CN107188953B - Glucagon-like peptide-1 analogs and uses thereof - Google Patents

Glucagon-like peptide-1 analogs and uses thereof Download PDF

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CN107188953B
CN107188953B CN201710559884.8A CN201710559884A CN107188953B CN 107188953 B CN107188953 B CN 107188953B CN 201710559884 A CN201710559884 A CN 201710559884A CN 107188953 B CN107188953 B CN 107188953B
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xaa
ser
artificial sequence
nucleic acid
ala
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CN107188953A (en
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李奎
裴杨莉
杨述林
安翠平
齐传翔
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Institute of Animal Science of CAAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

The invention discloses a glucagon-like peptide-1 analogue and application thereof. The general formula of the amino acid sequence of the glucagon-like peptide-1 analogue provided by the invention is as follows: xaa1‑Ser‑Glu‑Gly‑Thr‑Phe‑Xaa7‑Ser‑Asp‑Xaa10‑Ser‑Xaa12‑Xaa13‑Xaa14‑Xaa15‑Xaa16‑Xaa17‑Xaa18‑Xaa19‑Xaa20‑Xaa21‑Phe‑Xaa23‑Xaa24‑Trp‑Leu‑Xaa27‑Xaa28‑Xaa29‑Xaa30. The invention replaces the amino acid at the second position of GLP-1 with Ala to Ser and changes the amino acid at other positions, so that the glucagon-like peptide-1 analogue has the biological functions of GLP-1 and GIP, the dosage of the analogue is reduced, and the hypoglycemic effect is realized.

Description

Glucagon-like peptide-1 analogs and uses thereof
Technical Field
The invention belongs to the field of biomedicine, and relates to a glucagon-like peptide-1 analogue and application thereof.
Background
GLP-1 (glucagon-like peptide-1, GLP-1) has wide physiological effects, GLP-1 promotes the biosynthesis and secretion of insulin, the number of insulin β cells which react to glucose in pancreas is increased, that is, more insulin β cells can participate in the production of insulin, GLP-1 inhibits the secretion of glucagon, GLP-1 inhibits the apoptosis of insulin β cells and promotes the proliferation and differentiation of insulin β cells, GLP-1 inhibits the emptying of stomach and the secretion of gastric acid, the effect of GLP-1 on the feeding center, the influence of GLP-1 on heart and blood pressure, GLP-1 reduces endogenous gluconeogenesis, GLP-1 is an ideal treatment medicament for diabetes patients, especially obese type 2 diabetes patients due to the wide physiological effect of GLP-1, but GLP-1 produced by the organism itself is easily degraded by dipeptide IV (4) to inactivate DPP, the biological activity of GLP-1 is improved, the effective plasma concentration is maintained, and the effect of the diabetes patients is developed for a long time, and the GLP-1 analogue is researched by people in long-1.
The results of experiments to verify the therapeutic effects of GLP-1 and GIP in diet-induced diabetic mice show that mice using GLP-1 and GIP for two weeks all had better effects on weight loss, diet reduction, lipid deposition reduction, and blood glucose reduction than mice using GLP-1 or GIP alone (Finan, Ma et al.2013), and 3 nmol.kg-1The simultaneous effect of GLP-1 and GIP corresponds to 30nmol kg-1Therapeutic effects of Liraglutide. The research shows that the simultaneous use of GLP-1 and GIP not only can improve the treatment effect, but also can greatly reduce the dosage.
Disclosure of Invention
The invention aims to provide a novel glucagon-like peptide-1 analogue which can reduce the dosage on the basis of retaining the hypoglycemic activity and the application thereof.
The present invention first provides a protein comprising an amino acid sequence represented by the general formula 1.
General formula 1:
Xaa1-Ser-Glu-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30
wherein, Xaa1Is His or Tyr; xaa7Is Thr or Ile; xaa10Is Val or Tyr; xaa12Is Ser or Ile; xaa13Is Tyr or Ala; xaa14Is Met or Leu; xaa15Is Asp or Glu; xaa16Is Gly or Lys; xaa17Is Gln orIle；Xaa18Is Ala or His; xaa19Is Ala or Gln; xaa20Is Lys or Gln; xaa21Is Glu or Asp; xaa23Is Ile or Val; xaa24Is Ala or Asn; xaa27Is Val or Leu; xaa28Is Lys or Ala; xaa29Is Gly or Gln; xaa30Is Arg or Lys.
Furthermore, the amino acid sequence of the protein is shown in any one of sequences 1-30 in a sequence table.
To facilitate purification of the protein, tags as shown in the following table can be attached to the amino-or carboxy-terminus of the protein.
Table: sequence of tags
Figure BDA0001346800060000011
Figure BDA0001346800060000021
Nucleic acid molecules encoding such proteins are also within the scope of the invention.
The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be an RNA, such as an mRNA, hnRNA, or tRNA, and the like.
In the present invention, the nucleic acid molecule may specifically be any of:
(1) a DNA molecule shown in any one of sequences 31 to 60 in a sequence table;
(2) a DNA molecule which hybridizes with the DNA molecule defined in (1) under stringent conditions and encodes the protein;
(3) a DNA molecule which has a homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more with the DNA sequence defined in (1) or (2) and encodes the protein.
The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
Recombinant vectors, expression cassettes, transgenic cell lines or recombinant microorganisms containing the above-described nucleic acid molecules also belong to the scope of protection of the present invention.
In the invention, the recombinant vector is specifically a recombinant vector (named as pGAPZ α A-SGLP-1) obtained by connecting the 5 'end of the nucleic acid molecule with an enterokinase enzyme cutting site recognition sequence (sequence 61) and a Strep-II tag coding sequence (sequence 62) in sequence and inserting the nucleic acid molecule into a multiple cloning site of a pGAPZ α A vector after connecting a terminator sequence (TGATGA) at the 3' end of the nucleic acid molecule.
In the present invention, the recombinant yeast has a recombinant yeast obtained by transforming pGAPZ α A-SGLP-1 into a recipient yeast cell, and the recipient yeast may specifically be GS115, X-33, KM71, and SMD 1168.
The use of said protein or said nucleic acid molecule or said recombinant vector or said expression cassette or said transgenic cell line or said recombinant microorganism for the preparation of a product with a hypoglycemic effect also belongs to the scope of protection of the present invention.
The application of the protein or the nucleic acid molecule or the recombinant vector or the expression cassette or the transgenic cell line or the recombinant microorganism in the preparation of the medicine for treating and/or preventing diabetes also belongs to the protection scope of the invention.
The invention also provides a product, the active ingredient of which is the protein; the product has at least one of the following functions: (a) reducing blood sugar; (b) treating and/or preventing diabetes.
The diabetes may be type II diabetes.
In the invention, the amino acid at the second position of GLP-1 is replaced by Ser from Ala, and the amino acids at other positions are changed, so that the analogue has the biological functions of GLP-1 and GIP, the dosage of the analogue is reduced, and the hypoglycemic effect is realized. The invention has the advantages that a brand new blood sugar-reducing lipid-regulating peptide is constructed by amino acid substitution, and the dosage of the analogue is reduced on the basis of keeping the blood sugar-reducing activity.
Drawings
FIG. 1 is a design for GLP-1-like peptide expression purification.
FIG. 2 is an electrophoresis diagram of a GLP-1 analogue purified protein small molecule gel, lanes 1-6: SGP-1, SGP-5, SGP-13, SGP-19, SGP-23, SGP-25; m: and (5) a small-molecule Marker 26628.
FIG. 3 shows the results of measurement of the blood sugar-reducing ability of GLP-1 analogues. The control group was G + Vehicle, n-12, p < 0.05. In the figure, G represents glucose, and Vehicle represents physiological saline.
FIG. 4 shows the results of the test of the glucose-lowering ability of SGP-13 to male C57 mice. The control group was G + Vehicle, n-7, p < 0.05. In the figure, G represents glucose, and Vehicle represents physiological saline.
FIG. 5 shows the results of the test of the glucose-lowering ability of SGP-13 in female C57 mice. The control group was G + Vehicle, n-7, p < 0.05. In the figure, G represents glucose, and Vehicle represents physiological saline.
FIG. 6 shows the results of the measurement of the effective action time of SGP-13 in vivo. The control group was G + Vehicle, n-7, p < 0.05. In the figure, G represents glucose, and Vehicle represents physiological saline.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 design and preparation of glucagon-like peptide-1 analogs
The functional study of GIP shows that the function of GIP (1-42) is the same as that of GIP (1-30), and the amino acid of GIP (1-30) is 80% of that of GLP-1(7-36), so that the sequence of GIP (1-30) and GLP-1(7-36) is comprehensively considered in the subsequent amino acid mutation design of the invention, the common sequence of the two is kept, and the sequence of GLP-1(7-36) different from that of GIP (1-30) is replaced by the amino acid of GIP (1-30) in different combinations.
GLP-1(7-36)：HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR
GIP(1-30)：YAEGTFISDYSIAMDKI HQQDFVNWLLAQK
The amino acid sequence of the GLP-1 analogue designed by the invention is shown as a general formula 1.
General formula 1:
Xaa1-Ser-Glu-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30
wherein, Xaa1Is His or Tyr; xaa7Is Thr or Ile; xaa10Is Val or Tyr; xaa12Is Ser or Ile; xaa13Is Tyr or Ala; xaa14Is Met or Leu; xaa15Is Asp or Glu; xaa16Is Gly or Lys; xaa17Is Gln or Ile; xaa18Is Ala or His; xaa19Is Ala or Gln; xaa20Is Lys or Gln; xaa21Is Glu or Asp; xaa23Is Ile or Val; xaa24Is Ala or Asn; xaa27Is Val or Leu; xaa28Is Lys or Ala; xaa29Is Gly or Gln; xaa30Is Arg or Lys.
The amino acid sequence of the part of GLP-1 analogue obtained finally and the corresponding coding gene sequence are shown in Table 1.
To facilitate the concentration and purification of later period, an enterokinase enzyme cutting site recognition sequence (sequence 61) and a Strep-II label coding sequence (sequence 62) are connected to the 5 'end of a GLP-1 analogue coding gene in sequence, a terminator sequence (TGATGA) is connected to the 3' end of the GLP-1 analogue coding gene, so that the subsequent enzyme cutting purification is facilitated, the purified polypeptide is subjected to enzyme cutting without any label, and the base sequence is synthesized and cloned between enzyme cutting sites EcoR I and Xba I of pGAPZ α A vector and is sequenced to verify the correctness (figure 1).
TABLE 1 sequence Listing of GLP-1 analogs
Figure BDA0001346800060000031
Figure BDA0001346800060000041
Example 2 GLP-1 analogs Yeast transformation, expression screening and purification
Adding 20 μ l of the series of recombinant plasmids constructed in example 1 into 100 μ l of GS115 yeast competence, gently blowing and sucking for two times to uniformly mix the recombinant plasmids and the competence, placing on ice, adding the mixture into a precooled electric shock cup for electric shock, and immediately adding 1ml of precooled 1M sorbitol after electric shock; transferring the transformed bacterium liquid into a 1.5ml centrifuge tube, and putting the centrifuge tube into an incubator at 29 ℃ for standing for 3 hours; coating on YPDS plates with Zeocin concentrations of 0.5, 1 and 2mg/ml, culturing in 29 deg.C incubator, and PCR to identify the success of recombination.
Single clones on plates with different Zeocin concentrations were picked and inoculated into 100ml BMGY liquid medium, incubated continuously at 29 ℃ for three days at 220rpm, centrifuged at 13000rpm for 40min, and the supernatants were collected. Adjusting the pH of the collected supernatant to 8.0; filtering with 0.45 μm membrane to obtain sample solution.
Balancing Strep-tag II purification column with Binding Buffer of 5 times column volume; loading approximately 100ml of sample solution to a purification column; continuously washing the substrate by using a Binding Buffer until the UV is unchanged; washing the purification column with 5 column volumes of Digestion Buffer; loading 0.05ml enterokinase (EK enzyme) to a purification column, standing and digesting for 16h at 4 ℃; washing the purification column with a desaling Buffer, collecting the washing liquid, namely the target protein, Desalting the target protein solution with the desaling Buffer, and finally storing the target protein solution in a PBS solution.
Taking 30 mu L of target protein solution, and adding 10 mu L of 4 XLoading Buffer; boiling in water for 5 min; run small molecule gel and the results are shown in figure 2. The detected target protein is lyophilized, and 1 μ g is accurately weighed for subsequent experiments.
Example 3 functional verification of GLP-1 analogs
GLP-1 has wide physiological effects, wherein the most convenient and rapid detection index is the capability of reducing blood sugar in vivo. Therefore, in this experiment, male C57 mice of 6 weeks of age were used as subjects, and 12 mice were used per experimental group. All mice were injected with 2g/kg body weight of glucose solution. Mice in the experimental group are injected with glucose and simultaneously injected with GLP-1 analogues (SGP-1, SGP-5, SGP-13, SGP-19 or SGP-23, the concentration is 2 mmol/L); the control group is injected with 0.1mL of physiological saline, the positive control group is injected with Exendin-4(EX), GLP-1 and GIP (the concentration is 2mmol/L), and GLP-1 analogue and the control injection are injected according to the weight of 10nmol/kg of mice, and the blood sugar level of each group of mice is detected at 0, 20 and 40min after the injection respectively. 12 mice per group and the results were averaged.
The experimental results show that: SGP-13 was found to significantly lower blood glucose levels as was Exendin-4(EX) and GLP-1 (FIG. 3). Indicating that SGP-13 has the ability to lower blood glucose in male mice.
Example 4 SGP-13 functional verification
Some medicines can have the situation of drug effect difference of animals with different sexes, and in order to judge whether the SGP-13 has sex drug effect difference, related verification experiments on female mice are added when the original blood sugar reduction experiment on male mice is repeated again, and the SGP-13 blood sugar reduction effect verification experiment after the dosage is reduced is also added. All mice were injected with 2g/kg body weight of glucose solution. Injecting GLP-1 analogue SGP-13(2mmol/L) into mice of an experimental group while injecting glucose, and injecting GLP-1 analogue and a control injection into the mice according to the weight of 5nmol/kg and 10 nmol/kg; the control group was injected with 0.1mL of physiological saline, and the positive control group was injected with GLP-1(2mmol/L) at 10nmol/kg of mouse body weight. The blood glucose levels of the mice in each group were measured at 0, 20, 40 and 60min after injection, respectively. The results were averaged for 7 mice per group. The results on 6-week-old male mice are shown in FIG. 4. The results for 6-week-old female C57 mice are shown in fig. 5 (n ═ 7).
The results shown in fig. 4 and 5 show that: SGP-13 has the ability of reducing blood sugar on female and male mice, the effect of SGP-13 is obviously better than GLP-1 when the SGP-13 is injected according to the weight of 10nmol/kg mice, and the SGP-13 has the same blood sugar reducing effect as GLP-1 when only half dosage is used (the SGP-13 is injected according to the weight of 5nmol/kg mice).
Example 5 verification of effective duration of action of SGP-13 in vivo
Since SGP-13 had no sex difference in blood sugar lowering effect, 6-week-old female mice were used to verify the effective acting time in vivo. SGP-13 and GLP-1 are injected according to 10nmol/kg of mouse body weight at 0min, normal saline is injected into a control group, glucose is injected according to 2g/kg of mouse body weight at 0min, 30min, 60min and 90min, blood glucose values are detected at 0min, 30min, 60min, 90min and 120min, 7 mice in each group are obtained, and the results are averaged. The results are shown in FIG. 6.
As can be seen from the results in FIG. 6, SGP-13 was effective in reducing the blood glucose level in mice at 30min and 60min after injection, while GLP-1 in the control group was effective in reducing the blood glucose level only at 30 min. The results indicate that SGP-13 has a longer blood glucose lowering effect in vivo than GLP-1.
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> glucagon-like peptide-1 analogs and uses thereof
<130>GNCLN171250
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catagtgaag ggaccttcac cagtgatgta agttcttata tggaaggcat cgctgccaag 60
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catagtgaag ggaccttcac cagtgatgta agttcttata tggaaggcat cgctgccaag 60
gacttcgtga actggctgct ggccggcaag 90
<210>33
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>33
tacagtgaag ggaccttcat cagtgattac agtattgcct tggaaaagat tcaccaacaa 60
gacttcgtga actggctgct ggccggccga 90
<210>34
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>34
catagtgaag ggaccttcac cagtgattac agtatttatt tggaaggcca acacgcccaa 60
gacttcgtga actggctgct ggcccaaaag 90
<210>35
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>35
catagtgaag ggaccttcac cagtgatgta agttcttata tggacggcat tcacgcccaa 60
gacttcgtga actggctgct ggccggccga 90
<210>36
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>36
catagtgaag ggaccttcac cagtgatgta agttcttata tggacggcat tcacgcccaa 60
gacttcgtga actggctgct ggccggcaag 90
<210>37
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>37
catagtgaag ggaccttcac cagtgatgta agtatttata tggacggcat tgctgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>38
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>38
catagtgaag ggaccttcac cagtgatgta agttcttata tggacggcat tgctgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>39
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>39
catagtgaag ggaccttcac cagtgatgta agttcttata tggaaggcat tgctgccaag 60
gacttcgtga actggctggt ggccggccga 90
<210>40
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>40
catagtgaag ggaccttcac cagtgatgta agttcttata tggacaagat tgctgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>41
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>41
catagtgaag ggaccttcac cagtgatgta agttcttata tggacaagat tcacgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>42
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>42
catagtgaag ggaccttcac cagtgatgta agttcttata tggacaagat tcacgcccaa 60
gacttcgtga actggctggt ggccggcaag 90
<210>43
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>43
catagtgaag ggaccttcac cagtgatgta agttcttata tggaaggcat tgctgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>44
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>44
tacagtgaag ggaccttcac cagtgatgta agtatttata tggacggcat tgctgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>45
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>45
catagtgaag ggaccttcac cagtgatgta agtatttata tggaaggcat tgctgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>46
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>46
catagtgaag ggaccttcac cagtgatgta agttcttata tggaaaagat tgctgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>47
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>47
catagtgaag ggaccttcac cagtgatgta agttcttata tggacggcat tcacgcccaa 60
gacttcgtga actggctggt ggccggcaag 90
<210>48
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>48
catagtgaag ggaccttcac cagtgatgta agttcttata tggacaagat tcacgcccaa 60
gacttcgtga actggctggt ggccggcaag 90
<210>49
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>49
catagtgaag ggaccttcac cagtgatgta agtatttata tggacaagat tcacgcccaa 60
gacttcgtga actggctggt ggccggcaag 90
<210>50
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>50
catagtgaag ggaccttcac cagtgatgta agtatttata tggacaagat tcacgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>51
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>51
catagtgaag ggaccttcac cagtgatgta agtatttata tggacggcat tcacgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>52
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>52
catagtgaag ggaccttcac cagtgatgta agttcttata tggacggcat tcacgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>53
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>53
catagtgaag ggaccttcac cagtgatgta agttcttata tggacaagat tcacgcccaa 60
gacttcgtga actggctgct ggccggccga 90
<210>54
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>54
catagtgaag ggaccttcac cagtgatgta agtatttata tggacaagat tgtcgcccaa 60
gacttcgtga actggctggt ggccggcaag 90
<210>55
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>55
catagtgaag ggaccttcac cagtgatgta agttcttata tggacaagat tcacgcccaa 60
gacttcgtga actggctggt ggccggccga 90
<210>56
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>56
catagtgaag ggaccttcac cagtgatgta agtatttata tggacaagat tgtcgccaag 60
gacttcgtga actggctggt ggccggcaag 90
<210>57
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>57
catagtgaag ggaccttcac cagtgatgta agttcttata tggaaggcat tcacgccaag 60
gacttcgtga actggctgaa ggccggcaag 90
<210>58
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>58
catagtgaag ggaccttcac cagtgatgta agttcttata tggaaggcat tgctgcccaa 60
gacttcgtga actggctgaa ggccggcaag 90
<210>59
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>59
catagtgaag ggaccttcac cagtgatgta agttcttata tggacggcat tgctgccaag 60
gacttcgtga actggctgaa ggccggcaag 90
<210>60
<211>90
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>60
catagtgaag ggaccttcac cagtgatgta agttcttata tggaaggcat tcacgccaag 60
gacttcgtga actggctgaa ggccggccga 90
<210>61
<211>15
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>61
gacgacgacg acaag 15
<210>62
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>62
tggagtcatc cgcagttcga gaag 24

Claims (12)

1. A protein characterized by: the amino acid sequence of the protein is shown as a sequence 1 in a sequence table.
2. A nucleic acid molecule encoding the protein of claim 1.
3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown as a sequence 31 in a sequence table.
4. A recombinant vector comprising the nucleic acid molecule of claim 2 or 3.
5. An expression cassette comprising the nucleic acid molecule of claim 2 or 3.
6. A transgenic cell line comprising the nucleic acid molecule of claim 2 or 3.
7. A recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.
8. The recombinant vector according to claim 4, wherein: the recombinant vector is a recombinant expression vector or a recombinant cloning vector.
9. The recombinant microorganism according to claim 7, wherein: the recombinant microorganism is a recombinant yeast containing the nucleic acid molecule.
10. Use of a protein according to claim 1 or a nucleic acid molecule according to claim 2 or 3 or a recombinant vector according to claim 4 or 8 or an expression cassette according to claim 5 or a transgenic cell line according to claim 6 or a recombinant microorganism according to claim 7 or 9 for the preparation of a product with hypoglycaemic properties.
11. Use of a protein according to claim 1 or a nucleic acid molecule according to claim 2 or 3 or a recombinant vector according to claim 4 or 8 or an expression cassette according to claim 5 or a transgenic cell line according to claim 6 or a recombinant microorganism according to claim 7 or 9 for the preparation of a medicament for the treatment and/or prevention of diabetes.
12. A product, the active ingredient of which is the protein of claim 1; the product has at least one of the following functions: (a) reducing blood sugar; (b) treating and/or preventing diabetes.
CN201710559884.8A 2017-07-11 2017-07-11 Glucagon-like peptide-1 analogs and uses thereof Active CN107188953B (en)

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CN109730270B (en) * 2019-02-12 2022-06-28 西安培华学院 Sugar-reducing yeast fermented pumpkin powder and preparation method and application thereof
CN115947821B (en) * 2021-10-09 2023-06-30 合肥天汇生物科技有限公司 GLP-1 analogues

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1703424A (en) * 2002-10-11 2005-11-30 株式会社三和化学研究所 GLP-1 derivatives and transmicosal absorption preparations thereof
CN101119749A (en) * 2004-10-25 2008-02-06 赛托斯生物技术公司 Gastric inhibitory polypeptide (gip) antigen arrays and uses thereof
CN104582736A (en) * 2012-06-21 2015-04-29 印第安纳大学研究及科技有限公司 Incretin receptor ligand polypeptide Fc-region fusion polypeptides and conjugates with altered Fc-effector function
CN104829706A (en) * 2008-08-07 2015-08-12 益普生制药股份有限公司 Analogues of glucose-dependent insulinotropic polypeptide

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1703424A (en) * 2002-10-11 2005-11-30 株式会社三和化学研究所 GLP-1 derivatives and transmicosal absorption preparations thereof
CN101119749A (en) * 2004-10-25 2008-02-06 赛托斯生物技术公司 Gastric inhibitory polypeptide (gip) antigen arrays and uses thereof
CN104829706A (en) * 2008-08-07 2015-08-12 益普生制药股份有限公司 Analogues of glucose-dependent insulinotropic polypeptide
CN104582736A (en) * 2012-06-21 2015-04-29 印第安纳大学研究及科技有限公司 Incretin receptor ligand polypeptide Fc-region fusion polypeptides and conjugates with altered Fc-effector function

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