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Fusion protein with function of selective killing endothelial cells in tumor neogenetic blood vessels and use thereof

A technology of tumor neovascularization and fusion protein, which is applied in the field of fusion protein and its coding gene and application, and can solve the problem that vascular endothelial cells cannot be detected

Inactive Publication Date: 2010-10-13
SHANXI KANGBAO BIOLOGICAL PROD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been confirmed that the F1k1 / KDR receptor has tyrosine kinase activity and is overexpressed on the surface of vascular endothelial cells in various malignant tumor tissues, while it is almost undetectable in the vascular endothelial cells of adjacent normal tissues

Method used

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  • Fusion protein with function of selective killing endothelial cells in tumor neogenetic blood vessels and use thereof
  • Fusion protein with function of selective killing endothelial cells in tumor neogenetic blood vessels and use thereof
  • Fusion protein with function of selective killing endothelial cells in tumor neogenetic blood vessels and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0066] Example 1. The fusion protein VEGF having the effect of selectively killing tumor neovascular endothelial cells 121 / Acquisition of LuffinKDEL coding gene

[0067] 1. Human vascular endothelial growth factor VEGF 121 Acquisition of coding genes

[0068] 1. Extraction of total RNA from human liver cancer tissue

[0069] The total RNA of human liver cancer tissue was extracted using Trizol reagent from Invitrogen Company. The method was as follows: 100 mg of human liver cancer tissue (taken from the PLA General Hospital) was put into a sterile glass grinder, and 1 mL of Trizol reagent was added, quickly ground in an ice bath, and pipetted. Put the supernatant into an RNase-free centrifuge tube, centrifuge at 12000g for 10min at 4°C, absorb the supernatant, add 0.2mL chloroform / 1mL Trizol, mix well and let stand for 2-3min, then transfer the aqueous phase to a new centrifuge tube Add an equal volume of isopropanol, mix by inversion, precipitate at room temperature for 1...

Embodiment 2

[0082] Example 2, VEGF 121 -Expression and purification of LuffinKDEL fusion protein toxin in Escherichia coli

[0083] Inoculate 10mL of engineered bacteria cultured overnight into 1000mL LB liquid medium containing 25ug / mL kanamycin, cultivate at 37°C to OD=0.4-0.6, then add IPTG to a final concentration of 1mM, and at the same time, add IPTG without adding IPTG As a control, the induction culture was continued for 5 hours. After the cultivation, take 500ul of bacterial liquid, centrifuge at 10,000g for 2 minutes to collect the bacterial cells, add 50ul of loading buffer, denature at 100°C for 5 minutes, centrifuge at 12,000g for 1 minute, take 10ul for 10% denatured polyacrylamide gel electrophoresis detection, detection The result is as figure 1 As shown (lane 1 is the protein molecular weight standard, lane 2 is the bacterial protein before induction, and lane 3 is the bacterial protein after induction), after induction and expression with IPTG, a recombinant protein wi...

Embodiment 3

[0085] Embodiment 3, tumor suppression experiment

[0086] 5-week-old Balb / c nude mice, female, weighing 18-20 g, were randomly divided into 2 groups, 5 mice in each group. A375 cells were cultured in vitro, and each nude mouse was injected with 5×10 5 For human melanoma A375 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 -LuffinKDEL / kg body weight, with injection of the same volume of saline as a control. Measured at different times in the control and test groups (VEGF 121 -Luffin (KDEL) 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 - P121 -LuffinKDEL) tumor growth curves at different times after administration, the growth curves are as follows image 3 Shown, show that the fusion protein VEGF of the present invention 121 -LuffinKDEL has a significant in...

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Abstract

This invention relates to a fusion protein that possess action of selectively kill tumour rebirth blood vessel endotheliocyte, and its application. This fusion protein through connecting peptide connect VEGF121 with amido end of sponge gourd seed ribosome inactivating protein(RIP) Luffin Alpha. The vascular endothelial cell growth factor VEGF121 act as means of delivery, make this fusion toxin idiosyncratic incorporate with vascular endothelial cell growth factor receptor F1k1 / KDR, thereby optionally ingress tumour rebirth vascular endothelial cell; the polypeptide possess amphipathic molecule character, by destroy lysosome membrane to promote the release of lysosome inside dissociate toxin molecule; the toxin carboxyl terminal endoplasmic reticulum loacting signal led toxin molecule( sponge gourd seed RIP Luffin Alpha) to target site to exert toxic effect, so to destroy rebirth blood vessel of tumour organization, cut off tumour blood supply, achieve the end of restraining tumor.

Description

technical field [0001] The present invention relates to a fusion protein and its encoding gene and application, in particular to a fusion protein and its encoding gene capable of selectively killing tumor neovascular endothelial cells, as well as the expression method of the fusion protein and its application in the preparation of antitumor drugs. Background technique [0002] Cancer is one of the major diseases that seriously threaten human health. There are more than 10 million new cancer cases in the world every year, and more than 7 million deaths. At present, there are more than 40 million cancer patients worldwide. According to statistics from the Ministry of Health, the incidence of cancer in my country is on the rise. 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 4.5 million. [0003] Because traditional tumor treatment...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/16A61K48/00A61P35/00
Inventor 孙红琰周满祥申云飞
Owner SHANXI KANGBAO BIOLOGICAL PROD
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