Purifying and use for human vascular endothelial growth factor and granzyme B fusion protein

A vascular endothelium and growth factor technology, which can be applied to peptide/protein components, medical preparations containing active ingredients, hybrid peptides, etc., and can solve the problems of high blood drug concentration, unsuitable for clinical application, and poor vascular effect.

Inactive Publication Date: 2004-01-07
曾位森
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AI Technical Summary

Problems solved by technology

Known angiogenesis inhibitors, such as endostatin, angiostatin, etc., mainly inhibit the growth of vascular endothelial cells through competitive inhibition, and can only inhibit the further proliferation of vascular endothelium,

Method used

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Examples

Experimental program
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Embodiment Construction

[0005] Recombination of exon 2-5 of vascular endothelial growth factor (VEGF) and granzyme B (GraB) gene to make VEGF 2-5 It is connected with GraB through a flexible linker to form a fusion gene. The VEGF-GraB fusion gene was cloned into the multiple cloning site of the prokaryotic secretory expression vector PBAD / GIII, and its C-terminus was fused with the 6His tag on the vector to facilitate the purification of the recombinant by immobilized metal affinity chromatography fusion protein. Transform Escherichia coli with the recombinant vector PBAD / GIII-VEGF-GraB, induce the expression of the recombinant protein with arabinose, collect the bacteria by centrifugation, lyse the bacteria by osmotic shock, obtain the soluble recombinant fusion protein, and purify the recombinant VEGF by metal affinity purification combined with gel filtration -GraB fusion protein. The recombinant protein is detected by high performance liquid chromatography (HPLC), and the purity exceeds 95% for...

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Abstract

By adopting gene recombination technology to implement recombination of vascular endothelial growth factor (VEGF)2-5 exon and gran zyme B (GraB) gene and make VEGF2-5 and GraB be connected and formedinto fusion gene by means of a part of elastic connecon, and utilizing the specific combination of VEGF2-5 and VEGFR to guide GraB and make it target-act on the nescent vascular endothelial cell so as to attain the goal of target-resisting angiogenesis. Said invention uses prokaryocyte secretion type expression system to express recombinant VEGF-Gra fusion protein, and uses immobilized metal ion affinity chromatography and gel filtration method to purity the fusion protein, and uses GraB serine proteinase activity test experiment and chick embryo allantoic membrane angiogenesis anther membrane inhibition experiment to determine the bio-activity of recombinant VEGF-GraB fusion protein.

Description

technical field [0001] Biomedical Genetic Engineering Antiangiogenic Drugs Background technique [0002] Angiogenesis is a necessary condition for the growth and proliferation of solid tumors. Inhibiting or blocking angiogenesis in tumor tissue and cutting off the nutrient supply of tumor cells can effectively inhibit tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is the most specific and effective angiogenesis stimulating factor. VEGF and its receptor VEGFR are the marker molecules of angiogenesis. Many experiments have shown that inhibiting VEGF and VEGFR can effectively inhibit and block angiogenesis. Known angiogenesis inhibitors, such as endostatin, angiostatin, etc., mainly inhibit the growth of vascular endothelial cells through competitive inhibition, and can only inhibit the further proliferation of vascular endothelium, and have a poor effect on the formed blood vessels. Higher blood levels. The toxin mediated by VEGF is not suitable for c...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00C07K14/435C07K19/00C12N15/52C12N15/63
Inventor 曾位森
Owner 曾位森
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