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Exendin-4 analog fusion protein and its preparation method and use

A technology of fusion proteins and analogs, applied in the directions of drug combinations, peptide/protein components, chemical instruments and methods, etc., can solve the problems of no clinical use value, damage to cytotoxicity and complement activation, and small molecular weight. Prolonged circulating half-life in vivo, prolonged functional half-life in vivo, well-tolerated effect

Active Publication Date: 2017-11-28
AMPSOURCE BIOPHARMA (SHANGHAI) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Natural GLP-1 can be rapidly hydrolyzed and inactivated by dipeptidyl peptidase IV (DPP-IV), and its molecular weight is small, so it is easily eliminated by glomerular filtration, making its half-life in vivo circulation less than 5 minutes, and has no clinical significance. use value
Obviously, the cytotoxicity and complement activation mediated by the Fc segment of the fusion protein disclosed in the patent CN101891823 may cause certain damage to the body

Method used

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  • Exendin-4 analog fusion protein and its preparation method and use
  • Exendin-4 analog fusion protein and its preparation method and use
  • Exendin-4 analog fusion protein and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, construct the expression plasmid encoding Ex(1-45)-L-vFc fusion protein

[0053] The gene sequence encoding α1 microglobulin (α1microglobulin) leader peptide and mature Ex(1-45) polypeptide, the 10 amino acid flexible peptide linker (GlyGlyGlyGlySerGlyGlyGlyGlySer) and the fusion gene of human IgG2Fc variant (Pro331Ser / Thr250Gln / Met428Leu mutation) are all It is an artificially optimized CHO cell preferred codon, the full-length sequence is obtained by chemical synthesis (see the fusion protein nucleotide and amino acid sequence figure 1). In order to facilitate the insertion of the target fragment into the specific site of the expression vector, there is a restriction enzyme endonuclease site at the 5' and 3' ends of the synthesized fragment, respectively SpeI and EcoRI. After verification, the GLP-1 gene was digested with SpeI and EcoRI, and then inserted into the corresponding restriction site of the transformed plasmid pCDNA3.1 to obtain the fusion ge...

Embodiment 2

[0054] Embodiment 2, the expression of fusion protein in transfection cell line

[0055] The recombinant expression vector plasmid was transfected into a mammalian host cell line to express the Ex(1-45)-L-vFc fusion protein. 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, with a Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) set to a 240V electric field and a 1050 μFd capacitance, 3×10 cells in a cuvette were used. 7 Add 40 μg of plasmid linearized with PvuI to each cell. Two days after transfection, the medium was changed to growth medium containing 0.6 mg / mL G418. Transfectants were screened for resistan...

Embodiment 3

[0057] Embodiment 3, purification and qualitative of fusion protein

[0058] 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. with 0.4 volumes of 1M K 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 conditions, the molecular weight of the purified Ex(1-45)-L-vFc protein ranged from 40 to 45 kDa as determined by SDS-PAGE. Under reducing conditions, the purified protein migrates to approximately 90 k...

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Abstract

The invention discloses an Exendin-4 analogue fused with a human IgG2 Fc variation and a preparation method of the Exendin-4 analogue. The circulating half-life period of the fusion protein is remarkably prolonged, and the biological availability is greatly improved. The fusion protein can be used for treating diabetes, obesity and other diseases that are benefited by lowering plasma glucose, inhibiting gastric and / or intestinal motion and inhibiting gastric and / or intestinal emptying or inhibiting food intake.

Description

technical field [0001] The present invention relates to Exendin-4 analogues, more specifically, a fusion protein composed of a recombinant Exendin-4 analogue and a human IgG2 Fc variant and a preparation method thereof, and also relates to its role in treating diabetes, obesity and reducing plasma glucose, Use in other diseases benefiting from inhibition of gastric and / or intestinal motility and inhibition of gastric and / or intestinal emptying, or inhibition of food intake. Background technique [0002] Glucagon-like peptide-1 (GLP-1), also known as insulin-stimulating hormone, is a polypeptide hormone mainly secreted by intestinal mucosal L cells. GLP-1 has the function of regulating glucose metabolism and gastrointestinal secretion. It can effectively stimulate the glucose-dependent insulin secretion of pancreatic β-cells, promote insulin gene transcription, islet cell proliferation, and β-cell proliferation and regeneration. It has independent insulin-independent Hypogly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K38/22A61K47/42A61P3/10A61P3/04
Inventor 李强李媛丽王著董炤李子瑞武翠
Owner AMPSOURCE BIOPHARMA (SHANGHAI) INC
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