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STie2 fusion protein, carrier thereof and pharmaceutical composition containing sTie2 fusion protein

A technology of fusion protein and stie1-c, which is applied in the field of genetic engineering and protein engineering, can solve the problems of unclear mechanism and failure to predict thrombus formation, and achieve the effect of inhibition and promotion

Active Publication Date: 2017-08-11
CHENGDU NUOEN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism is unclear and thrombosis may be the result of platelet activation mediated through the gammaRIIa (IgG) receptor of Fc (Meyer, Rob

Method used

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  • STie2 fusion protein, carrier thereof and pharmaceutical composition containing sTie2 fusion protein
  • STie2 fusion protein, carrier thereof and pharmaceutical composition containing sTie2 fusion protein
  • STie2 fusion protein, carrier thereof and pharmaceutical composition containing sTie2 fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Recombinant Construction of Fusion Protein Expression Plasmids

[0066] The present invention carries out genetic engineering transformation on the basis of the human source Tie2 gene sequence (GenBank: NM_000459) template, and designs 5 kinds of partial expression sequences of the Tie2 gene containing different functional regions ( figure 2 ). 1. Tie2-781: Contains 1-781 amino acids of the Tie2 gene, has a transmembrane region, and does not have extracellular secretion ability. 2. sTie2-751.6His: contains amino acids 1-751 of Tie2 gene, without transmembrane region. Six His amino acid tags were added to the C-terminus. A soluble sTie2 mimic formed by proteolysis. 3. sTie2-738.SV.6His: Contains amino acids 1-738 of the Tie2 gene, this fragment is compatible with the extracellular binding region before the sTie1-C terminal sequence of soluble sTie1, and does not contain the transmembrane region. 11 amino acid sequences of sTie1-C and 6 His amino acid tags...

Embodiment 2

[0068] Example 2: Effect of C-terminal amino acid composition expressed in different cells on soluble protein expression and extracellular secretion

[0069] The cultured cells were transfected with 5 kinds of plasmid DNAs respectively containing Tie2-781, sTie2-738.6His, sTie2-738.SV.6His, sTie2-738.Fc and sTie2-544.SV.6His gene expression sequences, and determined by ELISA The content of Tie2 protein in cell culture supernatant and cell lysate was determined by method, and the effect of sTie1-C terminal on the yield and secretion of fusion protein was compared. The specific method is: inoculate 2.4x10^4 cells in a 6-well plate. Mix 2.5 μg with lipefectamine cell transfection reagent (Life Technology), add to the cultured cells, mix well, after 24 hours of transfection, replace into DMEM culture medium containing 1% FBS, incubate for 24 hours, take the supernatant, and collect the cells , the content of Tie2 in the sample was measured by ELISA quantitative assay method, and ...

Embodiment 3

[0071] Embodiment 3: Culture cell HEK293T produces fusion protein and Ni-NTA chromatographic column purification method

[0072] Collection of fusion protein: HEK293T cells were cultured with DMEM medium (HyClone) containing 10% FBS, and when the cell density grew to 60%-80%, fresh 10% FBS DMEM medium was replaced, and the recombinant plasmid and liposome mixture , transfected into the cells by liposome transfection, the medium was replaced with DMEM medium containing 1% FBS on the second day after transfection, and the culture was continued until the third day, and the culture medium was collected. For three consecutive days thereafter, the DMEM medium with 1% FBS was changed every day, and the supernatant of the culture solution was harvested after 24 hours of cultivation. The supernatant contains the fusion protein expressed by the cells. ELISA assay was used to identify and confirm the expression of sTie2 fusion protein.

[0073] Protein purification process: collect the...

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Abstract

The invention discloses sTie2 fusion protein and belongs to the field of genetic engineering and the field of protein engineering. The sTie2 fusion protein is sTie2-738. SV and preferably sTie2-544. SV and comprises a Tie2 extracellular ligand identification and combination key area and an sTie1-C terminal amino acid sequence. The invention further discloses a carrier of the sTie2 fusion protein, cells for producing the sTie2 fusion protein, a pharmaceutical composition containing the sTie2 fusion protein and a preparation of the pharmaceutical composition. The sTie2 fusion protein has the advantages that the protein can allow new blood vessels to atrophy and disappear, the treatment effect of angiogenesis diseases can be increased greatly, the protein is high in action efficiency, good in stability and high in yield, and the angiogenesis inhibiting ability of the protein is proved in various types of animal angiogenesis models.

Description

technical field [0001] The invention relates to the fields of genetic engineering and protein engineering, in particular to a sTie2 fusion protein, its carrier and a pharmaceutical composition containing the sTie2 fusion protein. Background technique [0002] The formation and maintenance of blood vessels are the basic guarantee for the formation and functioning of various tissues and organs in animals and humans. The dominant angiogenesis process includes endothelial cell proliferation, migration and differentiation, degradation of extracellular matrix, and capillary sprouting, etc., which are regulated by various factors. The stimulatory factors for vascular endothelial cell formation that have been discovered include: vascular endothelial growth factor (VEGF for short), vascular permeability factor (VPF), fibroblast growth factor (FGF) and angiopoietin (Angiopoietin for short). Ang) et al. These factors bind to receptors on the surface of endothelial cells, activate diff...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/17A61P9/10A61P35/00A61P19/02A61P29/00
CPCA61K38/00C07K14/71C07K2319/00C07K2319/21
Inventor 罗德伦徐凯吴秀锦
Owner CHENGDU NUOEN BIOLOGICAL TECH
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