgacgt 60

ccgaga 66

<210> 21

<211> 66

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

gatctctcgg acgtcaggct ctcggacgtc aggctctcgg acgtcaggct ctcggacgtc 60

aggctg 66

<210> 22

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

acccaagctg gctagcatgg ccggcgcccc cggc 34

<210> 23

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

gctgatcagc ggtttaaact cagctgcagg aggcctgg 38

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

ctattcggct atgactgggc 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

aatatcacgg gtagccaacg 20

<210> 26

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

acccaagctg gctagcgcca ccatgactac ctctccg 37

<210> 27

<211> 44

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

gctgatcagc ggtttaaacc tagcagtaac tttccaactc ccgg 44

Claims

1. A GLP-1R/GIPR double-target agonist fusion protein is characterized in that the fusion protein is formed by sequentially connecting three structural functional domains of exendin-4 mutant, human glucose-dependent insulinotropic polypeptide GIP mutant and Fc fragment of human immunoglobulin IgG from N-terminal to C-terminal through a connecting peptide linker, and the specific amino acid sequence of the fusion protein is shown as SEQ ID NO. 12.

2. The GLP-1R/GIPR dual target agonist fusion protein of claim 1, wherein the fusion protein has a coding gene sequence as shown in SEQ ID No. 15.

3. The method for preparing the GLP-1R/GIPR dual target agonist fusion protein according to claim 1, wherein the method comprises the following steps:

(1) Designing, synthesizing and cloning a coding gene of the fusion protein of claim 1;

(2) Constructing an expression plasmid of the fusion protein of claim 1, and transforming the expression plasmid into escherichia coli for expression;

(3) Isolation and purification of the expressed fusion protein of claim 1.

4. The use of the GLP-1R/GIPR dual target agonist fusion protein of claim 1 in the preparation of a medicament for treating diabetes, obesity, hyperlipidemia.