CN111944061B - Glucagon-like peptide-1 analogue monomer, dimer and application thereof - Google Patents
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Abstract
The invention discloses a glucagon-like peptide-1 analogue monomer. The amino acid sequence of the monomer is shown as SEQ ID NO.1, and the fusion protein obtained by fusing two-molecule mutant glucagon-like peptide-1 and human albumin binding domain ABD which are connected in series is named as (mGLP-1) 2 -ABD, represented by the general formula: 6 His-Linker-1-mGLP-1-Linker-2-ABD-Linker-2-Cys; also discloses a glucagon-like peptide-1 analog dimer prepared by the monomer. And the glucagon-like peptide-1 analog monomer and the pharmaceutically acceptable salt thereof or the glucagon-like peptide-1 analog dimer and the pharmaceutically acceptable salt thereof are applied to the preparation of the medicine with the effects of preventing and treating the non-insulin-dependent diabetes mellitus and the complications thereof.
Description
Technical Field
The invention belongs to the technical field of diabetes drugs, and particularly relates to a glucagon-like peptide-1 analog monomer, a dimer thereof and application thereof, in particular to a long-acting glucagon-like peptide-1 analog monomer with an extended half-life period and a dimer thereof, a preparation method of the dimer and application of the monomer and the dimer thereof in diabetes treatment drugs.
Background
According to the latest global Diabetes synopsis (IDF Diabetes Atlas) promulgated by the International Diabetes Federation (IDF) (9 th edition), in 2019, about 4.63 million adults worldwide suffer from Diabetes, with a gross prevalence of 9.3%; it is predicted that by 2030 and 2045 years, diabetic patients will reach 5.784 hundred million and 7.002 hundred million, respectively, with crude prevalence of 10.2% and 10.9%, respectively. Diabetes has become an important disease that endangers human health, including potential diabetes patients who have not been diagnosed, which is about 1.164 million people in China and has a rapidly increasing tendency of incidence.
Glucagon-like peptide-1 (GLP-1) is the second endogenous polypeptide following insulin for treating diabetes, GLP-1 is a polypeptide hormone secreted by L cells of human small intestine, and has two active forms of GLP-1 (7-37) and GLP-1 (7-36) NH 2 Although the contents of both in vivo are different, both have the same physiological activity. The active GLP-1 can be widely applied to a plurality of tissues and organs such as pancreas, liver, kidney, heart, muscle, digestive system, nervous system, adipose tissue and the like to participate in a plurality of physiological functions of human bodies. GLP-1 acts on pancreas, can stimulate the secretion of insulin in a sugar-dependent manner, obviously reduces postprandial blood sugar, and does not cause hypoglycemia; acting on pancreas, it can promote the transcription and synthesis of insulin, promote the proliferation of islet beta-cell, inhibit the apoptosis of islet beta-cell, and inhibit the secretion of glucagon; acting on liver, promoting glycogenolysis, and reducing fatty liver; acting on kidney, and promoting excretion of salt and urine; acting on muscle, and enhancing glucose intake and consumption; acting on heart, improving heart rate and blood pressure, and reducing incidence of cardiovascular diseases; the composition has effects in inhibiting gastrointestinal motility, prolonging gastric emptying, and increasing satiety; acts on fat tissue and promotes fat catabolism. GLP-1 has incomparable advantages in the treatment of type 2 diabetes due to its unique function.
However, in vivo, GLP-1 (7-37) and GLP-1 (7-36) NH 2 The two amino acids His-Ala at the N-terminal end are quickly cut off by DPP-4 enzyme widely distributed in organs and tissues to become inactive GLP-1 (9-37) and GLP-1: (9-36)NH 2 Because the molecular weight is only 3kDa, the GLP-1 is easily cleared by the kidney and is discharged out of the body, the two main metabolic pathways ensure that the half-life period of the GLP-1 is less than 2min, and the clinical application of the GLP-1 is greatly limited.
The GLP-1 in vivo stability is improved, the plasma circulation half-life period is prolonged, and the drug property is improved, so that the GLP-1 in vivo stability and the plasma circulation half-life period are important points and hot spots for research in the field of GLP-1 clinical application. At present, GLP-1 structural mutation and modification, fusion protein technology, slow-control drug delivery system and the like are adopted to prolong GLP-1 in vivo residence time in a few researches. For example, liraglutide and Semaglutide are subjected to molecular modification by coupling palmitic acid and oleic acid, and at an injection site, the two drugs form a polymer under the self-assembly action of amphiphilic fatty acid, and slowly release monomer molecules to enter a blood system. The drug binds to the fatty acid binding site of blood albumin via the fatty acid C chain, thereby greatly reducing renal clearance. Albiglutamide and Dulaglutide are used for degrading DPP-4 by mutating amino acid of key sites, the degradation of DPP-4 is greatly reduced by the steric hindrance effect of fusion protein through fusion with Fc regions of HSA and IgG and increasing the molecular size, meanwhile, HSA and IgG can be specifically combined with a neonatal Fc receptor (FcRn), and through an FcRn-mediated recirculation mechanism, the filtering effect of glomeruli is avoided, so that the half lives of HSA and IgG are respectively 21 days and 19 days. Compared with the site-directed chemical modification, the protein fusion technology does not need additional chemical modification, the product is uniform, the process is simple and mature, the yield is higher, the quality is easy to control, and the metabolite is safe and nontoxic. However, the molecular weight of HSA and IgG is large, the tertiary structure of protein is complex, and the correct renaturation of protein is a challenge, particularly, HSA has 35 cysteines to form 17 pairs of disulfide bonds, and the correct renaturation of protein makes the purification conditions complex and harsh, the difficulty is large, and the yield is low.
Disclosure of Invention
The invention aims to provide a glucagon-like peptide-1 analogue monomer.
The invention also aims to provide a glucagon-like peptide-1 analogue dimer.
The last purpose of the invention is to provide the application of the glucagon-like peptide-1 analogue monomer and the pharmaceutically acceptable salt thereof or the glucagon-like peptide-1 analogue dimer and the pharmaceutically acceptable salt thereof in preparing the medicines with the effects of preventing and treating the non-insulin-dependent diabetes mellitus and the complications thereof.
The first object of the present invention can be achieved by the following technical solutions: a glucagon-like peptide-1 analogue monomer, the amino acid sequence of which is shown in SEQ ID NO.1, the monomer is a fusion protein obtained by fusing serially connected mutant glucagon-like peptide-1 with a human albumin binding domain ABD, and the monomer is named as (mGLP-1) 2 -ABD, said monomer (mGLP-1) 2 -ABD composition is represented by the general formula:
6His—Linker-1—mGLP-1—Linker-2—mGLP-1—Linker-2—ABD—Linker-2—Cys；
wherein:
6His is a histidine purification tag, and the amino acid sequence of the histidine purification tag is HisHisHisHisHisHis;
linker-1 is connecting peptide-1, is hydrolysis site of thrombin and DPP-4 enzyme, and has amino acid sequence LVPRTP;
mGLP-1 is a mutant obtained by substituting Gly for Ala at position 2 of GLP-1 (7-37), and the amino acid sequence of the mutant is as follows:
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG；
linker-2 is connecting peptide-2, and the amino acid sequence is GGGGSGGGGSGGGGSA;
ABD is a Human Serum Albumin (HSA) binding peptide having the amino acid sequence:
LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP；
cys is cysteine.
GLP-1 referred to in the present invention is GLP-1 (7-37).
The glucagon-like peptide-1 analogue monomer is a mutant mGLP-1 obtained by substituting Gly for Ala at position 2 and Arg at position 30 of GLP-1 (7-37), and further inhibits degradation of GLP-1 by endogenous protease by mutating amino acid at key sites.
In addition, HAS is the most abundant protein in plasma, accounting for about 50% of total plasma protein, and HAS a molecular weight of 65kDa. HAS binds Fc receptors (FcRn) in a pH-dependent manner, avoiding lysosomal degradation through FcRn-mediated recycling mechanisms, thereby extending its half-life to 22d. HAS HAS a flexible and versatile structure, can bind many endogenous and exogenous small molecule compounds, fatty acids, polypeptides, proteins, etc., and is used as a drug delivery vehicle in vivo due to its good circulatory stability.
The Albumin Binding Domain (ABD) of the present invention is reversibly non-covalently bound to HAS, and the probability of clearance by proteases and kidneys is reduced by the steric hindrance of HAS.
The ABD of the present invention is derived from a partial sequence in the surface protein G of group C streptococci, which are bound to human HAS via the ABD domain, thereby avoiding clearance by the immune system. ABD was discovered by Olsson in 1987, and Jonsson in 2008 mutated 6 amino acids by genetic engineering, so that the affinity of the ABD with human albumin is greatly improved. The ABD in the invention is an optimized sequence, and the amino acid sequence of the ABD is as follows: LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP.
Therefore, the inventor of the present application finds that the strategy for prolonging the half-life of the glucagon-like peptide-1 analogue monomer mainly focuses on the following three aspects according to the metabolic characteristics of GLP-1: (1) Mutating a key site amino acid, particularly Ala at position 2, to inhibit degradation of GLP-1 by endogenous proteases; (2) Form fusion protein or form compound with macromolecule in vivo, produce the steric hindrance effect, reduce the elimination of the kidney; (3) by increasing molecular weight, glomerular filtration is reduced.
Moreover, compared with Fc region fusion proteins of HSA and IgG, the invention selects the polypeptide with smaller molecular weight to prepare the GLP-1 fusion protein, which can increase the molecular weight and the molecular size, and simultaneously, the polypeptide has simple structure and no renaturation trouble, so the process for preparing the GLP-1 fusion protein by using the polypeptide is simple and the yield is high.
The glucagon-like peptide-1 analogue monomer in the application is a fusion protein obtained by fusing serially connected mutant glucagon-like peptide-1 and a human albumin binding domain ABD, the half-life period of the glucagon-like peptide-1 analogue monomer can be prolonged through the three modes, and the glucagon-like peptide-1 analogue monomer can be further applied to medicines with effects of preventing and treating non-insulin-dependent diabetes mellitus and complications thereof, and has longer half-life period and better treatment effect compared with the traditional GLP-1.
The invention also provides a gene for coding the glucagon-like peptide-1 analogue monomer, and the nucleotide sequence of the gene is shown in SEQ ID NO.2.
The invention also provides a recombinant expression vector which comprises the gene.
Further, the recombinant expression vector is a recombinant prokaryotic expression vector.
As a preferred embodiment, the prokaryotic expression vector is pET30a, and the recombinant expression vector is pET30a- (mGLP-1) 2 -ABD。
The invention also provides recombinant expression engineering bacteria, which comprise the genes.
Further, the expression engineering bacterium is escherichia coli BL21 (DE 3).
The second objective of the present invention is achieved by the following technical solutions: a glucagon-like peptide-1 analog dimer, said dimer consisting of the monomer (mGLP-1) 2 -ABD are formed by disulfide bond linkage, said glucagon-like peptide-1 analog dimer being designated [ (glp-1) 2 -ABD] 2 。
The invention also provides a preparation method of the glucagon-like peptide-1 analogue monomer, which comprises the following steps:
(1) Glucagon-like peptide-1 analog monomer (mGLP-1) obtained by SOE-PCR amplification 2 The ABD gene is used for carrying out enzymolysis on the monomer gene and the expression vector by using two restriction enzymes respectively, and then the two restriction enzymes are connected by using ligase to form a recombinant expression vector containing the glucagon-like peptide-1 analogue monomer nucleotide sequence;
(2) Transforming the recombinant expression vector with the correct sequence in the step (1) into an expression host cell for induced expression;
(3) Purified glucagon-like peptide-1 analog monomer (mGLP-1) 2 -ABD。
Further, the preparation method of the glucagon-like peptide-1 analogue dimer comprises the following steps:
(1) Glucagon-like peptide-1 analog monomer (mGLP-1) obtained by SOE-PCR amplification 2 The ABD gene is used for carrying out enzymolysis on the monomer gene and the expression vector by using two restriction enzymes respectively, and then the two restriction enzymes are connected by using ligase to form a recombinant expression vector containing the glucagon-like peptide-1 analogue monomer nucleotide sequence;
(2) Transforming the recombinant expression vector with the correct sequence in the step (1) into an expression host cell for induced expression;
(3) Purified glucagon-like peptide-1 analog monomer (mGLP-1) 2 -ABD；
(4) Oxidized glucagon-like peptide-1 analog monomer (mGLP-1) 2 ABD to obtain a disulfide-linked glucagon-like peptide-1 analog dimer [ (mGLP-1) 2 -ABD] 2 。
In the preparation method of the glucagon-like peptide-1 analogue monomer and the glucagon-like peptide-1 analogue dimer:
preferably, the expression vector in step (1) is pET30a, the two restriction enzymes are Nde I and Xho I, respectively, and the recombinant expression vector is pET30a- (mGLP-1) 2 -ABD。
Thus, further, step (1) comprises: glucagon-like peptide-1 analog monomer (mGLP-1) obtained by SOE-PCR amplification 2 -the gene of ABD, said monomer (mGLP-1) 2 The ABD gene and pET30a vector are subjected to double enzyme digestion by Nde I and EcoR I respectively, and then are connected by T4 DNA ligase to construct a recombinant expression vector pET30a- (mGLP-1) 2 ABD, recombinant expression vector pET30a- (mGLP-1) 2 And converting ABD to DH5 alpha, preliminarily screening positive clones in LB solid culture medium containing kanamycin, selecting single clones to perform PCR and Nde I and EcoR I double-enzyme digestion identification, and identifying positive clones to perform DNA sequencing.
Preferably, the expression host cell described in step (2) is Escherichia coli BL21 (DE 3).
Therefore, further, the step (2) comprises: and (2) converting the positive clone with the correct sequencing in the step (1) into an expression host bacterium BL21 (DE 3), screening the positive clone by using an LB solid culture medium containing kanamycin, selecting a positive monoclonal to prepare a fresh culture solution, transferring the fresh culture solution to an LB liquid culture medium according to the proportion of 1:100, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression when a flora enters a logarithmic phase, inducing for about 6-8 hours, and collecting thalli.
Preferably, the purification in step (3) comprises: (3.1) centrifugation to collect the cells, ultrasonic lysis of cell release (mGLP-1) 2 -an ABD fusion protein; (3.2) Ni-NTA affinity chromatography purification, removing the background protein of the host bacteria; (3.3) alignment of glucagon-like peptide-1 analog monomer (mGLP-1) with C8 column 2 -desalting and purifying the crude ABD product.
Therefore, further, the step (3) includes: centrifuging the fermentation product, collecting thallus, washing and precipitating with phosphate buffer solution for 2 times, suspending thallus with urea-containing phosphoric acid buffer solution (pH8.0), ultrasonic crushing, centrifuging, and collecting supernatant; purifying with Ni-NTA affinity chromatography to obtain glucagon-like peptide-1 analog monomer (mGLP-1) 2 -crude ABD product; desalting with Sep-Pak C8 solid phase extraction column, rotary evaporating for concentration, freeze drying, collecting part of lyophilized powder, further removing impurities by HPLC, refining, concentrating, freeze drying to obtain high purity glucagon-like peptide-1 analog monomer (mGLP-1) 2 -ABD。
Preferably, the glucagon-like peptide-1 analogue monomer (mGLP-1) is oxidized by hydrogen peroxide or DMSO in the step (4) 2 ABD formation of glucagon-like peptide-1 analog dimer [ (mGLP-1) 2 -ABD] 2 。
Therefore, further, the step (4) includes: taking the concentrated solution in the step (3), and adding H 2 O 2 Or DMSO, shaking at 20 deg.C for 12h, oxidizing Cys in monomer with air to generate intermolecular disulfide bond, to obtain glucagon-like peptide-1 analog dimer [ (mGLP-1) 2 -ABD] 2 Further removing impurities by HPLC, refining, concentrating, and freeze drying to obtain high purity glucagon-like peptide-1 analog dimer [ (mGLP-1) 2 -ABD] 2 。
The second object of the present invention is achieved by the following technical solutions: the glucagon-like peptide-1 analog monomer and the pharmaceutically acceptable salt thereof or the glucagon-like peptide-1 analog dimer and the pharmaceutically acceptable salt thereof are applied to the preparation of the medicine with the effects of preventing and treating the non-insulin-dependent diabetes mellitus and the complications thereof.
The non-insulin dependent diabetes mellitus and the complications thereof are type II diabetes mellitus, impaired glucose tolerance, weight loss and metabolic syndrome.
Compared with the prior art, the invention has the following advantages:
(1) Compared with natural GLP-1, the glucagon-like peptide-1 analogue monomer (mGLP-1) 2 -dimer of ABD and glucagon-like peptide-1 analog [ (mGLP-1) 2 -ABD] 2 The mGLP-1 in the polypeptide mutates amino acid of a key site, inhibits the degradation of DPP-4 and slows down the metabolism speed of the DPP-4 in a blood circulation system;
(2) Compared with natural GLP-1, the aims of the series connection of mGLP-1, the fusion with ABD and the monomer polymerization to generate dimer are to increase the molecular weight and the molecular size of the GLP-1 analogue related by the invention and reduce the filtering effect of glomerulus;
(3) In contrast to native GLP-1, the fusion protein formed in the present invention (mGLP-1) 2 -ABD、[(mGLP-1) 2 -ABD] 2 The fusion protein-HSA complex can be formed by reversibly and non-covalently binding ABD and serum albumin HSA in blood to form the fusion protein-HSA complex, and then the fusion protein-HSA complex is prevented from clearing the kidney by virtue of an FcRn-mediated recycling mechanism through the specific binding of the HSA to a neonatal Fc receptor (FcRn);
(4) The invention modifies and modifies the molecular structure of GLP-1 through the three ways, so that the glucagon-like peptide-1 analogue monomer (mGLP-1) 2 The half-life of ABD in SD rat is improved to 8.2h, and the half-life of glucagon-like peptide-1 analogue dimer in SD rat is improved to 15.6h;
(5) The glucagon-like peptide-1 analog monomer (mGLP-1) of the present invention 2 -dimer of ABD and glucagon-like peptide-1 analog [ (mGLP-1) 2 -ABD] 2 Sugar on sugarThe obvious hypoglycemic effect is shown in an Oral Glucose Tolerance Test (OGTT) of a urine disease model rat, and the diabetes mellitus oral glucose tolerance test has advantages and potential to be applied to clinical treatment of diabetes mellitus.
(6) Compared with Fc region fusion protein of HSA and IgG, the present invention has increased molecular weight and molecular size, and the polypeptide has simple structure and no renaturation, so that the present invention has simple technological process and high yield of GLP-1 fusion protein.
Drawings
FIG. 1 shows glucagon-like peptide-1 analog monomer (mGLP-1) of example 1 2 -dimer of ABD and glucagon-like peptide-1 analog [ (mGLP-1) 2 -ABD] 2 A schematic structural diagram of (a);
FIG. 2 is a graph showing the results of PCR of E.coli DH5a transformed with pET30a-m (GLP-1) 2-ABD recombinant plasmid in example 2 to form a single clone;
FIG. 3 is a diagram showing the results of the single-cloning double-digestion of E.coli DH5a transformed with pET30a-m (GLP-1) 2-ABD recombinant plasmid in example 2;
FIG. 4 shows glucagon-like peptide-1 monomer m (GLP-1) of example 2 2 -an electropherogram of ABD;
FIG. 5 shows glucagon-like peptide-1 dimer [ (mGLP-1) of example 2 2 -ABD] 2 Time-of-flight mass spectrogram of (a);
FIG. 6 shows m (GLP-1) in example 2 2 -a chromatogram of ABD monomers;
FIG. 7 is example 2[m (GLP-1) 2 -ABD] 2 A chromatogram of the dimer;
FIG. 8 is a chromatogram of serum albumin of example 2;
FIG. 9 shows serum albumin and m (GLP-1) of example 2 2 -chromatograms after ABD action;
FIG. 10 shows serum albumin and [ m (GLP-1) of example 2 2 -ABD] 2 Chromatograms after dimer action;
FIG. 11 is the plasma concentration-time profile of the drug in example 3;
fig. 12 is a graph showing the results of oral glucose tolerance (OGTT) in example 3.
Detailed Description
The foregoing is a summary of the present invention, and the following is a detailed description of the preferred embodiments of the invention with reference to the accompanying drawings.
Example 1
According to the metabolic characteristics of GLP-1 in vivo, the invention carries out modification and modification on the GLP-1 from the following aspects: on the premise of ensuring the biological activity, mutating key amino acid of a protease DPP-4 action site, and substituting Ala at the 2 nd position with Gly to obtain a GLP-1 mutant mGLP-1; mGLP-1 is connected in series and fused with serum albumin binding peptide ABD, the molecular weight is increased, the clearance of kidney is reduced, meanwhile, the fusion protein is non-covalently combined with serum albumin HAS through ABD to form a compound, and the filtering effect of glomerulus is avoided by utilizing an FcRn-mediated recycling mechanism.
In the invention, in order to ensure the formation of the natural structures of the mGLP-1 and the ABD and the exertion of the efficacy, the serially connected mGLP-1 and ABD are connected through the flexible connecting peptide Linker-2, so that the natural structures of the functional parts are kept while the functional parts keep certain spaces. Cys at the end of the fusion monomer is used to form intermolecular disulfide bonds, linking the fusion protein monomers to form homodimers.
Therefore, the glucagon-like peptide-1 analogue monomer constructed in the embodiment has the amino acid sequence shown in SEQ ID NO.1, is a fusion protein obtained by fusing serially connected mutant glucagon-like peptide-1 with human albumin binding domain ABD, and is named as (mGLP-1) 2 ABD, this monomer (mGLP-1) 2 -ABD composition is represented by the general formula:
6His—Linker-1—mGLP-1—Linker-2—mGLP-1—Linker-2—ABD—Linker-2—Cys；
wherein:
6His is a histidine purification tag, and the amino acid sequence of the histidine purification tag is HisHisHisHisHisHis;
linker-1 is connecting peptide-1, is hydrolysis site of thrombin and DPP-4 enzyme, and has amino acid sequence LVPRTP;
mGLP-1 is a mutant obtained by substituting Gly for Ala at position 2 of GLP-1 (7-37), and the amino acid sequence of the mutant is as follows:
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG；
linker-2 is connecting peptide-2, and the amino acid sequence is GGGGSGGGGSGGGGSA;
ABD is a Human Serum Albumin (HSA) binding peptide having the amino acid sequence:
LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP；
cys is cysteine.
Example 2
The glucagon-like peptide-1 analogue monomer in the example 1 is adopted to construct glucagon-like peptide-1 analogue dimer, and the specific process comprises the following steps:
1. pET30a-m (GLP-1) 2 Construction of the ABD recombinant plasmid
pET30a-m(GLP-1) 2 Construction of the ABD recombinant plasmid comprises four steps:
SOE-PCR amplification (mGLP-1) 2 -ABD fusion gene
(mGLP-1) 2 507bp of ABD fusion gene, and obtaining the complete sequence of the fusion gene by three steps of SOE-PCR by using 3 pairs of overlapped complementary primers.
(1) First step SOE-PCR
According to (mGLP-1) 2 -amino acid sequence of ABD fusion protein, using E.coli preferred codons to design overlapping complementary primers P1 and P2, the primer sequences are as follows:
upstream primer P1 (SEQ ID NO. 3)
5’-GCGCCATATGCACCATCACCATCACCACCTGGTGCCACGCACTCCACACGGTGAAGGTACCTTCACTTCCGACGTTTCCTCTTACCTGGAAGGTCAGGCTGCAAAAGAATTTATCGCTTGGCTG-3’
Downstream primer P2 (SEQ ID NO. 4)
5’-AACGTCGGAAGTGAAGGTACCTTCACCGTGAGCGGAGCCTCCACCACCCGATCCACCGCCACCAGAGCCACCTCCTCCGCCACGACCTTTAACCAGCCAAGCGATAAATTCTTTTGC-3’
The bold inclined part at the 5' end of the upstream primer P1 is an Nde I restriction site, and the complementary primers P1 and P2 are used for amplification by SOE-PCRIncreasing yield (mGLP-1) 2 -nucleotide sequence of amino acid sequence 1-69 of the N-terminus of the ABD fusion protein.
And (3) PCR reaction system: the primers P1 and P2 each 2. Mu.L, 10 XH Buffer 5.0. Mu.L, dNTP 5.0. Mu.L, taq enzyme 1.0. Mu.L, ddH was added 2 0 to 50. Mu.L.
PCR reaction procedure: pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, extension at 72 ℃ for 30s, running for 30 cycles, and final extension at 72 ℃ for 5min.
(2) Second step SOE-PCR
Adopting the preferred codon of Escherichia coli, designing overlapping complementary primers P3 and P4, wherein the primer sequences are as follows:
upstream primer P3 (SEQ ID NO. 5)
5’-GGCTCCGCTCACGGTGAAGGTACCTTCACTTCCGACGTTTCCTCTTACCTGGAAGGTCAGGCTGCAAAAGAATTTATCGCTTGGCTGGTTAAAGGTCGT-3’
Downstream primer P4 (SEQ ID NO. 6)
5’-GTTAGCTAAGACTTTAGCTTCAGCTAATGCCGAACCACCGCCGCCAGAACCGCCACCGCCCGAACCTCCACCTCCGCCACGACCTTTAACCAGCCAAGCGAT-3’
Using PCR product of the first step as template, and using P1, P3 and P4 primers to amplify to obtain (mGLP-1) 2 -nucleotide sequence of amino acid sequence 1-115 of the N-terminus of the ABD fusion protein.
And (3) PCR reaction system: the first step PCR product 0.5. Mu.L, each of the primers P1, P3 and P4 2. Mu.L, 10 XH Buffer 5.0. Mu.L, dNTP 5.0. Mu.L, taq enzyme 1.0. Mu.L, add ddH 2 0 to 50. Mu.L.
PCR reaction procedure: pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, extension at 72 ℃ for 30s, running for 30 cycles, and final extension at 72 ℃ for 5min.
(3) Third step SOE-PCR
Adopting the preferred codon of Escherichia coli, designing overlapping complementary primers P5 and P6, wherein the primer sequences are as follows:
upstream primer P5 (SEQ ID NO. 7)
5’-TCGGCATTAGCTGAAGCTAAAGTCTTAGCTAACAGAGAACTTGACAAATATGGAGTAAGTGACTTCTACAAGCGCC TAATCAACAA GGCCAAAACTGTTGAAGGTG TAGAAGCA-3’
Downstream primer P6 (SEQ ID NO. 8)
5’-GCGCGAATTCTTAGCAGGCCGAGCCGCCTCCTCCGGAACCTCCGCCACCAGATCCGCCTCCGCCAGGTAATGCAGCTAAAATATGCAGCTTCAGTGCTTCTACACCTTCAACAGTTTTGGCCTT-3’
The 5' end of the downstream primer P6 is provided with a bold inclined part which is an Xho I enzyme cutting site, a PCR product of the second step is taken as a template, and the downstream primer P6 is amplified by using primers P1, P5 and P6 to obtain (mGLP-1) 2 -ABD fusion protein complete gene nucleotide sequence SEQ ID No.2.
And (3) PCR reaction system: the second step PCR product 0.5. Mu.L, each of the primers P1, P5 and P6 2. Mu.L, 10 XH Buffer 5.0. Mu.L, dNTP 5.0. Mu.L, taq enzyme 1.0. Mu.L, add ddH 2 0 to 50. Mu.L.
PCR reaction procedure: pre-denaturation at 95 ℃ for 4min, denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, extension at 72 ℃ for 30s, running for 30 cycles, and final extension at 72 ℃ for 10min.
The obtained PCR product is purified by a PCR product purification kit for later use.
2.(mGLP-1) 2 Double digestion of the-ABD fusion protein Gene, plasmid vector
Will (mGLP-1) 2 Carrying out double enzyme digestion on a PCR product of the ABD fusion protein, wherein the reaction system is as follows:
| fusion protein PCR product | 1μg |
| 10×Buffer | 3μL |
| Nde I | 2μL |
| EcoR I | 2μL |
| ddH20 | To 30 μ L |
The reaction conditions of the reaction system are as follows: react for 2h at 37 ℃. And after the reaction is finished, purifying the PCR cleaning kit for later use.
Double enzyme digestion of pET30a vector, the reaction system is as follows:
| pET30a vector | 1μg |
| 10×H Buffer | 3μL |
| Nde I | 2μL |
| EcoR I | 2μL |
| ddH20 | To 30 μ L |
The reaction conditions of the above reaction system are as follows: react for 2h at 37 ℃. And after the reaction is finished, purifying the PCR cleaning kit for later use.
3. Ligation of fusion protein Gene and plasmid vector
The system of the ligation reaction is as follows:
| pET30a vector | 1μg | |
| m(GLP-1) 2 | 2μL | |
| 10×Ligation Buffer | 2μL | |
| T4 ligase | 2μL | |
| ddH20 | To 20 μ L |
The reaction conditions are as follows: reacting for 12-16h at 16 ℃. Finally obtaining pET30a/m (GLP-1) 2 -ABD recombinant plasmid.
4.pET30a-m(GLP-1) 2 ABD recombinant plasmid transformation of E.coli DH5a
Recombinant plasmid pET30a/m (GLP-1) 2 Adding ABD into competent Escherichia coli DH5a suspension, ice-cooling for 30min, transferring into 42 deg.C water bath, maintaining for 90s, immediately transferring into ice bath, and standing for 5min. 0.5mLLB medium was added thereto, and cultured at 37 ℃ for 30min, and 50. Mu.L of the above-mentioned bacterial suspension was applied uniformly to LB solid medium containing 100. Mu.g/mL kanamycin. Shaking culture is carried out at 37 ℃ until colonies visible to the naked eye grow. Kan resistant monoclonals are picked and subjected to PCR and double enzyme digestion identification, the PCR result is shown in figure 2, and specific primers P1 and P6 can be used for amplifying bands with the sizes consistent with those of 530bp bands in positive clones. The positive clone was digested with Nde I and EcoR I, and the result is shown in FIG. 3, which also gives a single band of about 530bp, similar to the size of the target band. The results show that m (GLP-1) 2 The cDNA of the ABD fusion protein has been inserted directionally into the pET30a vector. DNA assay of positively identified clonesSequence, further confirmation of pET30a/m (GLP-1) 2 -ABD recombinant plasmid base sequence.
2. Glucagon-like peptide-1 monomer m (GLP-1) 2 Inducible expression of ABD
Extracting recombinant plasmid pET30a-m (GLP-1) from positive DH5a recombinant bacteria with correct identification 2 ABD, transformed into Escherichia coli BL21 (DE 3) according to the method (4) in the step (a), to obtain a recombinant expression strain. The overnight cultured recombinant expression bacteria were inoculated into 2L of LB liquid medium containing 100. Mu.g/mL kanamycin at a ratio of 1 600nm And (4) =0.5-0.6, adding IPTG (isopropyl-beta-D-thiogalactoside) to a final concentration of 0.6mmol/L, performing induced expression for 6-8h, finally centrifuging at 4 ℃ for 12000r/min for 20min, and collecting thalli. 10mg of the cells were added to 100. Mu.L of 1 XSDS loading buffer, and subjected to a boiling water bath for 5min, and 8. Mu.L of the treated sample was subjected to SDS-PAGE, and the results are shown in FIG. 4, in which lane 1 is a protein marker, lane 2 is a positive clone without an inducer, lane 3 is a positive clone with 0.6M IPTG inducer, and it can be seen that BL21 (DE 3) host cells expressed M (GLP-1) with a molecular weight of about 16.4kDa under the induction of IPTG 2 -an ABD fusion protein.
3. Glucagon-like peptide-1 monomer m (GLP-1) 2 -separation and purification of ABD
Lysis was performed with lysis buffer (400 mmol/L NaCl,50mmol/L NaH) 2 PO 4 10mmol/L imidazole, pH 8.0) suspending the collected cells by centrifugation; performing ultrasonic crushing for 10min on ice bath; centrifuging at 4 deg.C and 12000r/min for 20min, collecting thallus, and collecting supernatant; purifying the supernatant with Ni-NTA affinity resin, loading the sample on a column, washing with lysis solution containing 10mmol/L imidazole to remove impure protein, and eluting with lysis solution containing 250mmol/L imidazole to obtain target protein m (GLP-1) 2 -ABD。
M (GLP-1) 2 Desalting the eluate of the ABD fusion protein with WAT054525C8 column, comprising the steps of: the column was equilibrated with 20mL of a washing solution (5% acetonitrile in water, pH 3.5), and the column was loaded, 50mL of the washing solution was washed with salts and impurities, and 150mL of an eluent (40% acetonitrile in water, pH 3.5) was used to elute the target protein m (GLP-1) 2 -ABD; concentrating the eluate by rotary evaporation, and freeze drying.
Taking part of the dry powder to perform reversed phase HPLC refining, comprising the following steps: dissolving m (GLP-1) in a washing solution (5% acetonitrile in water, pH 3.5) 2 ABD dry powder, supersil SB-C8 column (200x4.6mm, 5um) after being equilibrated with washing solution, loading, washing with washing solution until the baseline is stable, eluting with eluent (40% acetonitrile in water, pH 3.5) at a flow rate of 1mL/min for eluting protein m of interest (GLP-1) 2 ABD, concentrating the eluent by rotary evaporation, and freeze-drying.
4. Glucagon-like peptide-1 dimer [ (mGLP-1) 2 -ABD] 2 Preparation of
Glucagon-like peptide-1 m (GLP-1) prepared in the step (three) 2 ABD was dissolved at 0.1mg/mL in 5%H 2 O 2 And 5% DMSO by shaking at room temperature to promote disulfide bond formation, oxidizing for 4h, concentrating by rotary evaporation, and freeze drying to obtain [ (mGLP-1) 2 -ABD] 2 The molecular weight of the fusion protein was analyzed by time-of-flight mass spectrometry (Bruker) from the dried powder, and the results are shown in FIG. 5, [ (mGLP-1) 2 -ABD] 2 The molecular weight of (A) is 32778, which is consistent with the theoretical molecular weight.
5. Glucagon-like peptide-1 monomer m (GLP-1) 2 -ABD and its dimer [ m (GLP-1) 2 -ABD] 2 Determination of affinity for human serum Albumin
Using 0.1mol/L phosphate buffer solution (containing 0.1mol/L Na) 2 SO 4 pH7.4) was prepared in the serum albumin solution at a concentration of 0.1mmol/L and m (GLP-1) at a concentration of 1. Mu. Mol/L, respectively 2 ABD monomer solution, 1. Mu. Mol/L of [ m (GLP-1) 2 -ABD] 2 Dimer solution and 0.1mmol/L human serum albumin + 1. Mu. Mol/L m (GLP-1) 2 -mixed solution of ABD monomer, 0.1mmol/L human serum albumin + 1. Mu. Mol/L [ m (GLP-1) ] 2 -ABD] 2 The mixture solution of dimer, 300. Mu.L each of the above 5 samples, was incubated at 37 ℃ for 2h, and GEL chromatography GEL 2000SW was performed XL (7.8 mmID. Multidot.30cm) analysis, 20. Mu.L of sample was added and the eluent was 0.1mol/L phosphate buffer solution (0.1 mol/L Na) 2 SO 4 ，0.05％NaN 3 pH 7.4), flow rate 1.0ml/min. The results are shown in FIGS. 6-10, and m (GLP-1) in FIG. 6 2 Chromatogram, retention time of ABD monomert R Is 13.89min; FIG. 7 shows [ m (GLP-1) 2 -ABD] 2 Chromatogram of dimer, t R Is 12.08min; FIG. 8 is a chromatogram of serum albumin, t R 9.64min; FIG. 9 shows serum albumin and m (GLP-1) 2 Chromatogram after ABD action, partial serum albumin with m (GLP-1) 2 ABD non-covalently bound to form a complex, t thereof R Is 7.26min; FIG. 10 is a chromatogram of serum albumin after reaction, a portion of serum albumin with [ m (GLP-1) 2 -ABD] 2 Formation of a complex, t R It is 5.95min. FIG. 9 and FIG. 10 shows surface m (GLP-1) 2 -ABD、[m(GLP-1) 2 -ABD] 2 The binding rates with serum albumin were 87% and 78%, respectively.
Example 3
3.1 glucagon-like peptide-1 monomer m (GLP-1) 2 -ABD and its dimer [ m (GLP-1) 2 -ABD] 2 Pharmacokinetics in SD rats
SD rats (provided by northern suing laboratory animal farm in white cloud region, guangzhou city) with a body mass of about 300g were randomly divided into two groups (m (GLP-1) 2 ABD group, [ m (GLP-1) 2 -ABD] 2 Groups) with 8 animals each, half a female. Phosphate buffer solution with pH of 6.8 is used to prepare (m (GLP-1) with concentration of 100nmol/mL 2 ABD solution, [ m (GLP-1) 2 -ABD] 2 Solution (prepared as in example 2). Each group of rats was injected subcutaneously at a dose of 30nmol/kg, respectively. Blood was collected from the orbital venous plexus and blood samples taken before and after dosing at0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 36, 48h were placed in EDTA-treated EP tubes. Centrifuging at 4 deg.C and 4000g for 10min, collecting supernatant, rapidly freezing with liquid nitrogen, storing at-80 deg.C, determining sample concentration in blood sample with rat GLP-1ELISA kit (SBJ-R0066) according to the experimental operation of the instruction, and drawing drug plasma concentration-time curve, wherein the result is shown in FIG. 11.
As can be seen from FIG. 11, (m (GLP-1) 2 -ABD、[m(GLP-1) 2 -ABD] 2 In rats, maximal plasma concentrations were achieved at 2h and 4h post-dose, respectively, (m (GLP-1) 2 ABD has a half-life of about 8.1h, [ m (GLP-1) 2 -ABD] 2 Has a half-life of about 15.4h. Half-life compared to native GLP-1: (<2min),(m(GLP-1) 2 -ABD、[m(GLP-1) 2 -ABD] 2 The half-life of (a) is prolonged by about 240 times and 460 times, respectively.
3.2 glucagon-like peptide-1 monomer m (GLP-1) 2 -ABD and its dimer [ m (GLP-1) 2 -ABD] 2 The hypoglycemic effect
SD rats (provided by northern ear experiment animal farms in white cloud district, guangzhou city) with the age of 4 weeks are fed with high-sugar high-fat diet, after 4 weeks, the SD rats are fasted overnight for 16 hours, the body mass is weighed, and rats with the body mass being more than or equal to the body mass mean value of a control group and 2s in a high-sugar high-fat diet group are selected for subsequent experiments, so that the SD rats with the body mass not reaching the standard are eliminated. Feeding each group for 4 weeks according to feeding mode of high sugar and high fat diet, fasting for 16h overnight, selecting 30 rats with highest body mass, and randomly dividing into Plambo group, m (GLP-1) 2 ABD group and [ m (GLP-1) 2 -ABD] 2 Groups (prepared in example 2) of 10 individuals, each of which was divided into male and female halves, were each administered by a single intraperitoneal injection of 0.5% STZ (0.1 mmol/L citric acid buffer as a solvent, pH 4.4) at a dose of 50mg/kg, and were fed 1h after injection and 0.5% aqueous glucose solution.
After 2 weeks of STZ induction, fasting was performed for 16h, tail vein bleeding was performed, and fasting blood glucose was measured by one touch ultra, followed by subcutaneous injection at a dose of 20nmol/kg (now-30 min). After 30min the blood glucose level was again measured (now noted as 0 min) and a 20% glucose solution was infused orally at a dose of 1.6g/kg (glucose mass/body weight), the blood glucose concentration was measured at 15, 30, 60, 120 and 180min after the administration of the sugar, and the blood glucose concentration-time curve was plotted, the results of which are shown in fig. 12.
As can be seen from FIG. 12, at each monitoring time point after sugar administration, m (GLP-1) 2 ABD group and [ m (GLP-1) 2 -ABD] 2 The average blood sugar values of the groups have no significant difference, but the blood sugar values of the two groups are significantly lower than those of the Planbo group, and the blood sugar value of the two groups shows good blood sugar reducing effect.
According to the above-mentioned contents of the present invention, according to the technical strategy and conventional method for modification and modification of GLP-1 and its analogues, other mutations, cascades or fusions of various forms can be made without departing from the basic technical idea of the present invention.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of Guangdong department of pharmacy
<120> glucagon-like peptide-1 analogue monomer, dimer and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 169
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
His His His His His His Leu Val Pro Arg Thr Pro His Gly Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly Gly Gly Gly Ser
35 40 45
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala His Gly Glu Gly Thr
50 55 60
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
65 70 75 80
Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Ala Glu Ala Lys Val
100 105 110
Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Phe Tyr Lys
115 120 125
Arg Leu Ile Asn Lys Ala Lys Thr Val Glu Gly Val Glu Ala Leu Lys
130 135 140
Leu His Ile Leu Ala Ala Leu Pro Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160
Gly Ser Gly Gly Gly Gly Ser Ala Cys
165
<210> 3
<211> 507
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
caccatcacc atcaccacct ggtgccacgc actccacacg gtgaaggtac cttcacttcc 60
gacgtttcct cttacctgga aggtcaggct gcaaaagaat ttatcgcttg gctggttaaa 120
ggtcgtggcg gaggaggtgg ctctggtggc ggtggatcgg gtggtggagg ctccgctcac 180
ggtgaaggta ccttcacttc cgacgtttcc tcttacctgg aaggtcaggc tgcaaaagaa 240
tttatcgctt ggctggttaa aggtcgtggc ggaggtggag gttcgggcgg tggcggttct 300
ggcggcggtg gttcggcatt agctgaagct aaagtcttag ctaacagaga acttgacaaa 360
tatggagtaa gtgacttcta caagcgccta atcaacaagg ccaaaactgt tgaaggtgta 420
gaagcactga agctgcatat tttagctgca ttacctggcg gaggcggatc tggtggcgga 480
ggttccggag gaggcggctc ggcctgc 507
<210> 3
<211> 124
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcgccatatg caccatcacc atcaccacct ggtgccacgc actccacacg gtgaaggtac 60
cttcacttcc gacgtttcct cttacctgga aggtcaggct gcaaaagaat ttatcgcttg 120
gctg 124
<210> 4
<211> 117
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aacgtcggaa gtgaaggtac cttcaccgtg agcggagcct ccaccacccg atccaccgcc 60
accagagcca cctcctccgc cacgaccttt aaccagccaa gcgataaatt cttttgc 117
<210> 5
<211> 99
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggctccgctc acggtgaagg taccttcact tccgacgttt cctcttacct ggaaggtcag 60
gctgcaaaag aatttatcgc ttggctggtt aaaggtcgt 99
<210> 6
<211> 102
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gttagctaag actttagctt cagctaatgc cgaaccaccg ccgccagaac cgccaccgcc 60
cgaacctcca cctccgccac gacctttaac cagccaagcg at 102
<210> 7
<211> 114
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tcggcattag ctgaagctaa agtcttagct aacagagaac ttgacaaata tggagtaagt 60
gacttctaca agcgcctaat caacaaggcc aaaactgttg aaggtgtaga agca 114
<210> 8
<211> 124
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gcgcgaattc ttagcaggcc gagccgcctc ctccggaacc tccgccacca gatccgcctc 60
cgccaggtaa tgcagctaaa atatgcagct tcagtgcttc tacaccttca acagttttgg 120
cctt 124
Claims (9)
1. A glucagon-like peptide-1 analog monomer, characterized by: the amino acid sequence of the monomer is shown as SEQ ID NO.1, the monomer is a fusion protein obtained by fusing serially connected mutant glucagon-like peptide-1 and human albumin binding domain ABD, and the monomer is named as (mGLP-1) 2 -ABD, said monomer (mGLP-1) 2 -ABD composition is represented by the general formula:
6His—Linker-1—mGLP-1—Linker-2—mGLP-1—Linker-2—ABD—Linker-2—Cys；
wherein:
6His is a histidine purification tag, and the amino acid sequence of the histidine purification tag is HisHisHisHisHisHis;
linker-1 is connecting peptide-1, is hydrolysis site of thrombin and DPP-4 enzyme, and has amino acid sequence LVPRTP;
mGLP-1 is a mutant obtained by substituting Gly for Ala at position 2 of GLP-1 (7-37), and the amino acid sequence of the mutant is as follows:
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG；
linker-2 is connecting peptide-2, and the amino acid sequence of the connecting peptide is GGGGSGGGGSGGGGSA;
ABD is human serum albumin binding peptide, and the amino acid sequence thereof is as follows:
LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP；
cys is cysteine.
2. A gene encoding the glucagon-like peptide-1 analog monomer of claim 1, wherein: the nucleotide sequence of the gene is shown in SEQ ID NO.2.
3. A glucagon-like peptide-1 analog dimer, characterized by: the dimer is composed of the monomer (mGLP-1) of claim 1 2 -ABD is formed by disulfide bond linkage, and the glucagon-like peptide-1 analog dimer is named [ (mGLP-1) 2 -ABD] 2 。
4. A method for preparing the glucagon-like peptide-1 analog dimer of claim 3, comprising the steps of:
(1) Glucagon-like peptide-1 analog monomer (mGLP-1) obtained by SOE-PCR amplification 2 The ABD gene is used for carrying out enzymolysis on the monomer gene and the expression vector by using two restriction enzymes respectively, and then the two restriction enzymes are connected by using ligase to form a recombinant expression vector containing the glucagon-like peptide-1 analogue monomer nucleotide sequence;
(2) Transforming the recombinant expression vector with the correct sequence in the step (1) into an expression host cell for induced expression;
(3) Purified glucagon-like peptide-1 analog monomer (mGLP-1) 2 -ABD；
(4) Oxidized glucagon-like peptide-1 analog monomer (mGLP-1) 2 ABD, obtaining a disulfide-linked glucagon-like peptide-1 analog dimer [ (mGLP-1) 2 -ABD] 2 。
5. The method of claim 4, wherein the glucagon-like peptide-1 analog dimer is selected from the group consisting of: the expression host cell in the step (2) is Escherichia coli BL21 (DE 3).
6. The method of claim 4, wherein the glucagon-like peptide-1 analog dimer is selected from the group consisting of: in the step (3)The purification comprises the following steps: (3.1) centrifugation to collect the cells, ultrasonic lysis of cell release (mGLP-1) 2 -an ABD fusion protein; (3.2) Ni-NTA affinity chromatography purification, removing the background protein of the host bacteria; (3.3) alignment of glucagon-like peptide-1 analog monomer (mGLP-1) with C8 column 2 -desalting and purifying the crude ABD product.
7. The method of claim 4, wherein the glucagon-like peptide-1 analog dimer is selected from the group consisting of: in the step (4), the glucagon-like peptide-1 analogue monomer (mGLP-1) is oxidized by hydrogen peroxide or DMSO 2 ABD formation of glucagon-like peptide-1 analog dimer [ (mGLP-1) 2 -ABD] 2 。
8. The use of the glucagon-like peptide-1 analog monomer of claim 1 and pharmaceutically acceptable salts thereof or the glucagon-like peptide-1 analog dimer of claim 3 and pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and treatment of non-insulin dependent diabetes mellitus and its complications.
9. The use according to claim 8, wherein the non-insulin dependent diabetes mellitus and its complications are type II diabetes, impaired glucose tolerance, metabolic syndrome.
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| Title |
|---|
| "Recombinant Fusion of Glucagon-Like Peptide-1 and an Albumin Binding Domain Provides Glycemic Control for a Week in Diabetic Mice";Parisa Yousefpour等;《advanced therapeutics》;20200720;第3卷(第10期);参见摘要，第1页右栏第2段-第10页左栏第2段 * |
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| CN111944061A (en) | 2020-11-17 |
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