Soluble VEGFR difunctional fusion receptors, preparation method and use thereof

A technology of VEGF-B and VEGF-C, applied in its preparation and preparation of anti-tumor drugs, in the field of bifunctional fusion receptors, can solve the problems of poor treatment effect of malignant tumors, inability to achieve metastasis, and poor prognosis.

Active Publication Date: 2009-11-11
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment effect of malignant tumors is poor, the late metastasis rate is high, and the prognosis is often poor
Although conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently used clinically, although the pain is relieved to a large extent and the survival time is prolonged, these methods have great limitations, and the curative effect is difficult to further improve.
[0003] The growth of tumor has two distinct stages, that is, from the slow growth period without blood vessels to the rapid proliferation period with blood vessels. The formation of blood vessels enables the tumor to obtain enough nutrition to complete the vascular switching period. If there is no angiogenesis, The growth of the primary tumor does not exceed 1 to 2 mm, and metastasis cannot be achieved

Method used

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  • Soluble VEGFR difunctional fusion receptors, preparation method and use thereof
  • Soluble VEGFR difunctional fusion receptors, preparation method and use thereof
  • Soluble VEGFR difunctional fusion receptors, preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Cloning of Human Antibody Light and Heavy Chain Constant Regions and Fc Region Genes

[0058] Isolate healthy human lymphocytes with lymphocyte separation medium, and extract total RNA with Trizol reagent (Invitrogen company product), according to literature (Cell, 1980, 22: 197-207) and literature (Nucleic Acids Research, 1982, 10: 4071-4079) Primers were designed to amplify the heavy chain and light chain constant region genes of the reported sequence, and the Fc region of the antibody was amplified with primers FC sense: GAAGCTTAATAATGGCCCGGGCGAGCCCAAATCTTGTGAC and FC antisense: CGAATTCTCATTTACCCGGAGACAGG. All PCR reactions were hot-started, and the reaction conditions were: 94°C for minutes; 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 1 minute and 10 seconds, 30 cycles; 72°C for 10 minutes. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector. After sequencing verification, it was confirmed tha...

Embodiment 2

[0059] Example 2: Construction of Soluble Bifunctional VEGFR Fusion Receptor

[0060] 用淋巴细胞分离液分离健康人淋巴细胞,用Trizol试剂(Invitrogen公司产品)提取总RNA,以引物VEGFR11有义:TAAGCTTGCCGCCACCATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTGTCTGCTTCTCACAGGATCTAGTTCAGGTTCAAAATTAAAAGATCCTG,VEGFR12反义:CAGATGGTGCAGCCACAGTGCCCGGGCCTGCTTTATCATATATATGCACTGAGG(Invitrogen公司合成),做RT-PCR获得VEGFR1基因Ig1 The -3 region was loaded into the PGEM-T vector (PGEM-T / VEGFR1). The VEGFR3 gene was synthesized according to the sequence in embl|AR860556|AR860556 by Shanghai Bioengineering Technology Service Co., Ltd., and the VEGFR 1 gene signal peptide (with primer VEGFR11 义:TAAGCTTGCCGCCACCATGGTCAGCTACTGGGAC;VEGFR11反义:ACGAAAGGTCTACCTCCGGAACTAGATCCTGTGAGAAG以PGEM-T / VEGFR1为模板扩增)和Ig 2-3区(以引物VEGFR12有义:TCCGGAGGTAGACCTTTCG;VEGFR12反义:CAGATGGTGCAGCCACAGTGCCCGGGCCTGCTTTATCATATATATGCACTGAGG,以PGEM-T / VEGFR1为模板扩 Increment) and human antibody light chain constant region (k constant region) gene (using primer LC sense: ACTGTGGCTGCACCATCTGT; LC an...

experiment example 1

[0068] Experimental Example 1: Affinity Detection Experiment of Soluble Bifunctional VEGFR Fusion Receptor-ELISA Method

[0069] Experimental procedure: Dilute VEGF and VEGF-C (products of R&D Company) with 0.05mmol / L sodium carbonate-sodium bicarbonate buffer (pH 9.6) to 2ug / ml, 100ul / well, and coat overnight at 4°C. After blocking with 3% skimmed milk at room temperature for 2 hours, different concentrations of soluble VEGFR fusion receptors VEGFR1Fc, VEGFR3Fc, VEGFRFc, VEGFRIg, VEGFRIgM were added, 100ul / well, and three parallel wells were taken for each concentration, and incubated at room temperature for 2 hours. Discard the supernatant, wash with PBS three times, add HRP-labeled mouse anti-human IgG monoclonal antibody (product of DAKO Company) diluted according to the titer, 50ul / well, and incubate at room temperature for 45min. After fully washed with PBS, OPD was protected from light for color development, and 2mol / L H 2 SO 4 After the reaction was terminated, the a...

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Abstract

The invention discloses soluble VEGFR difunctional fusion receptors, a preparation method thereof and the application thereof to antineoplastic preparation. Particularly, the invention discloses mutants VEGFRIg of the soluble VEGFR difunctional fusion receptors VEGFRFc, VEGFRIg and VEGFRIg and a method for preparing the difunctional fusion receptors. The difunctional fusion receptors can be combined with the VEGF and VEGF-B, and also can be combined with the VEGF-C or VEGF-D, and are used for preparing the antineoplastics.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses a kind of bifunctional fusion receptor, its preparation method and its application in the preparation of antitumor drugs. Background technique [0002] Tumors, especially malignant tumors, are diseases that seriously endanger human health in the world today, ranking second among the deaths caused by various diseases. And in recent years, its incidence has shown an obvious upward trend. The curative effect of malignant tumors is poor, the rate of advanced metastasis is high, and the prognosis is often poor. Although conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently used clinically, although the pain is relieved to a large extent and the survival time is prolonged, these methods have great limitations, and the curative effect is difficult to further improve. [0003] The growth of tumor has two distinct stages, t...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/13C12N15/63C12N5/10C12P21/02A61K38/17A61P35/00
Inventor 郭亚军王皓李博华张大鹏寇庚侯盛钱卫珠
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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