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Exendin-4 and analog fusion protein thereof

A technology of fusion proteins and analogues, applied in animal/human proteins, fusion polypeptides, drug combinations, etc., can solve the problems of inferior efficacy to exendin-4, short half-life, low binding activity, etc.

Active Publication Date: 2010-11-24
BEIJING DONGFANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] In response to the short half-life of GLP-1 or exendin-4 in vivo, researchers have developed corresponding fusion proteins, such as the GLP-1 albumin fusion protein or GLP-1-IgG4 described in WO 02 / 46227 or WO 05 / 000892 Fusion protein with low binding activity in vitro (Kim et al, Diabetes.2003; 52(3):751-759), and its efficacy is not as good as exendin-4

Method used

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  • Exendin-4 and analog fusion protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1, exendin-4 fusion protein expression gene synthesis and vector construction

[0098] The DNA construction of the Exendin-4 fusion protein is gene synthesized by ligase chain reaction, and its protein sequence composition is as follows:

[0099] HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGAGGGGV

[0100] ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN

[0101] WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV

[0102] SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP

[0103] SDIAVEWESNGQPENNYKTTPPMLLDSDGSFFLYSKLTVDKSRWQQGNVF

[0104] SCSVMHEALHNHYTQKSLSLSPGK* [SEQ ID NO: 4]

[0105] The fusion protein contains a vector encoding exendin-4 and human IgG2-Fc fusion protein. The IgG2-Fc region contains the CH2 and CH3 portions of the IgG2 constant heavy chain. The IgG secretion leader peptide sequence SP-IgGH was fused to exendin-4 in order to direct the secretion of the synthetic fusion protein into the culture medium. The cDNA encoding the SP-IgGH / e...

Embodiment 2

[0106] Example 2, exendin-4 fusion protein engineering cell line construction, expression and purification

[0107] 1. Construction of exendin-4 fusion protein engineering cell line

[0108] Chinese hamster ovary cells (CHO) were cultured in DMEM (purchased from Invitrogen) complete culture medium containing 10% (volume percentage) fetal calf serum (FCS), and evenly spread on 6-well plates one day before transfection, 3× per well. 10 5 . For the transfection method, refer to the instructions of LIPOFECTAMINE 2000. After 48 hours of transfection, the cells were placed in selective medium (MSX 25uM) and cultured under pressure for about a week. The empty cells basically died, and the surviving cells were seeded in 96-well plates (50 cells / well) and continued to be cultured under pressure. After the cells grew into clones, ELISA was used to detect the expression level of the protein in the culture supernatant, and the wells with high expression were selected and transferred to...

Embodiment 3

[0111] Example 3 Structural analysis of exendin-4 fusion protein

[0112] 1. Western Blot analysis

[0113] After the purified fusion protein was subjected to non-reduced SDS electrophoresis, the electrophoresis band was transferred to methanol-activated PVDF membrane (current: 25mA, time: 2h) using a membrane transfer device (GE). Block the PVDF membrane in 5% skimmed milk for 2 hours, then add the diluted alkaline phosphatase-labeled anti-IgG2 antibody to incubate for 1 hour, wash the membrane with TBST for 1 hour, replace with fresh TBST every 5 minutes, and add CDP after washing -star luminescent detection substrate, press film exposure and development. see attached results figure 2 , the fusion protein was positive for IgG2 antibodies.

[0114] 2. Isoelectric focusing electrophoresis analysis

[0115] The purified fusion protein was analyzed by isoelectric focusing electrophoresis using a Phast System (GE), and a precast gel with pH 3-10 was selected. After the focus...

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Abstract

The invention discloses exendin-4, an analog fusion protein thereof, the corresponding polynucleotide sequence, carrier, host cell and pharmaceutical composite and a preparation method and applications of the fusion protein. The fusion protein is prepared by fusing exendin-4 and analog thereof with human immunoglobulin IgG2-Fc through special connecting peptide and has better stability and loner half-life in vivo. The fusion protein can be administered by performing local delivery, using aerosol and using injection. The fusion protein can promote the regeneration and repair of islet beta cells, increase islet beta cells, promote the secretion of insulin and improve the sensitivity of organism to insulin. The fusion protein is used to cure diabetes, adiposity and other diseases which can be benefited by reducing plasma glucose and inhibiting gastrointestinal motility and gastric emptying.

Description

technical field [0001] The present invention relates to a long-acting Exendin-4 and its analogue fusion protein, corresponding polynucleotide sequence, carrier, host cell and pharmaceutical composition, its preparation method and application. It belongs to the technical field of biological genetic engineering. Background technique [0002] Diabetes mellitus is a disease that seriously endangers human health. According to the latest statistics from the World Health Organization, there were 171 million diabetics in the world in 2000, and it is estimated that the global number of diabetics will reach 366 million in 2030, resulting in 3.2 million deaths worldwide every year. in diabetes. As of March 2010, the number of diabetic patients in my country has reached 135 million, ranking first in the world's largest diabetic country. The incidence rates in urban and rural areas are 11.4% and 8.2%, respectively, and the age of onset tends to be younger (Wenying Yang et al, N Engl JMe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10A61K38/22A61K47/48A61P3/10A61P3/04
CPCC07K14/46C07K16/00C07K14/575A61K38/00C07K2319/30C07K14/57563C07K14/605A61P3/00A61P3/04A61P3/10C07K19/00C12N15/62C12N15/85
Inventor 白先宏涂划李先钟
Owner BEIJING DONGFANG BIOTECH
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