Rapid cytotoxicity detection method based on luciferase reporter gene, construction method of cell strain and application of cell strain

A technology of luciferase and reporter gene, which is applied in biochemical equipment and methods, microbial determination/inspection, measuring devices, etc., can solve problems such as cell death, achieve high sensitivity, simple detection method, and facilitate high-throughput Detection effect

Pending Publication Date: 2022-03-04
NANJING UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Many environmental pollutants cause severe toxic effects by inducing cell death

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
  • Rapid cytotoxicity detection method based on luciferase reporter gene, construction method of cell strain and application of cell strain
  • Rapid cytotoxicity detection method based on luciferase reporter gene, construction method of cell strain and application of cell strain
  • Rapid cytotoxicity detection method based on luciferase reporter gene, construction method of cell strain and application of cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A method for rapidly detecting cytotoxicity based on a luciferase reporter gene, comprising the following steps in sequence:

[0041] (1) Construction of a luciferase-based reporter gene cell line;

[0042] (2) The sample to be tested is prepared into a solution and added to the culture medium, then the culture solution is added to the culture medium containing the cell line, after the culture solution is absorbed, the luciferase activity is measured to detect the cytotoxicity.

Embodiment 2

[0044] A method for constructing a luciferase reporter gene cell line, comprising the following steps in sequence:

[0045] S1: Construction of the fthl29-pGL6-TA plasmid:

[0046] S1.1: Promoter synthesis: Obtain the base sequence 3kb upstream of the transcription initiation site of the zebrafish fthl29 gene from NCBI, which is a promoter fragment, and synthesize the fthl29 gene promoter fragment by chemical synthesis;

[0047] S1.2: Ligation transformation: first digest the pGL6-TA vector with KpnI and XhoI, then carry out enzyme-ligation between the digested pGL6-TA vector and the fthl29 gene promoter fragment, and transform into TOP10 competent cells after the enzyme-ligation middle;

[0048] S1.3: Screening verification: spread the transformed E. coli on a plate containing antibiotics, the antibiotic is ampicillin, culture upside down for 12-16 hours, pick bacteria for PCR identification, extract plasmids from positive colonies, and identify recombinant plasmids by enzym...

Embodiment 3

[0058] Example 3: Application of a luciferase-based reporter gene cell line in characterizing the toxicity of pollutants:

[0059] Sodium arsenite was selected as the pollutant. Firstly, the activity of HepG2 cells was detected by the CCK-8 kit. The exposure concentration was set to 0, 0.25, 0.5, 1, 2.5, and 5 μM. The maximum exposure concentration of sodium, the experimental results are as follows image 3 As shown, 0.5 μM was finally determined as the maximum exposure concentration of sodium arsenite.

[0060] Set 6 exposure concentrations: 0, 0.01, 0.05, 0.1, 0.25, 0.5 μM, and set a control group of untransfected HepG2 cells and uninfected sodium arsenite, and set 6 parallels for each group. Before exposure, stably transfer cells were inoculated in a 96-well plate for 12 hours, the original medium was removed, and 150 μL of culture solution containing sodium arsenite of various concentrations was added. After 48 hours of exposure, the culture solution was aspirated, the ce...

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 rapid cytotoxicity detection method based on a luciferase reporter gene. The rapid cytotoxicity detection method comprises the following steps: (1) constructing a cell strain based on the luciferase reporter gene; and (2) preparing a sample to be detected into a solution, adding the solution into the culture solution, then adding the culture solution into a culture medium containing a cell strain, sucking the culture solution, and detecting the luciferase activity to detect the cytotoxicity. The construction method of the cell strain comprises the following steps: S1, constructing an fthl29-pGL6-TA plasmid; s2, transfecting the cells by using the fthl29-pGL6-TA plasmid; and S3, screening a stable cell line. The cell strain is applied to the aspects of characterizing the toxicity of pollutants and the comprehensive toxicity of actual water samples. The reporter gene cell strain constructed by using the ferroptosis related gene promoter can quickly indicate early cytotoxicity, has high specificity and high sensitivity, and is convenient for high-throughput detection; the method is suitable for toxicity effect detection of most toxic pollutants, characterization of ferroptosis related cytotoxicity and toxicity characterization of complex actual water samples.

Description

technical field [0001] The invention belongs to the technical field of pollutant cytotoxicity detection, and in particular relates to a rapid detection method of cytotoxicity based on a luciferase reporter gene, a method for constructing a cell line and an application thereof. Background technique [0002] According to incomplete statistics, there are 250 million registered chemical substances in the world, about 130,000 to 140,000 chemical substances are sold, produced, and used, and more than 4,000 are frequently used, and the number is increasing at a rate of 20,000 each year. Chemical medicines and practical chemicals, food additives, pesticides, building decoration materials, cosmetics and other chemicals frequently appear in people's production and life, and the impact of these chemicals on human health and biology has been closely watched. When various chemical substances produced in people's daily life directly or indirectly enter the water body, exceeding the load o...

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
IPC IPC(8): C12Q1/02C12N15/85C12Q1/66
CPCG01N33/5014C12N15/85C12Q1/66C12N2800/107G01N2333/90241
Inventor 张宴易珍
Owner NANJING UNIV
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