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Exendin-4 fusion protein, preparation method and uses thereof

A fusion protein and sequence technology, applied in the fields of peptide/protein components, chemical instruments and methods, hybrid peptides, etc., can solve the problems of insufficient safety and insufficient stability of fusion proteins, achieve high clinical application value, improve blood sugar Level control, the effect of prolonging half-life

Active Publication Date: 2016-11-23
LANZHOU INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The technical problem to be solved by the present invention is that the stability of the existing fusion protein containing Exendin-4 polypeptide is not good enough and the problem of insufficient safety

Method used

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  • Exendin-4 fusion protein, preparation method and uses thereof
  • Exendin-4 fusion protein, preparation method and uses thereof
  • Exendin-4 fusion protein, preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 Design of E4F4 fusion protein and construction of recombinant expression vector

[0067] Fusion protein sequence design, the mutant human IgG4Fc fragment ((aa.99-327) was fused to the C-terminal of two Exendin-4 fragments. At the same time, in order to realize the secretory expression of the fusion protein, a mouse signal was added to the N-terminal Peptide sequence, the final fusion protein obtained is: ssmIg-Ex-4-(G4S) 3 -Ex-4-(G4S) 3 -human mutant IgG4Fc (aa.99-327).

[0068] The fusion protein sequence containing signal peptide is as follows (SEQ ID No.6):

[0069] MGWSCIILFLVATATGVHSHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0070] The corresponding coding n...

Embodiment 2

[0073] Example 2 Screening and identification of CHO stable expression strain (CHO-E4F4 cell)

[0074] Take 10 μg of the pAAV2-E4F4 plasmid prepared in large quantities in Example 1, and use liposome Lipofectamine 2000 (purchased from Invitrogen, USA) to transfect CHO cells (purchased from ATCC, USA). The expression of the fusion protein in the culture supernatant was detected by ELISA. After four rounds of screening, high-expression E4F4 cells were selected, and the cells with stable expression were named CHO-E4F4 cells.

[0075] The RNA of CHO-E4F4 cells was extracted, and the transcription of the inserted fragment was identified by RT-PCR. The result is as image 3 As shown, CHO-E4F4 is a stable cell line that correctly expresses E4F4 recombinantly.

Embodiment 3

[0076] Example 3 Preparation of rhE4F4 fusion protein

[0077] CHO-E4F4 cells were cultivated on a large scale, and the supernatant was purified by Protein A Sepharose F.F. (purchased from GE, USA) affinity chromatography column. The electrophoretic pattern of the purified protein is shown in Figure 4 , the purity can reach more than 90%.

[0078] Specific steps are as follows:

[0079] (1) Pretreatment of the expression supernatant: Slowly adjust the pH of the supernatant to 7.2 with 1M NaOH, centrifuge at 4000 rpm for 10 min at 4°C, and collect the supernatant.

[0080] (2) The centrifuged supernatant was passed through a Protein A Sepharose F.F. affinity chromatography column, and the fusion protein was washed with 0.1M citrate buffer (pH3.0), and the washed fusion protein was adjusted to pH 7.2 with 1M NaOH.

[0081] (3) The obtained rhE4F4 fusion protein was dialyzed with 10 mM phosphate buffer and stored at -20°C.

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Abstract

The technical problem to be solved by the present invention is to prolong the half-life of an Exendin-4 polypeptide and to improve the stability and the safety of the existing Exendin-4 polypeptide-containing fusion protein. The scheme for solving the technical problem is to provide a new fusion protein containing the Exendin-4 polypeptide, wherein the structure of the fusion protein sequentially comprises Exendin-4, a connecting peptide, Exendin-4, a connecting peptide and an IgG4Fc mutant from the nitrogen terminal. According to the present invention, the provided fusion protein containing the Exendin-4 has characteristics of good stability, long half-life and low toxic-side effect; and the fusion protein of the invention can be used for treatment of diabetes type I and diabetes type II, and has high clinical application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering drugs, and in particular relates to a long-acting Exendin-4 fusion protein and its preparation method and application. Background technique [0002] Diabetes is a chronic lifelong disease, which is currently considered to be caused by genetics and acquired living habits, and has become a worldwide public health problem that seriously threatens human health. [0003] Clinically, hyperglycemia is the main sign, and diabetes is divided into type 1 diabetes and type 2 diabetes. Diabetes mellitus is an increase in blood sugar due to defective insulin secretion or defective insulin action. Type 1 diabetes is caused by the lack of insulin secretion due to the immune destruction of pancreatic β cells, and relies on exogenous insulin supplementation; while the important mechanism of type 2 diabetes is insulin resistance and relative insufficient insulin secretion by pancreatic β cells, and the amount of...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/79A61K38/26A61P9/10
Inventor 杨莉
Owner LANZHOU INST OF BIOLOGICAL PROD
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