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Recombined vessel endothelium growth factor receptor, encoding genes thereof and application

A growth factor receptor, vascular endothelium technology, applied in gene therapy, recombinant DNA technology, microorganisms, etc., can solve the problems of easy exposure of endothelial cells, no research reports on anti-VEGF drugs, etc., and achieve high affinity and angiogenesis. Inhibition, obvious effect of angiogenesis

Inactive Publication Date: 2013-02-13
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

② Endothelial cells are easily exposed to drugs
[0013] Domestic research on VEGF / VEGFR mostly focuses on the expression and significance of VEGF / VEGFR in various tumor tissues, and there are also cloning and expression of VEGF / VEGFR family members, but there are no research reports on anti-VEGF drugs

Method used

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  • Recombined vessel endothelium growth factor receptor, encoding genes thereof and application
  • Recombined vessel endothelium growth factor receptor, encoding genes thereof and application
  • Recombined vessel endothelium growth factor receptor, encoding genes thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the cloning of VEGFR-1 / Fc fusion gene and the construction of expression vector

[0069] Breast cancer cell MCF-7: cell bank of Concord Institute; pUC19 vector: TaKaRa Company; pIRES2-EGFP vector: Invitrogen Company; pIG1 plasmid: recorded in Liu Hezhong, Mao Ning, Hou Chunmei, etc. Blocking CD40 / CD40L interaction prolongs graft resistance Survival time of mice with host disease. Chinese Clinical Pharmacology and Therapeutics, 2003, 8(1): 6-10, the public can obtain from the Institute of Bioengineering, Academy of Military Medical Sciences of the Chinese People's Liberation Army; Top10 Competent Escherichia coli: Transgen ; Taq enzyme, Pyrobest enzyme, and DNA ligase are all TaKaRa products, and restriction endonucleases are all products of NEB Company; plasmid extraction, PCR product recovery, and enzyme digestion product recovery were respectively used by Promega Company Wizard Plus SV Minipreps DNA purification System, Wizard PCR preps DNA purification ...

Embodiment 2

[0106] Example 2, Expression, purification and identification of VEGFR-1 / Fc fusion protein

[0107] Materials used for plasmid transfection and pressurized screening: CHO-K1 cells: ATCC Number CCL-61, cultured in DMEM / F12 medium (Gibco), containing 5% (v / v) newborn bovine serum (FBS) (Hyclone) , cultivated in 5% CO 2 37°C incubator (Thermo Forma). Lipofectamine TM 2000 cationic liposome transfection kit, and I low serum medium (Invitrogen). G418 (Roche).

[0108] Materials used for ELISA screening of highly expressed clones: bovine serum albumin (BSA) (Sigma). PBS-T (KCl 0.2g / l; KH 2 PO 4 0.2g / L; NaCl 8.0g / L; NaCl 2 HPO 4 ·7H 2 O 1.56g / L; Tween 20 0.05% (v / v); pH7.4). 96-well ELISA plate (Nunc TM ). Horseradish peroxidase (HRP)-labeled goat anti-human IgG (H+L) (imported by Zhongshan Biotechnology Company). TMB chromogenic solution (Xinjingke Biotechnology Co., Ltd.). Microplate reader (Thermo Labsystems).

[0109] Packing and equipment for purification: rPro...

Embodiment 3

[0151] Example 3, Detection of biological activity of VEGFR-1 / Fc fusion protein

[0152] 1. Determination of affinity constant of VEGFR-1 / Fc fusion protein

[0153] The affinity constant of the VEGFR-1 / Fc fusion protein obtained in Example 2 was determined by surface plasmon resonance (SPR) technique.

[0154] Instruments and reagents: BIAcore 3000 surface plasmon resonance instrument (GE healthcare), CM5 sensor chip (GE healthcare), SPR dedicated HBS-P buffer (10mmol / L HEPES pH 7.4, 150mmol / L NaCl and 0.005% ( v / v) Surfactant P20), Amine Coupling Kit, BIAcore Maitenance Kit, 10mmol / L NaAc, 50mmol / L NaOH, 10mmol / L Glycine HCl pH1.5 were all purchased from GE healthcare.

[0155] BIACORE operating procedure:

[0156] (1) Pre-binding test: The surface of the chip (CM5 sensor chip) is negatively charged, so the molecules to be coupled to the chip must be positively charged to facilitate the adsorption of molecules to the surface of the chip for covalent coupling completion of ...

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Abstract

The invention discloses a recombined vessel endothelium growth factor receptor, encoding genes thereof and application. The provided recombined blood vessel endothelium growth factor receptor is a fusion protein, and a structural domain 1-3 and an Fc section of a human IgG1 of the blood vessel endothelium growth factor receptor 1 are arranged sequentially from an amino terminal to a carboxyl terminal. An experiment proves that the fusion protein vessel endothelium growth factor receptor (VEGFR)-1 / Fc and a vessel endothelium growth factor (VEGF) have good affinity, the KD value reaches 265pm, the VEGFR-1 / Fc can inhibit proliferation and migration of vessel endothelium cells and chick embryo chorioallantoic membranes and vessel growth of naked mouse. The fusion protein VEGFR-1 / Fc has wide application in tumor therapy.

Description

technical field [0001] The invention relates to a recombinant vascular endothelial growth factor receptor and its encoding gene and application, in particular to a recombinant vascular endothelial growth factor receptor VEGFR-1 domain 1-3 fused with the Fc fragment of human IgG1 Recombinant vascular endothelial growth factor receptor and its coding gene and application. Background technique [0002] Malignant tumors are currently a serious threat to human health. More than 7 million people die from malignant tumors worldwide each year, and more than 10 million new patients with malignant tumors. WHO predicts that by 2020, the incidence of cancer worldwide will be twice as much now. In the past few decades, chemotherapy drugs have achieved remarkable results in the treatment of tumors. However, chemotherapy drugs have serious side effects and are prone to drug resistance. Therefore, people have been working hard to find a new generation of anti-tumor drugs. Among them, anti...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/21C12N7/01A61K38/17A61K47/48A61K48/00A61P35/00A61K47/42
Inventor 胡显文师明磊郗永义高丽华邵勇冯立陈惠鹏赵志虎
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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