Method for screening medicament taking focal adhesion kinase (FAK) transcriptional regulation as target spot and application

A technology for transcriptional regulation and screening methods, applied in biochemical equipment and methods, microbial assay/inspection, etc., can solve the problems of lack of major breakthroughs in carrier and delivery, and the inability of RNA interference therapy to be practically applied, and achieve the effect of convenient use.

Inactive Publication Date: 2010-09-29
JIANGSU TARGET BIOMEDICINE RES INST
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Although RNA interference methods are effective in experiments and animal models, RNA interference therapy is essentially a gene therapy, and the carrier and delivery of gene therapy have lacked major breakthroughs since the 1990s, so RNA interference therapy is not yet practical. application

Method used

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  • Method for screening medicament taking focal adhesion kinase (FAK) transcriptional regulation as target spot and application
  • Method for screening medicament taking focal adhesion kinase (FAK) transcriptional regulation as target spot and application
  • Method for screening medicament taking focal adhesion kinase (FAK) transcriptional regulation as target spot and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] 1. Construction of mouse FAK promoter reporter gene vector:

[0073] Using the TRANSFAC database and bioinformatics methods to analyze and predict the mouse FAK gene promoter and its transcription factor binding sites, on this basis the mouse FAK promoter and its core sequence were obtained. Design corresponding PCR primers, chemically synthesize upstream and downstream primers (upstream primer: 5'-CGGCTAAGGATCTCCTGTGACCGTGA-3'; downstream primer: 5'-CAGCGCAGAGCTCTACGCCTGCGCAG-3', use the extracted mouse genome as a template, and use the PCR method to amplify The mouse FAK promoter sequence was obtained. The conditions of the PCR reaction were: heat denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, extension at 72°C for 1 minute, and a total of 25 cycles. The PCR product was recovered by agarose gel electrophoresis Kpn I / Sma I double digestion was carried out, and the reporter gene carrier pGL3-Basic was also subjected to Kpn I / Smal I double digestion. ...

Embodiment 2

[0127] 1. Human genomic DNA extraction:

[0128] Human pancreatic cancer cells SW1990 in the logarithmic growth phase were digested and divided into 35mm culture dishes, and cultured overnight in a cell culture incubator at 37°C and 5% CO2, until the cell density grew to about 80-90%, washed with PBS for 2 The second time, the cells were collected with a cell scraper, and the cell pellet was collected by centrifugation. The genome was extracted according to the instructions of the genome extraction kit. The extracted genome was dissolved in ultrapure water and stored at -20°C for later use.

[0129] 2. Amplify the FAK promoter sequence and construct a reporter gene vector:

[0130] On the basis of the theoretical and experimental analysis of the mouse FAK promoter in Example 1, use the TRANSFAC database and bioinformatics methods to analyze and predict the human FAK gene promoter and its transcription factor binding site, and design and amplify the human FAK promoter Primers ...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method for screening a medicament taking focal adhesion kinase (FAK) transcriptional regulation as a target spot. The method mainly comprises the steps of: expressing a reporter gene in a tumour cell by an expression vector which contains an FAK promoter and the reporter gene driven by a core segment; and detecting an expression level of the reporter gene by using a cell model, and evaluating the regulation of the medicament for the FAK promoter and the influence on a cell function which is medicated by the FAK. By using the method, the medicament taking the FAK transcriptional regulation as the target spot can be screened, and a compound obtained by screening has the application prospect for preparing an anti-metastasis medicament.

Description

1. Technical field: [0001] The invention belongs to the field of biotechnology. 2. Background technology: [0002] Although surgery and chemoradiotherapy can effectively control many cancers in the initial stages, the disease prognosis of metastatic tumors is poor. Tumor metastasis is the most common cause of death in cancer patients, and 90% of cancer patients die from metastasis of malignant tumors. At present, antitumor drugs basically target tumor growth, but inhibiting tumor growth or promoting tumor apoptosis or necrosis cannot inhibit tumor metastasis. Inhibiting tumor metastasis is the key to improving the survival rate of cancer patients. Therefore, the study of anti-metastatic drugs is urgently needed by cancer patients, and it is also a difficult point in the research of anti-tumor drugs. At present, there is no anti-metastatic drug in clinical practice. [0003] Focal Adhesion Kinase (FAK) is a non-receptor protein tyrosine kinase, which was identified in 1992...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02
Inventor 华子春唐波黄启来李淑锋
Owner JIANGSU TARGET BIOMEDICINE RES INST
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