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Cell line screened by anti-oxidant or chemical preventive agent, construction and application

An antioxidant and chemoprevention technology, applied in the field of cell lines, can solve the problems of not obtaining stable cell lines, complex and cumbersome construction methods, and inability to save time and use immediately, and achieve high-throughput applications, excellent test sensitivity, The effect of enhancing the activity of luciferase

Inactive Publication Date: 2013-01-30
TIANJIN YAOYU BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the 200510118390.3 patent application, the inventor provided a method for constructing a cell model for screening antioxidant drugs. The cell model established by this method was established by transiently transfecting cells with recombinant plasmids, and no stable cell lines were obtained. When multiple samples are required and a large number of screenings are required Inconvenient to use, can not save time and use
In the 201010293566.X patent application, the chemoprevention drug screening model provided by the inventor is complex and cumbersome, and the selected reporter gene GEP has a certain influence on the normal activities of cells. Polyubiquitination, so GFP-based drug screening may have some unexpected interference factors

Method used

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  • Cell line screened by anti-oxidant or chemical preventive agent, construction and application
  • Cell line screened by anti-oxidant or chemical preventive agent, construction and application
  • Cell line screened by anti-oxidant or chemical preventive agent, construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: Construction of pARE-Luc-Neo recombinant plasmid

[0049] 1) According to the pHTS-MCS carrier (purchased from Biomyx Technology Company, the carrier has a luciferase gene such as the nucleotide sequence shown in SEQ ID NO.2 and a neomycin resistance gene as shown in SEQ ID NO.3 Nucleotide sequence) polyclonal restriction site map, Bgl Ⅱ and Bgl Ⅱ and Sa l Ⅰ enzyme cutting site for connection with the vector, the ARE gene sequence was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0050] 2) Design primers for PCR identification of positive clones

[0051] Upstream primer: 5′-AGTCAAGTCAGATCTCGGCCGCAATAAAATATCTTT-3′

[0052] Downstream primer: 5′-ATACAATCTCTCGAGGATTCTGCTGAGTCACTGTGAC-3′

[0053] 3) Insert the double-stranded ARE gene into the pHTS-MCS vector using Ligation high DNA ligation reagent B gl II and Sa l Ⅰ enzyme cutting site. Transform the ligation product into Escherichia coli DH5α, pick a single colony and amplify it, extract...

Embodiment 2

[0054] Example 2: Establishing a stable antioxidant or chemopreventive screening cell line Hek293-ARE

[0055] 1) Determination of the optimal screening concentration of antibiotics

[0056] Spread Hek293 cells in a 96-well plate, about 200 cells per well, and add G418 to make the final concentration of 100 μg / mL-1000 μg / mL, respectively, and set 3 replicates for each concentration, and set a blank control. After culturing for 7-15 days, the cell viability was detected by the MTT method to determine the minimum lethal concentration of G418 that caused all the cells to die. The optimal screening concentration is to increase a concentration gradient (that is, an order of magnitude) on the basis of the lowest lethal concentration that causes all cells to die. The screening results are shown in Figure 3, and the optimal screening concentration is 700 μg / mL.

[0057] 2) Transient transfection of pARE-Luc-Neo plasmid into Hek293 cells

[0058] After pARE-Luc-Neo was prepared in b...

Embodiment 3

[0065] Example 3: Antioxidant or chemopreventive agent screening cell line Hek293-ARE stability verification experiment

[0066] The antioxidant screening cell line Hek293-ARE obtained in Example 2 was subcultured, and the Hek293-ARE cells subcultured to 10 generations, 15 generations and 20 generations were respectively stimulated according to the method of stimulating and inducing TBHQ in Step 3 of Example 2. The relative activity of luciferase was detected, and the results are shown in Figure 7 (the negative control in the figure was induced without TBHQ stimulation). The results showed that the luciferase activity of Hek293-ARE could Relative activity was not affected, indicating that the cell line Hek293-ARE is very stable.

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Abstract

The invention discloses a recombinant plasmid and a construction method, the recombinant plasmid contains an antioxidation reaction element, luciferase gene and neomycin resistant gene. The invention also discloses a cell line screened by an anti-oxidant or a chemical preventive agent, a construction and an application, and the cell line is the cell integrated with the above recombinant plasmid. The provided cell line integrates the ARE gene in a recombinant plasmid carrier pARE-Luc-Neo, luciferase gene and neomycin resistant gene to a genome of a host cell Hek293, by following with subcultring of cell (more than 10 generation), the detection of a luciferase expression is stable, compared with a transient transfection method, and the construction provided by the invention is more convenient, sensitive and reliable.

Description

technical field [0001] The present invention relates to a cell line for screening antioxidants or chemopreventive agents. It specifically relates to an efficient and fast method for establishing cell lines for screening antioxidants or chemopreventive agents and its application. Background technique [0002] Studies have shown that oxidative stress plays a pivotal role in the occurrence and development of rheumatism, diabetes, cardiovascular disease, cancer and other chronic diseases, as well as aging. It has been confirmed that reactive oxygen species (ROS) are the main oxidant in the process of oxidative stress, which has a strong oxidizing ability, and is also the main seed that causes various damages, which is easy to produce chain reactions, and is harmful to proteins, nucleic acids, and lipids. etc. can produce damaging effects, thereby causing functional damage to the body. The Nrf2-ARE pathway is the most important endogenous anti-oxidative stress pathway discovere...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N5/10C12Q1/02
Inventor 张耀洲刘阳
Owner TIANJIN YAOYU BIOLOGICAL TECH
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