Trichosanthin suicide ENH-TCS, recombination vector and application thereof

A technology of trichosanthin and suicide gene, applied in the field of genetic engineering

Inactive Publication Date: 2006-07-26
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Based on the chemical nature of RIP in TCS, there is no report on the expression of trichosanthin in higher mammalian cells

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Trichosanthin suicide ENH-TCS, recombination vector and application thereof
  • Trichosanthin suicide ENH-TCS, recombination vector and application thereof
  • Trichosanthin suicide ENH-TCS, recombination vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Construction of expression vector pTRE-ENH-TCS

[0030] DNA extraction, enzyme digestion, purification and recombination were carried out using conventional methods well known to workers in the field of molecular biology; the tool enzymes involved were Gibco BRL products; RNA extraction was performed with Trizol reagent from Huashun Bioengineering Co., Ltd. Kits, reverse transcriptases and related reagents are also products of the center.

[0031] TCS gene: As explained above, the TCS gene can be obtained in a variety of ways. Our group has cloned the corresponding plasmid pET-TCS, (Nie, H.L., X.F.Cai, X.H.He.X.Y.Ke, Y.B.Ke, S.X.Tam, 1998, Position 120-123, a potential active site of trichosanthin. Life Sciences, 62 (6): 491-500), the plasmid can be obtained by double digestion with NcoI and BamHI to obtain a gene fragment of about 0.8 kb, which is purified by low-melting agarose gel electrophoresis.

[0032] Acquisition of AFP gene regulatory element ENH: ...

Embodiment 2

[0040] Example 2. Cell culture and pTRE-ENH-TCS transfection

[0041] Human hepatoma cell line (7721) and human lung adenocarcinoma cells (SPCA-1) were cultured in RM1640 medium containing 10% inactivated calf serum at 37°C and 5% CO2.

[0042] Transfection was performed according to the instructions of the Tet-On kit.

[0043] First, transfect the cells with the plasmid pTet-On provided in the Tet-on system (introduce the Tet On regulatory protein into the cells); culture it in the culture medium containing G418 (Hua Shun Bioengineering Co., Ltd.) in the presence of 400 μg / ml for two months , screened out stable tetracycline response factor, G418-resistant cell clones; the resistant cells continued to culture and proliferate without G418; according to the kit instructions, the plasmids pTRE-ENH-TCS and pTRE-ENH were mixed with Tet-on The plasmid PTK-Hyg provided in the system was co-transfected and introduced into liver cancer cells and lung cancer cells respectively.

[00...

Embodiment 3

[0045] Example 3. Induction and expression of TCS gene in hepatoma cells

[0046] Hepatoma cells and lung cancer cells into which pTRE-ENH-TCS or pTRE-ENH were introduced were added with different concentrations of tetracycline (2μg / ml, 10μg / ml, 50μg / ml) and continued to culture. Control liver cancer cells were also treated in the same way.

[0047] After 24 hours, the liver cancer cells were obviously dead by light microscopy, and the death situation was aggravated after 48 hours; the higher the concentration of tetracycline, the more serious the cell death; the growth of lung cancer cells and control liver cancer cells transfected with empty vector was not affected. obvious impact.

[0048] Figure 3 is an optical microscope photograph (×400) of the growth of hepatoma cells and control cells after induction with 10 μg / ml tetracycline for 24 hours. Figure 4 shows the apoptosis of cells detected by flow cytometry.

[0049] The hepatoma cells induced by tetracycline concentra...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a Trichosanthin suicide gene ENH-TCS, whose nucleic acid sequence is provided in the specification.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a trichosanthin suicide gene ENH-TCS, a recombinant vector thereof and its application in preparing a medicine for treating tumors. Background technique [0002] "Suicide gene" is a promising tumor gene therapy method developed in recent years, in which the research on tumor-specific regulatory elements shows its superiority. However, the expression products of the usual "suicide gene" can only improve the sensitivity of tumor cells to chemotherapeutic drugs and increase the specificity of drugs. It is often necessary to synergize with other drugs to kill cancer cells. For example, a chimeric gene composed of VZV-TK and alpha-fetoprotein (AFP) transcriptional regulatory sequences constructed by Huber et al. (Brain, E.H., A.Cynthia, A.K.Richards & Thomas, 1991, Retroval-mediated gene therapy of the treatment of hepatocellular carcinoma: an innovatie approach therapy. Pr...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/63A61K48/00A61P35/00
Inventor 聂慧玲柯一保朱丽华董晓琴
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap