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FGF21 and IGF-1 fusion protein and applications thereof

A technology of FGF21 and IGF-1, applied in the field of fusion protein of FGF21 and IGF-1, can solve the problem of difficulty in expression, and achieve the effect of reducing osteoporosis, overcoming short half-life and tissue distribution specific elimination.

Pending Publication Date: 2018-08-24
REYOUNG SUZHOU BIOLOGY SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] IGF-1 has the disadvantage of being difficult to express as a drug, and because it has the effect of promoting cell growth and anti-apoptosis, it is speculated that it may increase the occurrence of cancer. At present, some experiments have shown that the increase of free IGF-1 in the blood is associated with some cancers. relevant

Method used

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  • FGF21 and IGF-1 fusion protein and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Expression and purification of FGF21-IgG4(Fc)-IGF-1 fusion protein

[0086] FGF21-IgG4(Fc)-IGF-1 fusion protein was expressed by mammalian HEK 293F cells. The commercial eukaryotic expression vector pCDNA3.1 was used to construct the FGF21-IgG4(Fc)-IGF-1 expression vector, an IgGκ signal peptide was added to the N-terminal of the FGF21 fusion protein to enhance the secretion of the target protein, and the C-terminal of the peptide segment FGF21 was used to Pass through the N-terminal of IgG4 (Fc) peptide and the C-terminal of IgG4 (Fc) peptide and N-terminal of IGF-1 peptide (GlyGlyGlyGlySer) 3 connector to connect. The expression is carried out with strong promoter CMV. When using the PEI method, an appropriate amount of DNA is used to transfect HEK293F suspension cells of appropriate density for expression. Cells at 37°C, 6% CO 2 cultured in serum-free medium. Samples were collected around day 6 after transfection. Normally, the expression level shou...

Embodiment 2

[0089] Embodiment 2: FGF21-IgG4 (Fc)-IGF-1 fusion protein affinity determination

[0090] The FGF21-IgG4(Fc)-IGF-1 protein was immobilized on the chip by amino immobilization or Fc fragment capture method as a ligand, and FGFR1c-beta-Klotho or IGF1R was used as an analyte for affinity determination. The assay was performed with HBS-EP+ buffer solution at a flow rate of 30 μl / min. The binding time was 300 seconds and 600 seconds respectively, and the concentrations of FGFR1c-beta-Klotho were 0, 6.25, 12.5, 25, 50, 100, 200nM, and regenerated with 10mM Glycine·HCl, pH 2.0. The concentration of IGF1R was 0, 6.25, 12.5, 25, 50, 100, 200nM, and it was regenerated with 10mM Glycine·HCl, pH 2.0. Biacore analysis software was used to analyze the data, and the fitting model was "1:1 binding (1:1 Binding)" model.

Embodiment 3

[0091] Example 3: Determination of FGF21-IgG4(Fc)-IGF-1 half-life in vitro

[0092] Mix the fusion protein with mouse plasma, take samples at corresponding time points and store them at 4°C. After all the samples at all time points are taken, dilute the samples to a certain concentration with cell culture medium, and perform gradient dilution. Samples were processed from HEK293 cells stably transfected with beta-Klotho and HEK293 cells stably transfected with IGF-1R, and performed for pERK ICW assay.

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Abstract

The present invention provides a FGF21 and IGF-1 fusion protein, wherein fusion is performed through an appropriate linker peptide fragment in an optimal fusion manner, and the produced fusion proteinsuccessfully retains the respective biological activities. According to the present invention, specifically the fusion protein sequentially contains human fibroblast growth factor-21 (hFGF21), a flexible peptide linker (L), a human immunoglobulin Fc fragment, a flexible peptide linker (L) and an insulin-like growth factor (IGF-1) from the N terminal to the C terminal; and the fusion protein provides high regulation effect in glycolipid metabolism and the like compared to the single use of the proteins, enhances the treatment effects on metabolic disorders, cardiovascular diseases, liver fat lesions and other diseases, and overcomes the side effects (such as osteoporosis, cancer risk and the like).

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a fusion protein of FGF21 and IGF-1 and its application. Background technique [0002] Fibroblast growth factor (FGF) is a class of structurally related proteins encoded by members of the FGF gene family. They are polypeptides expressed in both developing and mature tissues. At least 22 members have been found so far. , and their central regions all contain an amino acid sequence with a homology of 30-70%. According to the sequence homology analysis, the current FGF family members can be divided into 7 subfamilies. FGF not only plays a role in the growth of fibroblasts, but also plays an important role in morphogenesis, angiogenesis, tumorigenesis, wound healing, neuron survival, metabolic regulation and cell differentiation. [0003] FGF21 is an important member of the FGF family, and a subfamily of FGF composed of FGF19 and FGF23. The homology with other subfamilies is ...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/18A61K38/30A61K47/65A61K47/68A61P3/04A61P3/10A61P3/06A61P9/12A61P1/16A61P35/00A61P1/18A61P1/00A61P25/00
CPCA61K38/00C07K14/50C07K14/65C07K2319/00C07K2319/30
Inventor 吴燕燕崔文俊郭树华张佳春
Owner REYOUNG SUZHOU BIOLOGY SCI & TECH CO LTD
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