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Fusion toxin with specificity of VEGFR2/KDR acceptor, and coding gene and application thereof

A technology encoding gene and receptor specificity is applied to fusion toxin with VEGFR2/KDR receptor specificity and its encoding gene and application field, which can solve the problems of normal tissue damage and poor specificity.

Inactive Publication Date: 2014-06-18
SHANXI KANGBAO BIOLOGICAL PROD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional chemotherapy drugs use carpet bombing to kill tumors in the body, which has poor specificity and causes serious damage to normal tissues such as bone marrow, digestive tract, liver, and kidney, which has great limitations

Method used

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  • Fusion toxin with specificity of VEGFR2/KDR acceptor, and coding gene and application thereof
  • Fusion toxin with specificity of VEGFR2/KDR acceptor, and coding gene and application thereof
  • Fusion toxin with specificity of VEGFR2/KDR acceptor, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Embodiment 1, fusion toxin rhVEGF 121KDR Expression and purification of rGEL30

[0109] 1. Construction of recombinant expression vector pTrx-rhVEGF 121KDR / rGEL30 and pET32a(+)-rhVEGF 121KDR / rGEL30

[0110] 1. Construction of recombinant expression vector pTrx-rhVEGF 121KDR / rGEL30

[0111] 1) The amino-terminal (N-terminal)-linked thioredox protein (Trx) mutant (mTrx) was synthesized by Beijing Aoke Company (the amino acid residue sequence of the thioredox protein mutant mTrx is shown in SEQ ID NO: 7 in the sequence listing The nucleotide sequence of its coding gene is shown in SEQ ID NO: 8 in the sequence listing. SEQ ID NO: 7 in the sequence listing consists of 109 amino acid residues, starting from the 33rd-36th (4aa) amino acid at the amino terminal The residues are Cys-Pro-Tyr-Cys, the 34th amino acid residue from the amino terminal is a mutant amino acid residue Pro34, and the 35th amino acid residue from the amino terminal is a mutant amino acid residue T...

Embodiment 2

[0128] Example 2, rhVEGF 121KDR Toxicity detection of / rGEL30 fusion toxin on PAE / KDR and PAE / FLT cells

[0129] detect rhVEGF 121KDR The toxic effect of / rGEL30 fusion toxin on PAE / KDR and PAE / FLT cells, the specific method comprises the following steps:

[0130] 1) Dilute PAE / KDR and PAE / FLT cells in logarithmic growth phase to 1.5×10 4 / mL, add to 96-well plate, 200 μl per well (3000 cells / well) (no cells in the first row), 37°C, 5% CO 2 Incubate overnight. Gradients in 96-well plates (10 -3 、10 -2 、10 -1 、10 0 、10 1 、10 2 、10 3 、10 4 ) release rhVEGF 121KDR / rGEL30 fusion toxin (initial concentration 1 μM) in a final volume of 200 μl per well. Pour off the medium in the cell culture wells, add fusion toxins of different concentrations (add fresh medium to the first row, and not add toxin to the second row), at 37°C, 5% CO 2 Incubate for 72 hours.

[0131] 2) Pour off the medium in the cell culture well, add 100 μl 0.5% crystal violet solution (prepared with ...

Embodiment 4

[0135] Example 4, rhVEGF 121KDR / rGEL30 Fusion Toxin Animal Antitumor Pharmacodynamics Observation

[0136]Take 5-week-old Balb / c nude mice, female, weighing 18-20 g. The colon cancer HT-29 cell line was inoculated subcutaneously in the back of nude mice, and the tumor grew to about 200 (mm) 3 After that, take it out and homogenize, so that it can be passaged twice in vivo, and then in 1×10 6 / 0.2mL concentration and inoculated subcutaneously in the back of nude mice respectively, and sent them back to the animal breeding room to continue feeding after inoculation. About a week after inoculation, hard nodules with a diameter of 2-3 mm can be felt subcutaneously at the inoculation site of the mice, indicating that the mouse xenograft tumor model was established successfully. The animals were randomly divided into 4 groups, 6 animals in each group. Start medication.

[0137] Dosage, Frequency and Duration of Administration:

[0138] Negative control group (negative control...

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Abstract

The invention discloses a fusion toxin (rhVEGF121KDR / Rgel30) with specificity of VEGFR2 / KDR acceptor, and coding gene thereof and application thereof to prepare antitumor medicines. The fusion toxin is obtained by connecting the amino terminal (N terminal) of gelonium multiflorum ribosome inactivating protein rGEL30 to recombinant human vascular endothelial growth factor VEGF121 mutant through a connecting peptide, and the carboxyl terminal (C terminal) of the fusion toxin possesses endoplasmic reticulum positioning sequence. VEGF121KDR taken as a carrying tool helps the fusion toxin to specifically combine with VEGFR2 / KDR through high affinity, to be internalized and to enter tumor newborn blood vessel endothelial cells, then ribosome inactivating protein rGEL30 gives a play to toxin effect and kills tumor newborn blood vessel endothelial cells and further destroys newborn blood vessels of tumor tissue for reaching the purpose of inhibiting tumor, and also an endoplasmic reticulum positioning signal at the C terminal of the fusion toxin is capable of promoting free rGEL30 toxin to be gathered on rough endoplasmic reticulum, so that the antitumor effect of the fusion toxin is further enhanced.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a 2 / KDR receptor-specific recombinant human vascular endothelial growth factor VEGF 121 Fusion toxin of mutants with ribosome-inactivating protein (rhVEGF 121KDR / rGEL30) and its coding gene, as well as the preparation method of the fusion toxin and its application in the preparation of antitumor drugs. Background technique [0002] Malignant tumors seriously threaten human health. According to statistics from the International Union Against Cancer (UICC), there are more than 10 million new cases and more than 7 million deaths worldwide every year. At present, there are about 20 million malignant tumor patients in my country, with about 1.8-2 million new cases and 1.4-1.5 million deaths each year. [0003] Traditional chemotherapy drugs use carpet bombing to kill tumors in the body, which has poor specificity and causes serious damage to normal tissues such as bone marrow, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/62C12N15/63C12N5/10C12N1/21C12N1/19A61K38/47A61K47/48A61P35/00
CPCA61K38/47A61K47/642A61K48/0058C07K14/415C07K14/485C07K2319/04C07K2319/33
Inventor 孙红琰周满祥申云飞
Owner SHANXI KANGBAO BIOLOGICAL PROD
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