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A glucagon-like peptide-1 receptor agonist fusion protein and applications thereof

A fusion protein and receptor technology, applied in the field of fusion proteins, can solve the problems of long medication time, poor patient compliance, and inconvenience

Inactive Publication Date: 2017-12-15
BEIJING BIOHELIX BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the huge population of people with diabetes (currently there are only 96 million diabetics in China) and the long duration of medication for chronic diseases, as well as the pain, inconvenience, and poor patient compliance of injection preparations, it is necessary to develop oral medicines for the treatment of diabetes with good curative effect and reasonable price. Protein drugs are very necessary and urgently needed

Method used

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  • A glucagon-like peptide-1 receptor agonist fusion protein and applications thereof
  • A glucagon-like peptide-1 receptor agonist fusion protein and applications thereof
  • A glucagon-like peptide-1 receptor agonist fusion protein and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1: Construction of expression vector

[0086] The gene shown in SEQ ID NO.2 and the linker gene shown in SEQ ID NO.9 and the GLP-1 gene shown in SEQ ID NO.4 are connected by a fully synthetic method as shown in SEQ ID NO.13 (nomenclature OPG-A (CTB-L1-G), placed in the vector pUC57, named pUC57-OPG-A. Using SEQ ID NO.16primer F and SEQ ID NO.17primer R as the upstream and downstream primers respectively, and using the pUC57-OPG-A plasmid as the template, PCR amplification was performed, and the PCR product was digested with Nde I and Not I. Ligate with pET42a recovered by Not I double enzyme digestion, transform the ligated product into competent DH5a, pick a single clone on an LB plate containing 50ug / ml kanamycin, and extract the plasmid after correct sequencing to be the pET-OPG-A expression vector.

[0087] The gene shown in SEQ ID NO.2 and the linker gene shown in SEQ ID NO.9 and the GLP-1-a gene shown in SEQ ID NO.6 are connected by a fully synthetic m...

Embodiment 2

[0090] Embodiment 2: the expression of fusion protein

[0091] Transform the correctly constructed expression vectors pET-OPG-A, pET-OPG-B, pET-OPG-C, and pET-OPG-D into BL21(DE3) competent bacteria, respectively, in the presence of 50ug / ml kanamycin Single clones were picked on LB plates. Single clones were inoculated in LB liquid medium containing 50ug / ml kanamycin, cultured with shaking at 200rpm at 37°C, when OD 600 When 1.0 was reached, expression was induced by adding IPTG at a final concentration of 0.5 mM. Continue culturing at 30°C for 4-6 hours, collect the bacteria by centrifugation, and freeze them at -20°C.

Embodiment 3

[0092] Embodiment 3: the purification of fusion protein

[0093] Dissolve the cells of OPG-A, OPG-B, and OPG-C in 20mM Tris-HCl buffer solution with pH 8.0 at a weight ratio of 1:15, where the NaCl concentration is 0.1M, crush twice under 600 bar pressure, 8000g Centrifuge to remove fragments and then filter through 0.4um and apply to QFF anion chromatography column. After equilibration, it is eluted with 20mM Tris-HCl of pH 8.0 containing 0.5M NaCl. The eluate was adjusted to 1.8M salt concentration with NaCl solid, then subjected to butyl hydrophobic chromatography, and eluted with 20 mM Tris-HCl at pH 7.5 after equilibration. The protein eluate was chromatographed on Superdex S100 gel with 10mMpH7.2PB buffer, and the main protein peak was collected, which was the target protein purification solution.

[0094] Dissolve the OPG-D bacteria in 20mM Tris-HCl buffer solution with pH 8.0 at a weight ratio of 1:15, where the NaCl concentration is 0.1M, crush twice under 600 bar pr...

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Abstract

The invention discloses a fusion protein. The fusion protein includes a protein therapeutic agent and a receptor binding agent. The protein therapeutic agent is a GLP-1 peptide or an analogue thereof. The receptor binding agent is a cholera toxin B subunit or an analogue thereof. The fusion protein can be used for treating diabetes, and the like through oral administration.

Description

technical field [0001] The present invention relates to a fusion protein, more specifically, the present invention relates to a fusion protein comprising a protein therapeutic agent and a receptor binding agent, wherein the protein therapeutic agent is a GLP-1 peptide or an analog thereof, the The receptor binding agent is cholera toxin B subunit or an analog thereof. The fusion protein of the present invention can be administered orally to treat diabetes and the like. Background technique [0002] Diabetes mellitus (Diabetes Mellitus) is a global disease, the number of patients has increased dramatically in recent years, and it is one of the main threats to human health. Diabetes is divided into type 1 diabetes and type 2 diabetes. Insulin supplementation is the mainstay of treatment for type 1 diabetes. Type 2 diabetes (Diabetes Mellitus, DM) is a disease that seriously endangers human health. The number of patients is increasing rapidly with the improvement of living s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/26A61K38/22A61K47/42A61P25/28A61P25/16A61P3/10
CPCA61K38/00C07K14/28C07K14/605C07K2319/55
Inventor 崔俊生张彦鹏吴首标刘立孙成龙杨立宪席丰品刘树涛
Owner BEIJING BIOHELIX BIOTECH CO LTD
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