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Screening model of medicaments related to TGF-Beta signal path

A vector and expression vector technology, which is applied in the field of screening models for drugs related to the TGF-β signaling pathway and its dedicated vectors, can solve problems such as inability to respond to BMP, and achieve the effects of simple operation, shortening the screening cycle, and speeding up the development process.

Inactive Publication Date: 2008-05-21
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

"CAGA-box" can respond to TGF-β and activin, but not to BMP (Dennler et al., 1998, Embo J 17:3091)

Method used

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  • Screening model of medicaments related to TGF-Beta signal path

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1. Construction of a screening model for drugs related to the TGF-β signaling pathway

[0021] 1. Construction of CAGA-EGFP reporter plasmid

[0022] (1) Acquisition of 12 "CAGA-box" sequences connected in series and the adenovirus major late promoter (MLP) sequence containing "TATA box"

[0023] 12 "CAGA-box" double-stranded DNA sequences and adenovirus major late promoter (MLP) sequences were artificially synthesized, and the MLP sequence was connected behind the 12 CAGA-box sequences, and its nucleotide sequence was as follows: Shown in sequence 1 in the list.

[0024] 5'-CACACGCGTCTCGAG AAA TTT CTCGAG AAA TTT CTCGAG AAA TTT CTCGAG AAA TTT C AAGCTTAGC-3'

[0025] (2) Acquisition of the gene encoding enhanced green fluorescent protein (EGFP)

[0026] pEGFP-N1 (BD Biosciences Clontech, Catalog #6085-1) was digested with restriction endonucleases HindIII and NotI, and a 776bp gene fragment encoding enhanced green fluorescent prot...

Embodiment 2

[0034] Example 2. Using the screening model obtained in Example 1 to screen the known drug SB-431542 that can inhibit the TGF-β signaling pathway and the unknown drug A

[0035] The application method of this drug screening model is as follows: evenly culture the screening model obtained in Example 1 in 96-well plates coated with various appropriate amounts of small molecule compounds, after culturing for a period of time, add TGF-β1 stimulation, and after 24 hours Observe the change in green fluorescence. If the green fluorescence weakens or disappears, it indicates that under the interference of small molecule compounds, the expression of signal transduction molecules on the TGF-β pathway is inhibited; if the green fluorescence increases, it indicates that under the interference of small molecule compounds, the expression of TGF-β signaling pathway is inhibited. The expression of some inhibitors was affected.

[0036] The following example illustrates the application method...

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Abstract

The invention relates to a medicine selection model and a special plasmid, in particular to a medicine selection model and a special plasmid thereof relevant to the TGF-Beta signal channel. The reported plasmid provided by the invention is a recombinant expression vector obtained after a serial sequence consisting of 12 CAGA-boxes, an adenovirus major later promoter sequence and a reported protein code gene sequence is inserted into a plurality of cloning points of the mammal cell expression vectors. The selection model of medicines relevant to the TGF- Beta signal channel provided by the invention is a human liver cancer cell system which contains the reported plasmid. The selection model of the medicines relevant to the TGF- Beta signal channel of the invention is reliable and stable and is characterized by greatly-promoted selection efficiency, reduced selection cost, shortened selection cycle, reduced tested compound amount and reduced radioactive pollution. Therefore, the medicine selection model of the invention can be applied to the development of relevant medicines to reduce development cost and accelerate the development process; in this way, the invention has broad application prospects.

Description

technical field [0001] The invention relates to a drug screening model and its special plasmid, in particular to a drug screening model related to TGF-β signaling pathway and its special carrier. Background technique [0002] At present, high-throughput drug screening is an important means of discovering new drugs. The high-throughput drug screening platform consists of compound library, target selection, establishment of test methods for target and compound reactions, high-throughput screening of target interactors and The data information processing system consists of several parts. The establishment of screening models is the core of the high-throughput drug screening platform. [0003] TGF-β plays an important role in regulating a wide range of biological processes such as cell growth and differentiation, development of germ layers, organ formation, epithelial tissue proliferation, extracellular matrix synthesis, and immune responses. Uncontrolled TGF-β signal transduc...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 陈晔光王强宁元亨
Owner TSINGHUA UNIV
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