CN111944061A

CN111944061A - Glucagon-like peptide-1 analogue monomer, dimer and application thereof

Info

Publication number: CN111944061A
Application number: CN202010782404.6A
Authority: CN
Inventors: 桑延霞; 李占潮; 张军军
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2020-08-10
Filing date: 2020-08-10
Publication date: 2020-11-17
Anticipated expiration: 2040-08-10
Also published as: CN111944061B

Abstract

The invention discloses a glucagon-like peptide-1 analogue monomer. The amino acid sequence of the monomer is shown as SEQ ID NO.1A fusion protein obtained by fusing two serially connected molecules of mutant glucagon-like peptide-1 with human albumin binding domain ABD 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

Glucagon-like peptide-1 analogue monomer, dimer and application thereof

Technical Field

The invention belongs to the technical field of diabetes drugs, and particularly relates to a glucagon-like peptide-1 analogue monomer, a dimer thereof and application thereof, in particular to a long-acting glucagon-like peptide-1 analogue monomer with prolonged half-life and a dimer thereof, a preparation method of the monomer and the dimer thereof, and application of the monomer and the dimer thereof in preparation of drugs with effects of preventing and/or treating non-insulin-dependent diabetes and complications thereof.

Background

According to the latest global Diabetes mellitus outline (IDF Diabetes Atlas) released by the International Diabetes Federation (IDF) (9 th edition), in 2019, about 4.63 million adults worldwide suffer from Diabetes mellitus, with a gross prevalence of 9.3%; it is expected that by 2030 and 2045 years, diabetics will reach 5.784 and 7.002 billion 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) -NH₂Although 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 insulin secretion in a sugar-dependent manner, remarkably reduces postprandial blood sugar, and does not cause hypoglycemia; acting on pancreas and promoting the transcription and synthesis of insulin, promoting the beta-cell of pancreatic isletProliferation, inhibition of apoptosis, inhibition of glucagon secretion; 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 an incomparable advantage in the treatment of type 2 diabetes mellitus due to its unique function.

However, in vivo, GLP-1(7-37) and GLP-1(7-36) -NH₂The two N-terminal amino acids His-Ala are rapidly cleaved by DPP-4 enzyme widely distributed in organs and tissues to become inactive GLP-1(9-37) and GLP-1(9-36) -NH₂The GLP-1 has a small molecular weight of about 3kDa, is easily cleared by the kidney and is discharged out of the body, and the two main metabolic pathways ensure that the half-life period of the GLP-1 is less than 2min, thereby greatly limiting the clinical application of the GLP-1.

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 research points and hot spots 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, GLP-1 analogues 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 monomer molecules are slowly released to enter a blood system. The drug binds to the fatty acid binding site of serum albumin (HSA) via the fatty acid C chain, thereby greatly reducing renal clearance. GLP-1 analogues Albiglutamide and Dulaglutide are subjected to DPP-4 degradation through mutation of amino acids at key sites, GLP-1 is fused with Fc regions of HSA and IgG to form fusion protein with large molecular weight, the molecular size is increased, the filtering effect of glomerulus is reduced, and the degradation efficiency of DPP-4 is greatly reduced through the steric hindrance effect of the fusion protein. HSA and IgG are selected as molecular chaperones of fusion proteins, based on the fact that the HSA and the IgG have long in vivo circulation half-lives, the HSA and the IgG can be specifically combined with a neonatal Fc receptor (FcRn), and are prevented from degradation of lysosomes and filtration of glomeruli through an FcRn-mediated recirculation mechanism, so that the half-lives of the HSA and the IgG are 21 days and 19 days respectively, and the HSA and the IgG are applied to in vivo transport carriers of a plurality of therapeutic polypeptides and small molecular compounds to prolong the in vivo half-lives of the functional molecules. 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 weights of HSA and IgG are large, the tertiary structure of protein is complex, and the accuracy and repeatability of protein are the key and difficult points of the technology, and especially, HSA has 35 cysteines, the tertiary structure thereof has 17 disulfide bonds and a free cysteine Cys34, and the accuracy and repeatability of protein makes the purification conditions complex and harsh, and is difficult and the yield is low. Therefore, GLP-1 is fused with albumin-binding polypeptide with certain molecular weight, so that the purpose of prolonging half-life period can be realized, and the preparation process is simpler. In addition, more GLP-1 analogues need to be actively explored, and more options are provided for treating diabetes.

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, is a fusion protein obtained by fusing two serially connected molecules of mutant glucagon-like peptide-1 with a human albumin binding domain ABD, and the monomer is a peptide of the human albumin binding domain ABDMonomer designation (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:

the 6His is a histidine purification tag, the amino acid sequence of which is HisHisHisHisHisHis, and is used for purifying the fusion protein;

linker-1 is connecting peptide-1, is hydrolysis site of thrombin and DPP-4 enzyme, has an amino acid sequence of LVPRTP, and is used for releasing active fusion protein;

mGLP-1 is a mutant obtained by substituting Gly for Ala at position 2 and Arg at position 30 of GLP-1(7-37), and the amino acid sequence of the mutant is as follows:

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGG；

linker-2 is connecting peptide-2, and the amino acid sequence is GGGGSGGGGSGGGGSA;

ABD is a Human Serum Albumin (HSA) binding domain 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, HSA is the most abundant protein in plasma, accounting for about 50% of the total plasma protein, and has a molecular weight of 65 kDa. HSA binds to Fc receptors (FcRn) in a pH-dependent manner, avoiding lysosomal degradation through FcRn-mediated recycling mechanisms, thereby extending its half-life to 21 d. HSA has flexible and changeable structure, can be combined with a plurality of endogenous and exogenous small molecular compounds, fatty acid, polypeptide, protein and the like, and is used as an in vivo transportation carrier of a medicament due to better circulation stability.

The Albumin Binding Domain (ABD) of the present invention reversibly binds to HSA non-covalently, and reduces the probability of clearance by endogenous proteases and kidneys due to the steric hindrance and longer blood circulation cycle of HSA.

The ABD of the present invention is derived from a partial sequence in the surface protein G of group C streptococci, which bind to human HSA via the ABD binding 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 are provided.

Therefore, the inventor modifies and optimizes glucagon-like peptide-1 analogue monomer from three aspects according to the metabolic characteristics of GLP-1 to achieve the aim of prolonging the half life: (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 with other polypeptide or protein, increase mass and molecular size, and reduce renal clearance; (3) the fusion of the binding peptide or domain non-covalent binding on albumin or other endogenous protein to form a complex, with the help of endogenous protein steric hindrance effect and long half-life, reducing endogenous protease degradation and glomerular filtration.

Moreover, the invention selects albumin binding domain ABD with smaller molecular weight to prepare GLP-1 fusion protein, which can increase molecular weight and molecular size, and simultaneously, because the polypeptide has simple structure and no renaturation trouble, the process for preparing GLP-1 fusion protein by albumin binding 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 two serially-connected molecules of mutant glucagon-like peptide-1 and a human albumin binding domain ABD, can prolong the half-life period of the glucagon-like peptide-1 analogue monomer through the three modes, and can be further applied to medicaments with effects of preventing and treating non-insulin-dependent diabetes mellitus and complications thereof, and compared with the traditional GLP-1, the half-life period is longer, and the treatment effect is better.

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 object 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 amplification₂The 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 amplification₂ABD gene, monomer gene and expression vector with two limits respectivelyCarrying out enzymolysis by using a manufacturing endonuclease, and then connecting by using a ligase to form a recombinant expression vector containing a glucagon-like peptide-1 analogue monomer nucleotide sequence;

(3) purified glucagon-like peptide-1 analog monomer (mGLP-1)₂-ABD；

(4) Oxidized glucagon-like peptide-1 analog monomer (mGLP-1)₂ABD, obtaining 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, in the step (1), the expression vector 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 amplification₂-the gene of ABD, said monomer (mGLP-1)₂The ABD gene and pET30a vector were digested with Nde I and Xho I respectively, and then ligated with T4 DNA ligase to construct recombinant expression vector pET30a- (mGLP-1)₂ABD, recombinant expression vector pET30a- (mGLP-1)₂ABD is transformed to DH5 alpha, positive clones are preliminarily screened by LB solid culture medium containing kanamycin, single clones are selected for PCR and Nde I and Xho I double enzyme digestion identification, and the positive clones are identified for 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) transforming the positive clone with the correct sequencing in the step (1) to an expression host bacterium BL21(DE3), screening the positive clone in 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 the flora enters a logarithmic growth phase, inducing for about 6-8h, and collecting the 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 to obtain (mGLP-1)₂-an ABD monomer fusion protein; (3.3) Using C8 column for chromatography of glucagon-like peptide-1 analog monomer (mGLP-1)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; Sep-Pak C8 solid phase extraction column desalination, rotary evaporation concentration, freeze drying, taking part of lyophilized powder, further removing impurities by HPLC, refining, concentrating, freeze drying to obtain high purity glucagon-like peptide-1 analogue 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)₂ABD, forming intermolecular disulfide bonds, forming 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 H₂O₂Or DMSO, shaking at 20 deg.C for 4-8h, oxidizing Cys in monomer with air to generate intermolecular disulfide bond, and obtaining glucagon-like peptide-1 analog dimer [ (mGLP-1)₂-ABD]₂Further removing impurities by HPLC, purifying, concentrating, and freeze-drying to obtain high-purity glucagon-like peptide-1 analog dimer [ (mGLP-1)₂-ABD]₂。

The third object of the present invention is achieved by the following technical solutions: 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 are applied to the preparation of the medicine with the effects of preventing and/or 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]₂The 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, one of the purposes of the serial connection of mGLP-1, the fusion with ABD and the monomer polymerization to generate dimer in the invention is to increase the molecular weight and the molecular size of the GLP-1 analogue related to the invention and reduce the filtering effect of glomerulus;

(3) compared with natural GLP-1, the fusion protein (mGLP-1) involved in the invention₂-ABD、[(mGLP-1)₂-ABD]₂Can form a fusion protein-HSA complex by reversibly and non-covalently combining ABDs on the molecule with serum albumin HSA in blood, and then the fusion protein-HSA complex is prevented from being cleared by lysosome and kidney by the aid 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)₂The half-life of ABD in SD rat is increased to 8.2h, and the half-life of glucagon-like peptide-1 analog dimer in SD rat is increased to 15.6 h;

(5) the glucagon-like peptide-1 analog monomer (mGLP-1) of the present invention₂-dimer of ABD and glucagon-like peptide-1 analog [ (mGLP-1)₂-ABD]₂The glucose-lowering oral liquid shows obvious glucose-lowering effect in an Oral Glucose Tolerance Test (OGTT) of a diabetes model rat, and has advantages and potential to be applied to clinical treatment of diabetes.

(6) Compared with fusion protein of HSA and IgG, the invention can increase molecular weight and molecular size, and has simple polypeptide structure and no renaturation, so that the GLP-1 fusion protein prepared from the polypeptide has simple process and high yield.

Drawings

FIG. 1 shows glucagon-like peptide-1 analog monomer (mGLP-1) of example 1₂-dimer of ABD and glucagon-like peptide-1 analog [ (mGLP-1)₂-ABD]₂Schematic structural diagram of (a);

FIG. 2 is a graph showing the results of PCR of a single clone obtained by transforming E.coli DH5a with pET30a-m (GLP-1)2-ABD recombinant plasmid in example 2;

FIG. 3 is a graph showing the double digestion results of a single clone obtained by transforming E.coli DH5a 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 2₂-SDS-PAGE electropherograms of ABD;

FIG. 5 shows glucagon-like peptide-1 dimer [ (mGLP-1) of example 2₂-ABD]₂Time-of-flight mass spectrogram of (a);

FIG. 6 shows m (GLP-1) in example 2₂-a chromatogram of ABD monomers;

FIG. 7 is example 2[ m (GLP-1)₂-ABD]₂A chromatogram of the dimer;

FIG. 8 is a chromatogram of serum albumin HSA of example 2;

FIG. 9 shows serum albumin and m (GLP-1) of example 2₂-chromatograms after ABD action;

FIG. 10 shows serum albumin and [ m (GLP-1) of example 2₂-ABD]₂Chromatograms 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 the Oral Glucose Tolerance Test (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 bioactivity of GLP-1, mutating the key amino acid of the DPP-4 action site of protease, and substituting Ala at the 2 nd position and Arg at the 30 th position with Gly to obtain a GLP-1 mutant mGLP-1; mGLP-1 is connected in series and fused with a serum albumin binding domain ABD, the molecular weight is increased, the clearance of the kidney is reduced, meanwhile, the fusion protein is combined with serum albumin HSA in a non-covalent way through the ABD to form a compound, and the filtering action of lysosomes and glomeruli is avoided by utilizing an FcRn mediated recycling mechanism.

In the invention, in order to facilitate the purification of the expressed fusion protein, a His purification tag is designed at the N end; in order to release active fusion protein, the enzyme cutting site Linker-1 of endogenous protease thrombin and DPP-4 is designed to release active fusion protein monomer or dimer. In order to ensure the formation of the natural structures of the mGLP-1 and the ABD and the exertion of the efficacy, the mGLP-1 and the ABD which are connected in series are connected through flexible connecting peptides Linker-2, so that each functional part keeps a certain space so as to facilitate the formation of the natural structures. Cys at the end of the fusion monomer is used to generate intermolecular disulfide bonds that link 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 two serially-connected molecules of mutant glucagon-like peptide-1 with a human albumin binding domain ABD, and is named as (mGLP-1)₂ABD, this monomer (mGLP-1)₂-ABD composition is represented by the general formula:

wherein:

the 6His is a histidine purification tag, the amino acid sequence of which is HisHisHisHisHisHis, and is used for purifying the fusion protein monomer;

linker-1 is connecting peptide-1, is hydrolysis site of thrombin and DPP-4 enzyme, has an amino acid sequence of LVPRTP, and is used for releasing active fusion protein monomer or dimer;

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGG；

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:

one, pET30a-m (GLP-1)₂Construction of the ABD recombinant plasmid

pET30a-m(GLP-1)₂Construction of the ABD recombinant plasmid comprises four steps:

SOE-PCR amplification (mGLP-1)₂-ABD fusion gene

(mGLP-1)₂507bp 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, with the following primer sequences:

upstream primer P1(SEQ ID NO.3)

Downstream primer P2(SEQ ID NO.4)

5’-AACGTCGGAAGTGAAGGTACCTTCACCGTGAGCGGAGCCTCCACCACCCGATCCACCGCCACCAGAGCCACCTCCTCCGCCACGACCTTTAACCAGCCAAGCGATAAATTCTTTTGC-3’

The bold inclined part at the end of the upstream primer P15' is an NdeI restriction site, and the upstream primer is obtained by adopting SOE-PCR and amplifying by using P1 and P2 complementary primers (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: p1 and P2 primers 2. mu.L, 10 XH Buffer 5.0. mu.L, dNTP 5.0. mu.L, Taq enzyme 1.0. mu.L, ddH was added₂0 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 5 min.

(2) Second step SOE-PCR

The primers P3 and P4 were designed to be complementary to each other using E.coli preferred codons and have the following sequences:

upstream primer P3(SEQ ID NO.5)

5’-GGCTCCGCTCACGGTGAAGGTACCTTCACTTCCGACGTTTCCTCTTACCTGGAAGGTCAGGCTGCAAAAGAATTTATCGCTTGGCTGGTTAAAGGTCGT-3’

Downstream primer P4(SEQ ID NO.6)

5’-GTTAGCTAAGACTTTAGCTTCAGCTAATGCCGAACCACCGCCGCCAGAACCGCCACCGCCCGAACCTCCACCTCCGCCACGACCTTTAACCAGCCAAGCGAT-3’

Using the PCR product of the first step as a template, and performing amplification by using primers P1, P3 and P4 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 and Taq enzyme 1.0. mu.L, ddH was added₂0 to 50. mu.L.

(3) Third step SOE-PCR

The primers P5 and P6 were designed to be complementary to each other using E.coli preferred codons and have the following sequences:

upstream primer P5(SEQ ID NO.7)

5’-TCGGCATTAGCTGAAGCTAAAGTCTTAGCTAACAGAGAACTTGACAAATATGGAGTAAGTGACTTCTACAAGCGCC TAATCAACAA GGCCAAAACTGTTGAAGGTG TAGAAGCA-3’

Downstream primer P6(SEQ ID NO.8)

The 5' end of the downstream primer P6 is added with a coarse inclined part which is an Xho I enzyme cutting site, the PCR product of the second step is used as a template, and the PCR product 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 primers P1, P5 and P6 2. mu.L, 10 XH buffer 5.0. mu.L, dNTP 5.0. mu.L and Taq enzyme 1.0. mu.L, ddH was added₂0 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 10 min.

The obtained PCR product is purified by a PCR product purification kit for later use.

2.(mGLP-1)₂Double digestion of-ABD fusion protein Gene, plasmid vector

Will (mGLP-1)₂Carrying 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
ddH
	₂0	To 30 μ L

The reaction conditions of the reaction system were 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
ddH
	₂0	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 ABD Gene	2μL
10×Ligation Buffer	2μL
		T4 ligase	2μL
ddH
	₂0	To 20 μ L

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)₂ABD recombinant plasmid transformation of E.coli DH5a

Recombinant plasmid pET30a-m (GLP-1)₂Adding 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 5 min. 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. Selecting Kan resistant monoclone, performing PCR and double enzyme digestion identification, wherein the PCR result is shown in figure 2, and the specific primers P1 and P6 can be used for detecting the Kan resistance in positive gramA band corresponding to the band size of 530bp was amplified from the clone. 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)₂The cDNA of the ABD fusion protein has been inserted in a targeted manner into the pET30a vector. DNA sequencing was performed on the positively identified clones to confirm further that pET30a-m (GLP-1)₂-ABD recombinant plasmid base sequence.

II, glucagon-like peptide-1 monomer m (GLP-1)₂Inducible expression of ABD

Extracting recombinant plasmid pET30a-m (GLP-1) from correctly identified positive DH5a recombinant bacteria₂ABD, transformed into Escherichia coli BL21(DE3) according to the method (4) in step (a), to obtain a recombinant expression strain. Inoculating overnight cultured recombinant expression bacteria at a ratio of 1:100 into 2L LB liquid medium containing 100. mu.g/mL kanamycin, and shake-culturing at 37 deg.C for about 2h to OD_600nmAdding IPTG to final concentration of 0.6mmol/L, inducing expression for 6-8 hr, centrifuging at 4 deg.C and 12000r/min for 20min, and collecting thallus. 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(DE3) host cells expressed M (GLP-1) with a molecular weight of about 16.4kDa under the induction of IPTG₂-an ABD fusion protein.

Glucagon-like peptide-1 monomer m (GLP-1)₂-separation and purification of ABD

Lysis was performed with lysis buffer (400mmol/L NaCl,50mmol/L NaH)₂PO₄10mmol/L imidazole, pH8.0) 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)₂Desalting the eluate of ABD fusion protein with WAT054525C8 column: 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 dry powder to carry out reversed phase HPLC refining, and the steps are as follows: dissolving m (GLP-1) in a washing solution (5% acetonitrile in water, pH 3.5)₂ABD dry powder, Supersil SB-C8 column (200X4.6mm,5um) equilibrated with washing solution, loading, washing with washing solution until baseline is stable, and eluting with eluent (40% acetonitrile in water, pH 3.5) at flow rate of 2mL/min for eluting protein of interest m (GLP-1)₂ABD, concentrating the eluent by rotary evaporation, and freeze-drying.

Glucagon-like peptide-1 dimer [ (mGLP-1)₂-ABD]₂Preparation of

Glucagon-like peptide-1 m (GLP-1) prepared in the step (three)₂ABD dissolved in 5% H at 0.1mg/mL₂O₂And 5% DMSO aqueous solution, shaking and oxidizing at room temperature to promote generation of disulfide bonds, oxidizing for 6h, performing rotary evaporation and concentration on reaction liquid, and freeze-drying. The dimer was again purified using a Supersil SB-C8 column (200X4.6mm,5um) under the following conditions: equilibrating the column to baseline stability with 5% acetonitrile in water (pH 3.5), loading, eluting the target protein with 35% acetonitrile in water (pH 3.5) at a flow rate of 1.5mL/min, concentrating the eluate by rotary evaporation, and freeze-drying. Obtained [ (mGLP-1)₂-ABD]₂The 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]₂The molecular weight of (A) is 32778, which is consistent with the theoretical molecular weight.

V. glucagon-like peptide-1 monomer m (GLP-1)₂-ABD and its dimer [ m (GLP-1)₂-ABD]₂Determination of affinity for human serum Albumin

Using 0.1mol/L phosphate buffer solution (containing 0.1mol/L Na)₂SO₄pH7.4) was prepared as a human serum albumin solution at a concentration of 0.1mmol/L and m (GLP-1) at a concentration of 1. mu. mol/L, respectively₂ABD monomer solution, 1. mu. mol/L of [ m (GLP-1)₂-ABD]₂Dimer 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]₂The dimer mixture solution, 300. mu.L each of the above 5 samples, was incubated at 37 ℃ for 2 hours and subjected to GEL chromatography GEL 2000G 2000SW_XL(7.8mmID 30cm) analysis, loading 20. mu.L, 0.1mol/L phosphate buffer (0.1mol/L Na) as eluent₂SO₄，0.05％NaN₃pH7.4), flow rate 1.0 ml/min. The results are shown in FIGS. 6-10, and m (GLP-1) in FIG. 6₂Chromatogram of ABD monomer, retention time t_RIs 13.89 min; FIG. 7 shows [ m (GLP-1)₂-ABD]₂Chromatogram of dimer, t_RIs 12.08 min; FIG. 8 is a chromatogram of serum albumin, t_R9.64 min; FIG. 9 shows serum albumin HSA and m (GLP-1)₂Chromatogram after ABD action, part of HSA with m (GLP-1)₂ABD non-covalently bound to form a complex, t thereof_RIs 7.26 min; FIG. 10 is a chromatogram of serum albumin HSA after the action, a part of HSA and [ m (GLP-1)₂-ABD]₂Formation of a complex, t_RIt is 5.95 min. FIGS. 9 and 10 show that m (GLP-1) is measured in an in vitro simulation test₂-ABD、[m(GLP-1)₂-ABD]₂The binding rates with serum albumin were 78% and 87%, respectively.

Example 3

3.1 glucagon-like peptide-1 monomer m (GLP-1)₂-ABD and its dimer [ m (GLP-1)₂-ABD]₂Pharmacokinetics 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)₂ABD group, [ m (GLP-1)₂-ABD]₂Groups) of 8, each with male and female halves. Phosphate buffer solution with pH of 6.8 is used to prepare (m (GLP-1) with concentration of 100nmol/mL₂ABD solution, [ m (GLP-1)₂-ABD]₂Solution (prepared as in example 2). Each group of rats was injected subcutaneously at a dose of 30nmol/kg, respectively. Collecting blood from orbital venous plexus, and collecting 0h before administration and 1, 2, 3, 4, 5, 6, 8, 10, 1h after administration2. Blood samples of 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, and measuring in blood sample (m (GLP-1) with rat GLP-1ELISA kit (SBJ-R0066) according to the experimental operation of the instruction₂-ABD、[m(GLP-1)₂-ABD]₂The plasma concentration-time curve of the drug was plotted, and the results are shown in FIG. 11.

As can be seen from FIG. 11, (m (GLP-1)₂-ABD、[m(GLP-1)₂-ABD]₂In rats, maximal plasma concentrations were achieved at 2h and 4h post-dose, respectively, (m (GLP-1)₂ABD has a half-life of about 8.1h, [ m (GLP-1)₂-ABD]₂Has a half-life of about 15.4 h. Half-life (about 2min), (m (GLP-1) compared to native GLP-1₂-ABD、[m(GLP-1)₂-ABD]₂The 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]₂The hypoglycemic effect of

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 overnight for 16h, selecting 30 rats with highest body mass, and randomly dividing into Plambo group, m (GLP-1)₂ABD group and [ m (GLP-1)₂-ABD]₂Groups (prepared as described in example 2) of 10 animals each, male and female halves, each group were administered a single intraperitoneal injection of 0.5% streptozotocin (0.1mmol/L citrate buffer as solvent, pH 4.4) at a dose of 50mg/kg, 1h post-injection with food and 0.5% aqueous glucose.

After 2 weeks of STZ-induced modeling, fasting for 16h, Oral Glucose Tolerance Test (OGTT), tail vein blood sampling, fasting blood glucose determination by one touch ultra, and then m (GLP-1)₂ABD group and [ m (GLP-1)₂-ABD]₂Groups were each injected subcutaneously with a dose of 20nmol/kgMonomer or dimer drug (now-30 min) was administered to the Plambo group in the same volume of solvent (0.1mmol/L phosphate buffer, pH6.8) as a control. The blood glucose values of the groups were measured again after 30min, and immediately after oral infusion of a 20% glucose solution (now noted 0min) at a dose of 1.6g/kg (glucose mass/body weight), blood samples were taken, and blood glucose concentrations at 15, 30, 60, 120 and 180min after sugar administration were determined, and blood glucose concentration-time curves were plotted, and the results are shown in fig. 12.

As can be seen from FIG. 12, at each monitoring time point after sugar administration, m (GLP-1)₂ABD group and [ m (GLP-1)₂-ABD]₂The average blood sugar values of the groups have no significant difference, but the blood sugar values of the two groups of administration groups are significantly lower than those of the Plambo group, and the blood sugar-reducing effect is good.

Therefore, medicaments containing the glucagon-like peptide-1 analogue monomer and pharmaceutically acceptable salts thereof or the glucagon-like peptide-1 analogue dimer and pharmaceutically acceptable salts thereof can be prepared and used for preventing and/or treating non-insulin-dependent diabetes mellitus and complications thereof.

Wherein the non-insulin dependent diabetes mellitus and its complications can be type II diabetes, impaired glucose tolerance, weight loss, metabolic syndrome, etc.

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 changes, modifications, substitutions, combinations, and simplifications 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

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 two-molecule mutant glucagon-like peptide-1 and human albumin binding domain ABD which are connected in series, and the monomer is named as (mGLP-1)₂-ABD, said monomer (mGLP-1)₂-ABD composition is represented by the general formula:

wherein:

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGG；

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 as 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 1₂-ABD are formed by disulfide bond linkage, said glucagon-like peptide-1 analog dimer being designated [ (glp-1)₂-ABD]₂。

4. The method of preparing the glucagon-like peptide-1 analog dimer of claim 3, comprising the steps of:

(3) purified glucagon-like peptide-1 analog monomer (mGLP-1)₂-ABD；

5. The method of claim 4, wherein the glucagon-like peptide-1 analog dimer is selected from the group consisting of: the expression vector in the step (1) is pET30a, the two restriction enzymes are NdeI and XhoI respectively, and the recombinant expression vector is pET30a- (mGLP-1)₂-ABD。

6. 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).

7. Pancreatic hypertension according to claim 4The preparation method of the dimer of the glucide-like peptide-1 analogue is characterized in that: 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 to obtain (mGLP-1)₂-an ABD monomer fusion protein; (3.3) Using C8 column for chromatography of glucagon-like peptide-1 analog monomer (mGLP-1)₂-desalting and purifying the crude ABD product.

8. 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 DMSO₂ABD, forming intermolecular disulfide bonds, forming glucagon-like peptide-1 analog dimer [ (mGLP-1)₂-ABD]₂。

9. 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/or treatment of non-insulin dependent diabetes mellitus and complications thereof.

10. The use according to claim 9, wherein the non-insulin dependent diabetes mellitus and its complications are type II diabetes, impaired glucose tolerance, weight loss, metabolic syndrome.