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Fusion protein of human vascular endothelial growth factor and epidermal growth factor-like domain 7

A technology of epidermal growth factor and fusion protein, applied in the field of fusion protein of human vascular endothelial growth factor and epidermal growth factor-like domain 7, can solve problems such as cardiovascular abnormalities and vascular defects, and achieve the effect of improving the effect

Inactive Publication Date: 2015-12-23
连祺周
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In mice, deletion of the vegf gene is lethal as it leads to vascular defects and cardiovascular abnormalities

Method used

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  • Fusion protein of human vascular endothelial growth factor and epidermal growth factor-like domain 7
  • Fusion protein of human vascular endothelial growth factor and epidermal growth factor-like domain 7
  • Fusion protein of human vascular endothelial growth factor and epidermal growth factor-like domain 7

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of VEGF-EGFL7 fusion gene expression system in yeast

[0033] In this example, the gene coding sequences of VEGF and EGFL7 pro-angiogenic active regions were analyzed, and the rigid and elastic linker peptides were used to fuse them together, and the gene coding sequences of serum protein binding domains were added at the ends.

[0034] It mainly includes the following steps:

[0035] 1. Obtain VEGF protein sequence from NCBI

[0036] (http: / / www.ncbi.nlm.nih.gov / protein / 76781480?report=fasta),

[0037] Extract its functional area:

[0038] NHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKD (SEQ ID NO. 1).

[0039] 2. Get the EGFL7 protein sequence from NCBI

[0040] (http: / / www.ncbi.nlm.nih.gov / protein / 7705889?report=fasta),

[0041] Extract its functional area:

[0042] GAAICQPPCRNGGSCVQPGRCRCPAGWRGDTCQ (SEQ ID NO. 2).

[0043] 3. Obtain serum protein sequence from NCBI

[0044](http: / / w...

Embodiment 2

[0073] Example 2: Application of VEGF and EGFL7 fusion protein to promote angiogenesis in vitro

[0074] Human umbilical cord vascular endothelial cells (HUVEC) were divided into 1×10 6 The density of each hole was inoculated with Matrigel in 6-well plate, and different concentrations of EGFL7, VEGF and VEGF-EGFL7 fusion protein (0 ng / ml to 100 ng / ml) were added to the serum-free medium, and then the cells were cultured for 3- 6 hours (21% O under normoxia 2 or hypoxic 1% O 2 conditions) cultured. The results are shown in Figure 4. Without the addition of VEGF and EGFL7, the human umbilical cord vascular endothelial cells did not present a vascular network structure ( Figure 4A iv diagram). After adding VEGF or EGFL7 or VEGF-EGFL7 fusion protein in the medium, the cells formed a vascular network structure ( Figure 4A Figures i to iii), the density of vascular network formation was photographed and counted. The results in Fig. 4 show that after adding VEGF or EGFL7 or ...

Embodiment 3

[0075] Example 3: Application of VEGF and EGFL7 fusion protein to promote angiogenesis in vivo

[0076] Establish a rat model of acute myocardial ischemia by ligating the left anterior descending coronary artery, and inject the same concentration (1 to 100 μg / kg) of VEGF or EGFL7 or VEGF-EGFL7 fusion protein around the left anterior descending coronary artery Afterwards, the heart was taken out on the 14th day, and sections were immunohistochemically stained. CD31 was used to mark the vascular endothelial network, and DAP was used to mark the nuclei. As shown in the figure (i-iv of 4C), the immunohistochemical fluorescent staining showed that the density of the vascular network in each group The differences among the groups were obvious; statistics were taken to check the density of vascular network formation, and the degree of difference between the groups was compared. Such as Figure 4D The results showed that, compared with VEGF or EGFL7, VEGF-EGFL7 fusion protein could s...

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Abstract

The present invention relates to a synthetic fusion protein, and particularly to a fusion protein of human vascular endothelial growth factor (VEGF) and epidermal growth factor-like domain 7 (EGFL7). The fusion protein of human vascular endothelial growth factor (VEGF) and epidermal growth factor-like domain 7 (EGFL7) comprises an amino acid sequence from a VEGF protein, an amino acid sequence from an EGFL7 protein, a linker peptide and a serum protein binding domain fragment at the end. The fusion protein provided by the present invention has significantly improved angiogenesis promoting effect on endothelial cells, has clinical application advantages in terms of vascular endothelial neovascularization compared to VEGF or EGFL7, and can be used as a novel type of vascular endothelial growth factor in the field of neovascularization.

Description

technical field [0001] The invention relates to an artificially synthesized fusion protein, in particular to a fusion protein of human vascular endothelial growth factor (VEGF) and epidermal growth factor-like domain 7 (EGFL7). Background technique [0002] Vascular endothelial growth factor (VEGF) is a cytokine secreted by tumors. It is very important in both normal and tumor-associated angiogenesis. It has been a hot spot in the study of angiogenesis regulation mechanism in the past ten years. [0003] The VEGF gene is located on the short arm of chromosome 6 and consists of 8 exons and 7 introns. Different exon splicing methods can differentiate into four mature isoforms, namely VEGF121, VEGF165, VEGF189 and VEGF206. Different numbers represent different amino acid numbers, among which VEGF165 is the main homologous isomer. [0004] VEGF produces its biological effects by interacting with cell surface receptors. These receptors are transmembrane tyrosine kinase recepto...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/515C07K14/485C12N15/62C12N15/81C12N1/19
Inventor 连祺周
Owner 连祺周
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