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Dual-target fusion protein and preparation method and use thereof

A fusion protein, dual-target technology, applied in the field of biomedicine to achieve the effect of prolonging half-life, enhancing therapeutic effect and reducing clinical treatment cost

Inactive Publication Date: 2017-10-27
殷跃云
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Simultaneous dual-targeted blocking of VEGF / PDGFβ or receptor-specific biological targeted drugs has not been reported clinically

Method used

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  • Dual-target fusion protein and preparation method and use thereof
  • Dual-target fusion protein and preparation method and use thereof
  • Dual-target fusion protein and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 expression vector construction

[0050] VEGFR contains 7 immunoglobulin-like extracellular domains, and PDGFRβ contains 5 immunoglobulin-like extracellular domains. Human VEGFR1 and VEGFR2 gene sequences were obtained from NCBI (Reference Sequence: NP_001153392.1, NP_002244.1).

[0051] The PDGFRβ gene sequence was obtained from NCBI (Reference Sequence: NM_002609.3).

[0052] PDGFRβD1-D3, VEGFR1II-VEGFR2III, and Fc sequences (SEQ ID No. 1-3) were respectively obtained by gene synthesis. AA0 plasmid construction

[0053] Using primer 1 and primer 2, using primer 3 and primer 4 to take synthetic gene PDGFRβ and Fc as templates respectively, PCR amplification amplification, after its product is purified with plasmid purification reagent (Qiagen company), use primer 1 and primer 4 to bridge PCR They are spliced ​​together, digested with XhoI / EcoRI double enzymes, purified, and connected to the pcDNA3.1S-puro expression vector with a secretion signal peptide...

Embodiment 2

[0090] Example 2 Cell Transfection, Screening of Recombinant Clones and Expression of Fusion Protein

[0091] When the fusion protein in the present invention is transiently expressed, the recombinant plasmid is purified with a DNA purification kit (Qiagen), and then transfected into HEK293 (ATCC) with liposome 2000 (Invitrogen) or with polyethylenimine (Polysciences) # CRL-1573) cells. After 4 hours, the medium in the culture dish was replaced with serum-free CD Opti-MEMI medium, and the supernatant was collected after continuing to culture in serum-free medium for 3 days. The expressed protein concentration was determined by enzyme-linked immunosorbent assay (ELISA).

[0092] The specific method of the ELISA determination is as follows: use the Fc sandwich enzyme-linked immunoassay ELISA detection kit of Bethyl Company to determine the Fc concentration of each fusion protein. Add the fusion protein diluted with 1% BSA to the 96-well ELISA plate that had been diluted with t...

Embodiment 3

[0094] Example 3 Fusion protein combined with VEGF165 (V165) and PDGFββ

[0095] The binding ability of the fusion protein to V165 and PDGFββ in solution was determined by ELISA. Different concentrations of fusion proteins (0-25000 pM) were incubated overnight at room temperature with 10 pM of V165 (R&D Systems) or 10 pM of PDGFββ (R&D Systems). Add 100 microliters of the above overnight incubation samples to 96-well plates previously coated with anti-human VEGF antibody (R&D Systems) and anti-human PDGFβ antibody (R&D Systems), and incubate at room temperature for 2 hours. Then add 100 microliters of anti-human VEGF-HRP or 100 microliters of anti-human PDGFβ-HRP chimeric antibody respectively, and react for 1 hour at room temperature. Add 100 microliters of substrate TMB solution for color development, stop the reaction by adding 100 microliters of 1M HCl, read at a wavelength of 450 nm with a microplate reader (Molecular Device380) and use software (Graphpad Prism5) to anal...

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Abstract

The invention provides a dual-target fusion protein, which contains VEGF receptors and cell membrane fragments of PDGF beta receptors. Through the optimized combination of functional fragments, the present invention improves the binding ability of the fusion protein and the dual-target VEGF and PDGF β, can effectively inhibit the proliferation of new blood vessels, and achieve the treatment of neovascular proliferation diseases related to VEGF and PDGF β, such as tumor and wet age-related macular degeneration (AMD).

Description

technical field [0001] The present invention relates to the fields of biomedicine and technology, in particular to a dual-target fusion protein and its preparation method and application. The dual-target fusion protein can simultaneously bind PDGFβ and VEGF cytokines, and can be used to treat PDGFβ and VEGF-mediated related diseases. Background technique [0002] The formation of new blood vessels requires the participation of various cell growth factors, among which vascular endothelial growth factor (VEGF) is particularly important. [0003] VEGF binds to the amino acid kinase receptor on the cell membrane, activates the descending signal transmission pathway, and cooperates with other vascular endothelial growth factors to participate in a series of physiological or pathological effects. Among them, VEGF plays a vital role in the growth, migration, survival and vascular permeability of vascular endothelial cells. [0004] VEGF and its receptor (VEGFR) not only play an i...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/63C12N1/19C12N5/10C12N1/21A61K39/395A61P35/00A61P27/02A61P9/14
CPCC07K16/468A61K2039/505C07K16/18C07K16/2863C07K2317/24C07K2317/52
Inventor 不公告发明人
Owner 殷跃云
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