Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Synergistic bifunctional protein for regulating blood sugar and lipid

A bifunctional protein, amino acid technology, applied in glucagon, peptide/protein components, animal/human proteins, etc., can solve the problems of short half-life and unstable structure in vivo

Active Publication Date: 2019-07-19
AMPSOURCE BIOPHARMA (SHANGHAI) INC
View PDF27 Cites 18 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims to provide a bifunctional protein comprising human GLP-1 analogs and human FGF21 that has synergistic effects on blood sugar and lipid regulation, its preparation method and use, while solving the structural instability of natural GLP-1 and FGF21 , short half-life in vivo and other defects, retain the strong hypoglycemic effect of GLP-1 and the physiological effects of FGF21 in the treatment of insulin sensitization, weight loss, fatty liver and hypercholesterolemia, and alleviate the gastrointestinal tract caused by GLP-1 to a certain extent Adverse reactions

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Synergistic bifunctional protein for regulating blood sugar and lipid
  • Synergistic bifunctional protein for regulating blood sugar and lipid
  • Synergistic bifunctional protein for regulating blood sugar and lipid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1, construction of synergistic bifunctional protein expression plasmid

[0124] Gene sequences encoding α1 microglobulin leader peptide, GLP-1 analog, L1, FGF21 mature protein, L2 (both flexible and rigid peptide units) and human IgG Fc variants are artificially optimized CHO cell-preferred codes The full-length sequence was 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 synergistic bifunctional protein gene was digested with SpeI and EcoRI, and then inserted into the corresponding restriction site of the plasmid pXY1A1 transformed by pDNA3.1 to obtain the fusion gene expression plasmid ( figure 1 The nucleotide sequence and translated amino acid sequence of the potentiating bifunctional protein FP4I-2 are exemplarily listed). The plasmid co...

Embodiment 2

[0129] Example 2, expression of synergistic bifunctional protein in transfected cell lines

[0130] The recombinant expression vector plasmid is transfected into a mammalian host cell line to express a potent bifunctional 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 can also be used. In electroporation, use a Gene Pulser electroporator (Bio-Rad Laboratories, Hercules, CA) set to a 300V electric field and a 1500 μFd capacitance, 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 for resistance to the selective dr...

Embodiment 3

[0132] Example 3. Purification and characterization of synergistic bifunctional protein

[0133] The purification and characterization methods of FP4I-2 are exemplarily described in this example. The cell culture supernatant was clarified by high-speed low-temperature centrifugation and 0.22 μm sterile filtration, and then purified by three-step chromatography of protein A affinity, anion exchange and hydrophobicity. The specific method is as follows: the first step captures the protein A affinity layer Analysis, the balance solution is PBS buffer, the eluent is citrate buffer with pH 3.5, and the eluted target protein is then neutralized with 1M Tris solution. For intermediate purification, high-resolution anion exchange filler Q Sepharose HP (GE Company) was selected to remove residual impurity proteins. In binding mode, rinse with 20mM Tris-HCl, 0.2M NaCl, pH 7.5 solution and elute with 20mM Tris-HCl, 0.3M NaCl, pH 7.5 solution. In the purification step, Butyl Sepharose F...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a synergistic bifunctional protein for regulating blood sugar and lipid. The synergistic bifunctional protein comprises a human GLP-1 analog and human FGF21. In one aspect, the invention provides a method for preparing the synergistic bifunctional protein, and on the other hand, the invention also provides an application of the synergistic bifunctional protein for treatment of type 2 diabetes, obesity, dyslipidemia, fatty liver disease and / or metabolism syndrome drugs. The synergistic bifunctional protein provided by the invention can synergistically regulate blood sugar and lipid levels in vivo, and meet the multiple needs of hypoglycemia, alleviation of liver fatty degeneration, weight reduction and improvement of circulating lipid metabolism disorder in type 2 diabetic patients.

Description

technical field [0001] The present invention relates to a GLP-1-FGF21 synergistic bifunctional protein and a pharmaceutical composition thereof, and also relates to the use of the synergistic bifunctional protein in preparing and treating type 2 diabetes, obesity, hyperlipidemia, fatty liver disease and / or metabolism Use in Syndromic Medicine. Background technique [0002] Glucagon-like peptide-1 (GLP-1), a 36-amino acid incretin secreted by mammalian intestinal L cells, binds and activates the GLP-1 receptor (GLP-1R) to Glucose-dependent way stimulates islet β cells to secrete insulin, inhibits islet α cells to release glucagon, exerts biological effects of controlling blood sugar, and at the same time exhibits biological effects of inhibiting gastrointestinal motility and controlling appetite (Knudsen LB, J Med Chem, 2004, 4128-4134). Natural human GLP-1 is easily inactivated by dipeptidyl peptidase IV (DDP-IV) in vivo, making its half-life short. Exendin (Exendin-4) is...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K38/26A61K38/18A61P1/16A61P1/18A61P3/04A61P3/06A61P3/10A61P9/04A61P9/10A61P9/12A61P13/12
CPCC07K14/605C07K14/503C12N15/85C07K2319/30A61K38/26A61K38/1825A61K47/65A61P3/04A61P3/10A61P1/18A61P3/06A61P1/16A61P5/50A61P3/00A61P9/10A61P9/12A61P9/14A61P9/04A61P13/12A61P25/00C12N2800/107
Inventor 董炤周弛冯雄张吉余贾世香李强
Owner AMPSOURCE BIOPHARMA (SHANGHAI) INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com