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Hyperglycosylated Extendin-4, fusion protein of analogue thereof, and preparation method and application of fusion protein

A fusion protein and analogue technology, applied in biochemical equipment and methods, chemical equipment and methods, animal/human proteins, etc., can solve the problems of strong immunogenicity and short half-life of Exendin-4

Active Publication Date: 2016-11-16
AMPSOURCE BIOPHARMA (SHANGHAI) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to provide a fusion protein of hyperglycosylated Exendin-4 and its analogs, its preparation method and application, so as to solve the defects of Exendin-4 such as short half-life and strong immunogenicity

Method used

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  • Hyperglycosylated Extendin-4, fusion protein of analogue thereof, and preparation method and application of fusion protein
  • Hyperglycosylated Extendin-4, fusion protein of analogue thereof, and preparation method and application of fusion protein
  • Hyperglycosylated Extendin-4, fusion protein of analogue thereof, and preparation method and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Embodiment 1, construct the expression plasmid encoding fusion protein

[0120] The gene sequences encoding α1 microglobulin (α1 microglobulin) leader peptide and mature Exendin-4 and its analogues, flexible peptide linker, CTP rigid unit and human IgG Fc variants are all artificially optimized CHO cell preferred codons. Long sequences were obtained by chemical synthesis. In order to facilitate the insertion of the target fragment into the specific site of the expression vector, there is a restriction enzyme site at the 5' and 3' ends of the synthesized fragment, respectively SpeI and EcoRI. The verified Exendin-4 and its analog fusion protein genes were digested with SpeI and EcoRI, and then inserted into the corresponding restriction sites of the plasmid PXY1A1 transformed by PCDNA3.1 to obtain the fusion gene expression plasmid. The plasmid contains the early promoter of cytomegalovirus, which is an enhancer required for high-level expression of foreign genes in mam...

Embodiment 2

[0124] Embodiment 2, the expression of fusion protein in transfection cell line

[0125] The recombinant expression vector plasmid is transfected into a mammalian host cell line to express the fusion protein of Exendin-4 and its analogues. In order to stably express at a high level, the preferred host cell line is DHFR enzyme-deficient CHO-cells (see US Pat. No. 4,818,679). In this example, the host cell line is the CHO-derived cell line DXB11. A preferred method of transfection is electroporation, although other methods including calcium phosphate co-sedimentation, lipofection, and protoplast fusion can also be used. In electroporation, use a Gene Pulser electroporator (Bio-Rad Laboratories, Hercules, CA) set to a 300V electric field and a capacitance of 1050 μFd, in a 5×10 cell in a cuvette. 7 Add 50 μg of high-purity expression plasmid to each cell. Two days after transfection, the medium was changed to growth medium containing 0.6 mg / mL G418. Transfectants were screened...

Embodiment 3

[0127] Embodiment 3, purification and qualitative of fusion protein

[0128] The conditioned medium containing the fusion protein was titrated to pH 7-8 with 1N NaOH and filtered through a 0.45 micron nitrocellulose filter. The filtrate was loaded onto a Protein A column equilibrated in phosphate buffered saline (PBS). After the fusion protein is bound to the Protein A column, discard the effluent fraction. The column was washed with PBS until the OD value at 280nm was below 0.01. The bound fusion protein was then eluted with 0.1 M citrate buffer, pH 3.75. Eluate with 0.4 volumes of 1MK 2 HPO 4 For neutralization, fractions containing purified protein were pooled and dialyzed against PBS. Then filter through a 0.22 μm nitrocellulose filter and store at 4 °C. Under non-reducing and reducing conditions, the protein products were identified and analyzed by SDS-PAGE. The fusion protein was quantified by BCA protein assay using BSA as a standard.

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Abstract

The invention discloses hyperglycosylated Extendin-4, fusion protein of analogue thereof, and a preparation method and the application of fusion protein. The fusion protein comprises Extendin-4, the analogue of the Extendin-4, a flexible peptide joint, at least one human chorionic gonadotropin beta carboxyl terminal peptide rigid unit and a human immunoglobulin Fc fragment. The invention also discloses the preparation method and the application of the fusion protein. The fusion protein has optimal biological activity, obviously prolonged circulation half-time, lowered immunogenicity and improved bioavailability. The fusion protein can be used for treating diabetes, obesity and other diseases benefited by lowering fasting plasma glucose, inhibiting stomach and / or bowel movement and inhibiting and / or bowel evacuation or inhibiting food intake.

Description

technical field [0001] The present invention relates to long-acting GLP-1 analogues, more specifically, to fusion proteins of Exendin-4 and analogues thereof, and also to its preparation method and its role in the treatment of diabetes, obesity and by lowering plasma glucose, inhibiting gastric and / or Use in bowel motility and other diseases benefiting from inhibition of gastric and / or intestinal emptying, or inhibition of food intake. Background technique [0002] Exendin-4 is isolated from the saliva of giant monster lizard. It has 53% homology with mammalian GLP-1 amino acid sequence, and is an effective GLP-1 receptor agonist. The physiological activity of Exendin-4 is similar to that of GLP-1, but the second position of Exendin-4-NH2 is replaced by Gly in the Ala in GLP-1, so that it has a certain resistance to DPP-IV enzyme and is not easy to be detected by DPP-IV. IV is degraded, and the half-life in the circulation in the body is relatively long, which can reach 60-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K38/22A61K47/48A61P3/04A61P3/10
CPCA61K38/00C07K14/57563C07K14/59C07K2319/30C12N15/85C12N2800/107
Inventor 李强董炤王著李媛丽冯雄李子瑞
Owner AMPSOURCE BIOPHARMA (SHANGHAI) INC
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