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Angiogenesis inhibiting fusion protein and its use

A technology of fusion protein and protein, which is applied in the field of gene recombinant protein, can solve the problems of large side effects, no further development, instability, etc.

Active Publication Date: 2005-12-14
CHENGDU KANGHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] US patents 6,100,071, 5,952,199, and 6,383,486 describe several proteins fused with partial fragments of KDR and partial fragments of FLT-1, but they have not been further developed due to their instability and large side effects

Method used

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  • Angiogenesis inhibiting fusion protein and its use
  • Angiogenesis inhibiting fusion protein and its use
  • Angiogenesis inhibiting fusion protein and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of fusion protein and its plasmid.

[0043] Except for the immunoglobulin Fc fragment, the original sequences for constructing various fusion proteins in the present invention come from the corresponding cDNAs of FLT-1 and KDR. Since the expression of FLT-1 and KDR is mainly found in vascular endothelial cells, the present invention extracts total RNA from human umbilical vein vascular endothelial cells (HUVEC) with an RNA purification kit (QIAGEN). cDNA is then synthesized from RNA using reverse transcriptase. Then different primers were used to amplify the required FLT-1 and KDR fragments by polymerase chain reaction (PCR). Finally, PCR was used to fuse sequences from FLT-1, KDR, and human immunoglobulin Fc (IgG1 Fc) to construct DNA sequences of different fusion proteins. The structures of the six fusion proteins are shown in figure 1 .

[0044] Construction of FP3 gene:

[0045] Human umbilical vein vascular endothelial cells (HUVEC cel...

Embodiment 2

[0056] Example 2: Expression of fusion protein in cells.

[0057] One of the components of the present invention is the expression of the constructed fusion protein in cells. After completing the construction of each plasmid, high-purity plasmid DNA was extracted with a plasmid DNA purification kit (QIAGEN). Then, the plasmid DNA was introduced into 293 cells by using the FUGEN6 plasmid transfection kit (ROCHE Company). According to the amount of required protein, two different plasmid transfection methods were used to express the fusion protein.

[0058]The first method is the transient transfection method, by which a small amount of fusion protein can be obtained. Firstly, 293 cells were cultured in a cell culture dish with DMEM complete medium containing 10% fetal bovine serum. When the cells grow to cover 60-80% area, the complex of plasmid DNA and FUGEN6 reagent is added to the cell culture medium. The next day, replace the medium with serum-free DMEM medium. The cul...

Embodiment 3

[0061] Example 3: Binding experiment of fusion polypeptide and VEGF.

[0062] The present invention uses a VEGF binding test to measure the binding ability of each fusion polypeptide to VEGF. In this assay, recombinant VEGF (Chemicom) protein was first coated on a 96-well ELISA plate. Non-specific protein binding sites were then blocked with a 5% milk powder solution. Add various fusion proteins containing different concentrations to each well, and incubate at 37 degrees for two hours. After washing, rabbit anti-human Ig antibody-HRP was added. Finally, the color is developed with a peroxidase substrate. The OD value of each well of the 96-well plate was measured with an ELISA reader. High OD values ​​represent the binding signal of the fusion protein to VEGF.

[0063] Such as figure 2 As shown, the six fusion proteins constructed and expressed in the present invention all have the ability to combine with VEGF. Their binding signal to VEGF can be detected at a concentra...

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Abstract

The present invention relates to one kind of angiogenesis inhibiting fusion protein and its use. The fusion proteins of the present invention are produced through fusing FLT-1 segment, KDR segment and immunoglobulin Fc segment, and are named as FP1, FP2, FP3, FP4, FP5 and FP6. The amino acid sequences FLT-1 D2, FLT-1 D4, KDR, D1, KDR D3, KDR D4 and FP3 in the immunoglobulin-like area of FLT-1 and KDR are shown in the sequence lists 1-6, and FP3 coded DNA sequence is shown in the sequence list 7.

Description

technical field [0001] The invention relates to a series of gene recombinant proteins capable of effectively inhibiting angiogenesis. Angiogenesis is the process of growing new blood vessels from pre-existing blood vessels. Most of the blood vessels in the adult body are in a quiescent state, and angiogenesis is only seen in a few pathological or physiological states, such as tumors, retinal lesions in diabetics, arthritis, anemic organs, and endometrium in the proliferative period. During tumorigenesis, angiogenesis plays a key role in the rapid growth of tumors (Hanahan and Folkman: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996, 86: 353-364). Studies on animal tumor models and human clinical trials have proved that inhibiting the formation of new blood vessels in tumors can effectively prevent the growth and development of tumors, thereby prolonging the life of patients [0002] Angiogenesis is regulated and controlled by a vari...

Claims

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Application Information

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IPC IPC(8): C07K19/00
Inventor 刘征
Owner CHENGDU KANGHONG BIOTECH
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