Preparation and application of tumour vascular targeting agent VEGF*/RIP30fusion toxin

A new blood vessel and toxin technology for tumors, which is applied in the direction of anti-tumor drugs, drug combinations, recombinant DNA technology, etc., can solve the problems of normal tissue and cell damage, large toxic and side effects, and poor tumor specificity

Inactive Publication Date: 2009-02-04
SHANXI KANGBAO BIOLOGICAL PROD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Traditional anticancer drugs have poor tumor specificity. While killing cancer cells, they also cause damage to normal tissues and ...

Method used

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  • Preparation and application of tumour vascular targeting agent VEGF*/RIP30fusion toxin
  • Preparation and application of tumour vascular targeting agent VEGF*/RIP30fusion toxin
  • Preparation and application of tumour vascular targeting agent VEGF*/RIP30fusion toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, fusion protein Trx-VEGF 121 Construction of / RIP30KDEL expression vector

[0060] The following method is used to construct the fusion toxin VEGF capable of destroying neovascular endothelial cells of tumors 121 The coding gene of / RIP30KDEL and its prokaryotic expression vector, the specific method comprises the following steps:

[0061] 1. VEGF 121 Cloning of the G4S gene

[0062] 1. Primer design

[0063] Designed PCR to amplify VEGF 121 Peptide G 4 S fusion gene (VEGF 121 G4S), and the recognition site of restriction endonuclease Kpn I is added at the 5' end of the forward primer, and the coding sequence of connecting peptide G4S and the recognition site of restriction endonuclease BamH I are added on the reverse primer, The primer sequences are as follows:

[0064] Upstream primer F: 5'-ggTACCgACgACgACgACAgggCACCgATggCAgAAggTggCggg, downstream primer R: 5'-ggATCCgCCACCgCCCCgCCTCggCTTgTCACATTTTTC.

[0065] 2. Extraction of total RNA from human...

Embodiment 2

[0103] Embodiment 2, fusion protein VEGF 121 Preparation of / RIP30KDEL

[0104] 1. Fusion protein Trx-VEGF 121 / Expression of RIP30KDEL in Escherichia coli

[0105] 1. Add pET32-VEGF 121 The / RIP30KDEL plasmid was transformed into Escherichia coli Origami (DE3), cultured at 37°C for 30 hours, and positive clones were screened.

[0106] 2. Inoculate the positive single colony in 10 mL of LB liquid medium (containing carbenicillin 200 ug / mL, kanamycin 30 ug / mL and tetracycline 25 ug / mL), and culture overnight at 37 °C and 250 rpm.

[0107] 3. Transfer 10 mL of the overnight culture to 1 L of LB liquid medium (containing carbenicillin 200ug / mL, kanamycin 30ug / mL and tetracycline 25ug / mL), and culture to OD at 37°C and 250rpm 600 ≈0.6 (about 4 hours).

[0108] 4. Add IPTG to a final concentration of 0.1mM (add 100ul of 1M IPTG solution), and continue culturing overnight at 23°C and 250rpm; after the culture, centrifuge at 6000rmp for 5 minutes to harvest the bacteria.

[010...

Embodiment 3

[0124] Example 3, VEGF 121 / RIP30KDEL tumor inhibition experiment

[0125] 5-week-old Balb / c nude mice, female, weighing 18-20 g, were randomly divided into 2 groups, 5 mice in each group. A375M cells were cultured in vitro (purchased from the Cell Center of Chinese Academy of Medical Sciences), and each nude mouse was injected with 5×10 5For human melanoma A375M cancer cells, the day of tumor inoculation is regarded as the first day, and the third day is administered with a dose of 15 mg VEGF 121 / RIP30KDEL / kg body weight, with the same volume of saline injected as a control. Measured at different times in the control and test groups (VEGF 121 / RIP30KDEL) mouse tumor size. A two-factor design ANOVA with repeated measures was carried out on the outcome data, and the results were compared with the control group (saline), VEGF 121 The P121 / RIP30KDEL) tumor growth curves at different times after administration, the growth curves are as follows Figure 7 Shown, show that fu...

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Abstract

The invention discloses a fusion toxicant having the function of selectively killing new blood vessel endothelial cells of tumors and an application thereof. The fusion toxicant is a fusion protein which is prepared in the way that the amido ends of ribosome inactivating proteins RIP30 of balsam pear seeds are connected with VEGF121 through connecting peptides. As a carrier, VEGF121 can internalize and bond the fusion toxicant to blood vessel endothelial cell nutrilite receptors F1k1/KDR in a specific way before the fusion toxicant enters blood vessel endothelial cells and plays a role as a toxicant to kill new blood vessel endothelial cells of tumors, to destroy the new blood vessels of tumor tissues and cut off the blood supply of tumors, thus inhibiting tumors. In addition, the expression conditions of the fusion toxicant are simple; the fusion toxicant is purified easily and can be produced in large scale. Accordingly, the fusion toxicant can be used as an active ingredient for preparing antineoplastic drugs. The fusion protein can play an important role in the medical and biological pharmacy field.

Description

technical field [0001] The invention relates to a tumor blood vessel targeting agent in the field of biotechnology, in particular to a fusion toxin VEGF with the effect of selectively killing tumor neovascular endothelial cells 121 / RIP30KDEL fusion toxin and its coding gene and its application in the preparation of antitumor drugs. Background technique [0002] Cancer is one of the major diseases that seriously threaten human health. Since 1996, there have been more than 10 million new cases of cancer in the world every year, and more than 7 million deaths; by the end of 1999, there were more than 40 million cancer patients worldwide. According to statistics from the Ministry of Health, in the past 20 years, the incidence of cancer in my country has shown an overall upward trend. In recent years, there have been 2 million new cancer patients and more than 1.3 million deaths each year. At present, the total number of cancer patients in the country is estimated to be about 4...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A61K38/16A61K48/00A61P35/00
Inventor 孙红琰周满祥申云飞
Owner SHANXI KANGBAO BIOLOGICAL PROD
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