Preparation method of GLP (Glucagon-Like Peptide) -1 or analogue thereof and antibody Fc fragment fusion protein

A GLP-1, fusion protein technology, applied in the biological field, can solve problems such as uneconomical, reducing the degree of degradation of the N-terminal of GLP-1-Fc molecule, and can not really solve the problem of degradation

Active Publication Date: 2015-01-21
SYNDEGEN SHANGHAI BIOTECH
View PDF6 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the same article, it was mentioned that protease inhibitors (benzamidine hydrochloride) were added to control the degradation during the cell culture process, but the risk of drugging the product still increased, and it was also necessary to inactivate and eradicate the protease inhibitors in the subsequent process. It will make the preparation process of the whole product more complicated and uneconomical, and the addition of protease inhibitors can only reduce the degree of degradation of

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
  • Preparation method of GLP (Glucagon-Like Peptide) -1 or analogue thereof and antibody Fc fragment fusion protein
  • Preparation method of GLP (Glucagon-Like Peptide) -1 or analogue thereof and antibody Fc fragment fusion protein
  • Preparation method of GLP (Glucagon-Like Peptide) -1 or analogue thereof and antibody Fc fragment fusion protein

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0043] The invention provides a method for preparing a fusion protein of GLP-1 or its analogue and an antibody Fc fragment, comprising the following steps:

[0044] 1) Cloning the coding sequence of GLP-1 or its analogue and antibody Fc fragment fusion protein (GLP-1-Fc) into an expression vector;

[0045] 2) Transfect the expression vector into CHO-DXB11 cells, cultivate and screen positive cell lines;

[0046] 3) After using the cells obtained in step 2 to express and purify, the fusion protein is obtained.

[0047] In the preparation method of the fusion protein of GLP-1 or its analogue and antibody Fc fragment provided by the present invention, the fusion protein of GLP-1 or its analogue and antibody Fc fragment is composed of recombinant human glucagon-like peptide-1 (GLP- 1) or its analogues are formed by linking a linking peptide with an antibody Fc fragment that can prolong the half-life in the human body.

[0048] In the fusion protein of GLP-1 or its analogue and a...

Embodiment 1

[0081] Coding sequence of recombinant human GLP-1-Fc fusion protein

[0082] 1. Amino acid composition of GLP-1

[0083] Natural GLP-1 and any modified GLP-1 molecules can form fusion proteins by linking different flexible linking peptides with different Fc fragments, such as IgG1, IgG2, IgG3, IgG4 and modified ones. These fusion proteins Both have biological activity and long-term effect.

[0084] The amino acid sequence of native GLP-1 is as follows:

[0085] HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID No: 14).

[0086] The fusion protein molecule of the present invention is a variant modified on the basis of the original GLP-1 molecule, specifically two variants modified on the basis of the 31 amino acids of the original GLP-1 molecule. Its name and molecule The structures are:

[0087] Molecular name: G1 (1 amino acid modification in the 31 amino acid GLP-1 molecule)

[0088] Modification: Gly8-GLP-1 (7-37), the first amino acid number of the original GLP-1 is 7 (position...

Embodiment 2

[0114] Expression of GLP-1-Fc fusion protein in CHO-DXB11 and CHO-DG44 cells:

[0115] Two kinds of proteins (G1-L5-Fc and G3-L5-Fc) coding sequence SEQ ID No: 12 and SEQ ID No :13 was cloned into the expression vector sequence containing IRES-DHFR, and the expression vector expressed GLP-1-Fc under the drive of CMV promoter.

[0116] The coding sequence of G1-L5-Fc is shown in SEQ ID No: 12: (signal peptide containing 19aa)

[0117] atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttaccagtgatgtaagttcttatttggaaggacaagctgccaaggagttcattgcttggctggtgaaaggccgtggaggatccggtggcggttccggcggtggaggatcaggaggcggtggctctggaggtggtggaagtggtggcggcggttcgtctaagtacgggcccccttgccctccttgcccagctcctgaatttgagggcggacccagcgtgttcctgttccccccaaagcccaaggacaccctgatgatcagcagaacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcc...

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 the technical field of biology and particularly relates to a preparation method of a GLP (Glucagon-Like Peptide) -1 or analogues thereof and an antibody Fc fragment fusion protein. The preparation method of the GLP-1 or the analogues thereof and the antibody Fc fragment fusion protein comprises the following steps: cloning sequences for encoding the GLP-1 or the analogues thereof and the antibody Fc fragment fusion protein into an expression vector; transfecting the expression vector with CHO-DXB11 cells, culturing and screening a positive cell strain; expressing the obtained cell strain and purifying to obtain the fusion protein. By virtue of the preparation method provided by the invention, when the GLP-1-Fc molecule is expressed in DXB11 of the CHO cell line, the problem of degradation cannot be caused, the subsequent purification yield is greatly improved, the biological activity is not decreased and thus the preparation method is very suitable for the requirement of industrial production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of fusion protein of GLP-1 or its analogue and antibody Fc fragment. Background technique [0002] Glucagon-like peptide-1 (GLP-1) promotes insulin release, reduces plasma glucagon levels, reduces the rate of gastric emptying, promotes satiety and stimulates islet biosynthesis and β-cell proliferation , can be used for the treatment of type 2 diabetes. Type 2 diabetes is common in our country. If it is not treated in time, it will easily transform into type 1 diabetes, which will cause a great burden to patients and the country. Therefore, the demand for developing such drugs is extremely urgent. At present, Exenatide (Byetta) of Eli Lilly and Company and Liraglutide (Novoli) of Danish Novo Nordisk Pharmaceutical Company have obtained import registration in my country. Both drugs have the effect of lowering blood sugar and reducing weight. effect, but requires ...

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): C12N15/85C07K19/00C12P21/02A61K38/26A61K47/48A61P3/10
Inventor 许必雄郭颀然赵红阳
Owner SYNDEGEN SHANGHAI BIOTECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap