CN118620086A - Fusion protein for treating diabetes and application thereof - Google Patents
Fusion protein for treating diabetes and application thereof Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to the field of fusion proteins, in particular to a fusion protein for treating diabetes and application thereof. The fusion protein containing Exendin-4 provided by the invention is formed by fusing Exendin-4-C1, exendin-4-C2 with specific mutation and Fc fragment mutant of human immunoglobulin IgG1 with specific mutation. The fusion protein has higher biological activity of Exendin-4 and therapeutic effects on type I, type II diabetes and obesity, and has stronger glucose tolerance activity and longer half-life, and the blood sugar reducing effect of the fusion protein freeze-dried powder preparation containing Exendin-4 provided by the invention is obviously better than that of E4F4 fusion protein by directly pulmonary administration.
Description
Technical Field
The invention relates to the field of fusion proteins, in particular to a fusion protein for treating diabetes and application thereof.
Background
Diabetes is a chronic disease involving multiple causes, and involves inheritance, metabolism, nutrition, lifestyle habits, and the like. The pathogenesis of the diabetes mellitus is familial aggregation, inheritance is an important pathogenesis factor of diabetes mellitus, and simultaneously, the inheritance external factors are controlled by dietary structure, living habit, physical activity and weight.
Exendin-4 is a short-intestine insulinotropic polypeptide, which belongs to a glucagon-like peptide-1 (GLP-1) receptor long-acting agonist. In recent years, the research shows that Exendin-4 can act on islet beta cells through various ways to play a role in treating diabetes. In one aspect, exendin-4 may modulate insulin secretion by specifically binding to GLP-1 receptors on islet beta cells; on the other hand, the Exendin-4 can also promote proliferation of islet beta cells, regulate and control autophagy of cells, inhibit apoptosis of cells and protect islet functions. Exendin-4 can stimulate insulin secretion when blood sugar is high, so that blood sugar is reduced; this effect is not present when the blood sugar is low, so that it does not cause hypoglycemia. Exendin-4 can obviously reduce fasting and postprandial blood sugar and HbA1c of a T2DM patient, obviously reduce weight and improve lipid metabolism, and animal researches show that the Exendin-4 increases the number of beta-cells and has better curative effect on type II diabetes.
With the rapid development of biotechnology, it has become a recent research hotspot to extend the half-life of drugs in vivo and prevent them from being rapidly cleared. Established techniques for increasing half-life of drugs include chemical modification, microencapsulation, glycosylation, protease resistant mutants, protein fusion, etc., and many long-acting protein drugs have been developed and applied to clinical treatment. Meanwhile, the Fc fusion proteins currently on the market often use a fusion of a target protein and an Fc fragment of IgG, which reduces the in vitro activity of the target protein, especially the polypeptide with smaller self molecular weight. In particular to the field of fusion proteins of exendin-4 and Fc, the fusion protein containing exendin-4 has poor stability, low biological activity, unobvious treatment effect and insufficient safety, and further cannot meet the market demand.
Disclosure of Invention
The invention aims to solve the technical problems of unobvious effect, poor glucose tolerance and short half life of the existing fusion protein containing the Exendin-4 polypeptide on diabetes and obesity.
The technical problem to be solved by the invention is to provide a novel fusion protein containing Exendin-4 polypeptide, which is characterized in that the fusion protein has the following structure: the Fc segment mutant sequentially comprises Exendin-4-C1, a first connecting peptide, exendin-4-C2, a second connecting peptide and IgM from the nitrogen end. Namely, the structure of the fusion protein containing the Exendin-4 polypeptide is an Fc segment mutant of Ex 4-C1-first connecting peptide-Ex 4-C2-second connecting peptide-IgG 1-Fc from the nitrogen end.
Wherein, the amino acid sequence of the Fc segment mutant of the human IgG1 is shown in SEQ ID No. 5;
further, the amino acid sequence of Exendin-4-C1 is shown as SEQ ID No. 2;
Ex4-C1：HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO:2)；
Further, the amino acid sequence of Exendin-4-C2 is shown as SEQ ID No. 3;
Ex4-C2：HGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPS(SEQ ID NO:3)；
the fusion proteins are recombinant from Exendin-4-C1, exendin-4-C2 and human IgG1Fc mutant, and the connecting peptide between the fusion proteins needs to have enough length and better flexibility so as to ensure that 2 connected molecules can be folded correctly and ensure the biological activity of the fusion proteins. Flexible peptide stretches of 5-25 amino acids are typically used.
The amino acid sequence of the connecting peptide can be shown as SEQ ID No. 6;
Linker：GGGGSGGGGSGGGGS(SEQ ID NO:6)；
further, the amino acid sequence of the fusion protein is shown as SEQ ID No. 9.
Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant：HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSHGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPSGGGGSGGGGSGGGGSPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHRAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCSVSNKALPAPIQKTISKDKGQPREPQKYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDGSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO:9)
Furthermore, the fusion protein also comprises a signal peptide at the nitrogen end;
ssmIg：MGWSCIILFLVATATGVHS(SEQ ID NO:7)；
Furthermore, the nucleotide sequence of the fusion protein is shown as SEQ ID No.10 or a degenerate sequence thereof;
the invention also provides a gene vector containing a nucleotide sequence shown as SEQ ID No. 10;
the invention also provides a host cell containing the gene vector;
In addition, the invention also provides a medicine for treating diabetes or obesity, which is prepared by taking the fusion protein as a main active ingredient, wherein the fusion protein is further a freeze-dried powder preparation;
The invention also provides application of the medicine in preparing medicines for treating diabetes or obesity, and preferably, the diabetes is type 1 diabetes or type 2 diabetes.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the fusion protein containing Exendin-4 provided by the invention is formed by fusing Exendin-4-C1, exendin-4-C2 and Fc fragment mutants of specific mutated human immunoglobulin IgG 1. The fusion protein has higher biological activity of Exendin-4 and therapeutic effect for type I, type II diabetes and obesity, and has stronger glucose tolerance activity and longer half-life, even if a freeze-dried powder preparation of the fusion protein is used for pulmonary administration, the short-time blood sugar reducing effect of the fusion protein containing Exendin-4 provided by the invention is obviously better than the prior art.
Drawings
FIG. 1 shows the result of gel electrophoresis of pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant gene fragment
FIG. 2 elution pattern, SDS-PAGE and Western Blot results of G-25 gel filtration column of Ex4C1-Linker-Ex4C2-Linker-IgG 1-Fc-variant fusion protein
FIG. 3 changes in body weight and food intake of mice in NS group, ex4C1-Ex4C2-F1 group, E4F4 group, control group
FIG. 4 shows glucose tolerance in mice plotted as a blood glucose concentration-time curve based on blood glucose values measured at different time points
FIG. 5 half-life of Ex4C1-Linker-Ex4C2-Linker-IgG 1-Fc-variant fusion proteins in vivo
Detailed Description
While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Example 1ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein expression Gene Synthesis and vector construction
Fusion protein sequence design, mutant human IgG1-Fc fragments were fused to the C-terminus of Ex4-C1 and Ex 4-C2. Meanwhile, in order to realize secretory expression of the fusion protein, a mouse signal peptide sequence is added at the N-end, so that the final fusion protein is obtained as follows: ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG 1-Fc-variant.
Exendin-4 parent peptide: HGEGTFTSDLSKQMEEAVRLFEIEWLKNGPSSGAPPPS (SEQ ID NO: 1);
Ex4-C1：HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO:2)；
wherein the amino acid sequence of Ex4-C1 is completely the same as that of Exendin-4 parent peptide;
Ex4-C2：HGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPS(SEQ ID NO:3)；
IgG1-Fc：PTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHHAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKALPAPIQKTISKDKGQPREPQVYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO:4);
IgG1-Fc-Mutant：PTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHRAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCSVSNKALPAPIQKTISKDKGQPREPQKYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDGSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO:5);
Linker：GGGGSGGGGSGGGGS(SEQ ID NO:6)；
ssmIg：MGWSCIILFLVATATGVHS(SEQ ID NO:7)；
ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant：MGWSCIILFLVATATGVHSHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSHGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPSGGGGSGGGGSGGGGSPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHRAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCSVSNKALPAPIQKTISKDKGQPREPQKYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDGSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO:8);
Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant：HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSHGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPSGGGGSGGGGSGGGGSPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHRAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCSVSNKALPAPIQKTISKDKGQPREPQKYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDGSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO:9)
The corresponding coding nucleotide sequence of ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG 1-Fc-variant fusion protein is shown below:
atgggctggagctgcattattctgtttctggtggcgaccgcgaccggcgtgcatagccatggcgaaggcacctttaccagcgatctgagcaaacagatggaagaagaagcggtgcgcctgtttattgaatggctgaaaaacggcggcccgagcagcggcgcgccgccgccgagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagccatggcgaaggcaccgcgaccagcgatctgagcaaacagatggaacgcgaagcggtgcgcctgtttattgaatggctgaaaaacggcggcccgagcaccggcgcgccgccgccgagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcccgacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccaggaagatccggatgtgaaatttaactggtatgtgaacggcgcggaagtgcatcgcgcgcagaccaaaccgcgcgaaacccagtataacagcacctatcgcgtggtgagcgtgctgaccgtgacccatcaggattggctgaacggcaaagaatatacctgcagcgtgagcaacaaagcgctgccggcgccgattcagaaaaccattagcaaagataaaggccagccgcgcgaaccgcagaaatataccctgccgccgagccgcgaagaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgtggtggaatgggaaagcagcggccagccggaaaacacctataaaaccaccccgccggtgctggatagcgatggcagctattttctgtatagcaaactgaccgtggatggcagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcgtgagcccgggcaaa(SEQ ID NO:10);
Obtaining a DNA fragment (SEQ ID No. 10) of ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein containing a mouse signal peptide by adopting a total gene synthesis method, and directly inserting the synthesized fragment into an expression vector pcDNA3.1 to obtain a recombinant expression plasmid pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant; the PCR amplified plasmid product is analyzed by 2% agarose gel electrophoresis, and the pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant gene fragment with the size consistent with the expected size is seen; about 5427bp of vector fragment and about 1053bp of target gene fragment (FIG. 1) can be seen by double cleavage with NotI and EcoRI, wherein Lane1：pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant;Lane2：pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant is subjected to double cleavage with NotI and EcoRI; the results of Lane1 and Lane2 are respectively consistent with the theoretical values, the recombinant plasmid is sent to the Shanghai worker organism for sequencing, and the sequencing result is completely consistent with the ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein gene sequence, which indicates that the recombinant expression plasmid pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant is successfully constructed.
Example 2 expression and purification of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion proteins
CHO-S suspension cells were cultured and passaged in ExpiCHO Expression Medium medium at 37 ℃, 8% co 2 saturated humidity and additional shaker speed of 10 9r·min-1 in a thermostated incubator. When the cell growth density reaches 7X 10 10L-1, the transfected cells are diluted to 6X 10 10L-1, under the mediation of ExpiFectamine TM CHO Reagent, 25ug of recombinant plasmid pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant is transferred into CHO-S suspension cells, 150ul ExpiCHOTM Enhancer and 6mL ExpiCHOTM Feed are respectively added after 20h of transfection, and the culture is continued.
During the culture, the medium was changed every 3 days until cell clones formed. Monoclonal cells were isolated and expanded into stable cell lines and supernatants of tissue cultures grown on 24-well plates were assayed for fusion proteins by SDS-PAGE or Elisa. Cells were selected for secretion of large amounts of the fusion protein and stable passage was used for later characterization. Inoculating the CHO engineering cell strain with stable passage into 25cm 2T-shaped bottles, shaking and culturing for 4-5 days, amplifying into triangular flasks, culturing for 7-10 days, separating cell culture solution containing fusion Protein, purifying by Protein A affinity chromatography medium, sepharose SP XL chromatography column, butyl Sepharose FF chromatography column and DEAE Sepharose FF chromatography column, replacing with G-25 gel filtration column into preparation buffer solution, eluting with G-25 gel filtration column of Ex4C1-Linker-Ex4C2-Linker-IgG 1-Fc-instant fusion Protein, SDS-PAGE and Western Blot results (figure 2);
FIG. 2 shows that the elution diagram of the filter column shows that the Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein has fewer impurities, and the target protein is mainly concentrated in peak (1); SDS-PAGE shows that the molecular weight results of the fusion proteins of lane (1) are all consistent with the theoretical 35 kDa; western Blot shows that the fusion protein is secreted and expressed in a CHO engineering cell strain culture medium and can be specifically identified by an anti-IgG 1-Fc antibody and an anti-Exendin-4 antibody, and the results show that the Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein is successfully expressed and purified.
Example 3 body weight and food intake therapeutic Effect of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion proteins on obese mice diabetes model
Establishing a mouse diet-induced obesity model (DIO) to study the treatment effect of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein on a mouse diabetes model in vivo, and experimental methods: C57/B6 male mice with the weight of 40-50 g are selected and randomly divided into 4 groups:
saline group (NS group): feeding with high fat fodder for 10 weeks to form obesity;
ex4C1-Linker-Ex4C2-Linker-IgG 1-Fc-variant fusion protein experimental group prepared in example 2 (Ex 4C1-Ex4C2-F1 group): feeding with high fat fodder for 10 weeks to form obesity;
E4F4 fusion protein control group (E4F 4 group) prepared according to the method described in CN106146667B patent document: feeding with high fat fodder for 10 weeks to form obesity;
normal group (Control group): high-fat feed is not fed in the same period;
The administration was performed as follows:
Saline group (NS group): subcutaneous administration, 320 μg kg-1 in physiological saline once a week;
Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein experimental group (Ex 4C1-Ex4C2-F1 group): subcutaneous administration, 320 μg kg-1 once a week;
E4F4 fusion protein control group (E4F 4 group) prepared according to the method described in CN106146667B patent document: subcutaneous administration, 320 μg kg-1 once a week; four groups of body weight and food intake were weighed daily (fig. 3).
The results of fig. 3 show that: the E4F4 fusion protein group and the Ex4C1-Ex4C2-F1 fusion protein group can obviously inhibit the weight increase of mice by adopting 1-time administration of one week; and the effect of inhibiting the weight gain of the Ex4C1-Ex4C2-F1 group is far better than that of the E4F4 group; the feeding amount of the mice is weighed every day, and compared with the NS group, the Ex4C1-Ex4C2-F1 fusion protein group has a certain inhibiting effect on the feeding amount of the mice, and the inhibiting effect on the feeding amount is far better than that of the E4F4 fusion protein group.
Example 4 glucose tolerance Activity experiment of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein
18 Normal C57BL/6 mice were taken and randomly divided into 3 groups according to body mass, 6 mice in each group were numbered and administered in the following manner:
To the E4F4 fusion protein group (E4F 4 group) prepared according to the method described in CN106146667B patent document: dose 320 mug.kg-1;
ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein group (Ex 4C1-Ex4C2-F1 group) prepared according to example 2: dose 320 mug.kg-1;
Blank Control group (Control group): 10 mmol.L-1 phosphate buffer, pH7.4;
the mice were evaluated for glucose tolerance by intraperitoneal injection of 40% glucose (0.2 mL) at 12h after administration, blood collection from tail veins at 0, 15, 30, 60, 90, 100, and 120min before and after injection, and blood glucose level measurement by a blood glucose meter. The graph of blood glucose concentration versus time is shown in FIG. 4, which is plotted against blood glucose values measured at different time points.
The results in FIG. 4 show that the E4F4 fusion protein group and the Ex4C1-Ex4C2-F1 fusion protein group still have significant hypoglycemic effects when glucose is injected for 15min and 30min 12h after administration compared with the blank and the cable Ma Lutai, and that the Ex4C1-Ex4C2-F1 fusion protein group can maintain a lower blood glucose concentration compared with the E4F4 fusion protein group, and the maintenance time of the low blood glucose concentration is longer, i.e., the glucose tolerance activity of the Ex4C1-Ex4C2-F1 fusion protein group is far better than that of the E4F4 fusion protein group.
Example 5 in vivo half-life detection of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion proteins
4 SD male rats of 8 weeks old were selected and 200. Mu.g/kg of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein prepared according to example 2 was injected into the Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein group; detecting the change of the content of the Exendin-4 within 49 days, taking blood at different time points 0,1, 2, 4, 6, 14, 21, 28, 35, 42 and 49 days, and detecting the content of the Exendin-4 in serum by using an Exendin-4 detection kit. Serum of the Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein group can be continuously detected for 49 days at the content of Exendin-4 (as shown in figure 5), and the half-life t1/2 of the Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein in vivo is calculated to be about 10 days.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (10)
1. A fusion protein for use in the treatment of diabetes, the fusion protein having the structure: from the nitrogen end, the sequence is shown as SEQ ID NO:2, a first connecting peptide, an Exendin-4-C1 polypeptide shown as SEQ ID NO:3, a second connecting peptide, and a polypeptide of Exendin-4-C2 shown as SEQ ID NO:5, an Fc fragment mutant of IgG 1; optionally, the first and second connecting peptides may be identical.
2. The fusion protein of claim 1, wherein the nitrogen terminus further comprises a signal peptide.
3. The fusion protein of claim 2, wherein the signal peptide is as set forth in SEQ id no: 7.
4.A fusion protein according to any one of claims 1 to 3, wherein the first and second connecting peptides are as set forth in SEQ ID NOs: 6, and a polypeptide having the amino acid sequence shown in FIG. 6.
5. The fusion protein of claim 1, wherein the fusion protein is as set forth in SEQ ID NO: 9.
6. A nucleic acid encoding the fusion protein of any one of claims 2-5, wherein the nucleic acid sequence is SEQ ID No.10 or a degenerate sequence thereof.
7. A genetic vector comprising the nucleic acid of claim 6.
8. A host cell comprising the genetic vector of claim 7.
9. A pharmaceutical composition for the treatment of type 2 diabetes or diet-induced obesity comprising a lyophilized powder of the fusion protein of claim 1 or 5 and a pharmaceutically acceptable adjuvant.
10. Use of the fusion protein of any one of claims 1-5 and the pharmaceutical composition of claim 9 in the manufacture of a medicament for the treatment of type 2 diabetes or diet-induced obesity.
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| CN101525386A (en) * | 2008-03-05 | 2009-09-09 | 浙江华阳药业有限公司 | Fusion protein of Exendin-4 tandem polypeptide and human serum albumin, preparation and application thereof |
| CN106146667A (en) * | 2015-03-27 | 2016-11-23 | 四川大学华西医院 | Exendin-4 fusion protein and preparation method and application thereof |
| US20240150423A1 (en) * | 2019-10-12 | 2024-05-09 | Nanjing Finepeptide Biopharmaceutical Co.,Ltd | Glp-1 analogue-modified dimers with different configurations, preparation method thereof, and application thereof in treatment of type ii diabetes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101525386A (en) * | 2008-03-05 | 2009-09-09 | 浙江华阳药业有限公司 | Fusion protein of Exendin-4 tandem polypeptide and human serum albumin, preparation and application thereof |
| CN106146667A (en) * | 2015-03-27 | 2016-11-23 | 四川大学华西医院 | Exendin-4 fusion protein and preparation method and application thereof |
| US20240150423A1 (en) * | 2019-10-12 | 2024-05-09 | Nanjing Finepeptide Biopharmaceutical Co.,Ltd | Glp-1 analogue-modified dimers with different configurations, preparation method thereof, and application thereof in treatment of type ii diabetes |
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| 王圣钧;郁慧丽;翟琳;王倩;梁泉峰;祁庆生;: "人GLP-1-IgG Fc融合蛋白在毕赤酵母中的高效表达", 生物技术, no. 04, 15 August 2011 (2011-08-15), pages 31 - 35 * |
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