Recombinant cell strain for detecting dioxin compounds according to behaviour of leuciferinase gene

A technology of recombinant cells and recombinant vectors, which can be applied to cells modified by introducing foreign genetic material, recombinant DNA technology, and the determination/inspection of microorganisms.

Inactive Publication Date: 2006-03-22
CHENG SHIU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0021] To sum up, among the current dioxin detection methods, the chemical analysis method is the most accurate, but the disadvantages are complicated operation process, long test time and high cost
As for the enzyme-linked immunosorbent assay based on immunochemical methods, it has the advantages of specificity and sensitivity of biological detection methods, but it is still quite cumbersome in operation.
However, the biosensor based on the immunoassay method is not mature enough in the whole technical level and has not been widely used.

Method used

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  • Recombinant cell strain for detecting dioxin compounds according to behaviour of leuciferinase gene
  • Recombinant cell strain for detecting dioxin compounds according to behaviour of leuciferinase gene
  • Recombinant cell strain for detecting dioxin compounds according to behaviour of leuciferinase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1. Construction of recombinant plasmids M4P2 and MP containing dioxin-response elements containing dioxin-response elements

[0146] According to the nucleotide sequence shown in the accession number (accession number) AF210905 [Mus musculus (Mus musculus) CYP1A1 gene, promoter region and partial sequence] registered in the NCBI website and the accession number X01681 [Mus musculus (Mus musculus) musculus) CYP1A1 gene], and use the software Primer3 Version to design the following two sets of primer pairs for the dioxin-reactive fragments (DREs) in the Mus musculus (Mus musculus) CYP1A1 gene sequence (Mus musculus) CYP1A1 gene sequence ( primer pairs), wherein the nucleotide sequences of these primers were synthesized by Bost Biotechnology Co., Ltd.:

[0147] Forward primer 1

[0148] 5'-cagagagcacctgcaaaaca-3' (SEQ ID NO: 1)

[0149] Reverse primer 1

[0150] 5'-ggctacaaagggtgatgctt-3' (SEQ ID NO: 2)

[0151] forward primer 2

[0152] 5'- ctcgag gagggcaggt...

Embodiment 2

[0172] Example 2. Transfection of mouse hepatoma cells Hepa-1C1C7 with recombinant vectors M4P2 and MP using recombinant vectors M4P2 and MP

[0173] The Hepa-1C1C7 cell line can be transfected with the recombinant vector MP or M4P2 according to the following preparation procedure A or preparation procedure B, wherein the culture method of the Hepa-1C1C7 cell line is as described in the section "Cell Lines and Culture Conditions" above operating procedures.

[0174] Preparation Procedure A:

[0175] 1. Mix 172 μl of basic DMEM with 28 μl of GenePORTER TM 2. Mix Transfection Reagent (GST Inc.) evenly to form a first solution, and add 200 μl of New DNA diluent B (GST Inc.) and 8 μg of DNA [wherein the selected recombinant vector DNA and pcDNA (InvitrogenCooperation) are each 4 μg ] mixing uniformly to form a second solution, and allowing the first and second solutions to stand separately for 5 minutes; then, mixing the two solutions uniformly, and then allowing the resulting ...

Embodiment 3

[0190]Example 3. Establishment of the correlation of the correlation of dioxin concentration vs. the light intensity caused by luciferase with respect to the light intensity caused by luciferase

[0191] A. Expression of luciferase gene in transfected cells after the stimulation of dioxin:

[0192] The transfected cells (4×10 5 cells / well) were placed in a 24-well culture plate, and 1ml of medium (DMEM+10%FCS) was added to each well, and then the culture plate was placed at 37°C, 5% CO 2 placed in a constant temperature incubator for 2 hours to allow the cells to attach.

[0193] Dioxin (2,3,7,8-TCDD) stock solution (stock solution) with a concentration of 10 μg / ml was made 10-fold serial dilution (10-fold serial dilution) with DMSO, and 3.22 μl was taken for each dilution And added to each well of the culture plate, so that the final concentration of dioxin in each experimental group was 10nM, 1nM, 100pM, 50pM, 10pM, 1pM and 0.1pM, and each experimental group was repeated 4...

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Abstract

The present invention relates to one recombinant cell strain with foreign leuciferinase gene, and is especially one DNA segment of transfected mouse liver cancer cell strain Hepalcle7-M4P2 and Hepale7-MP and with dioxin reaction segment specific nucleotide sequence í‹cacgcíŒ and í‹gcgtgíŒ. The DNA segment is designed based on Mus musculus CYP1 A1 gene promoter area and partial fore end sequence of the structural gene. Therefore, the recombinant cell strain and the cultured descendant may be used in detecting dioxin compounds from the sample collected from natural environment by means of biological analysis process.

Description

field of invention [0001] The present invention is mainly related to a recombinant cell line with an exogenous luciferase gene (exogenous luciferase gene), especially the transfected mouse liver cancer cell (transfected mouse hepatoma cell) Hepa1c1c7-M4P2 and Hepa1c1c7 -MP, wherein upstream of the luciferase gene is located a DNA fragment containing the nucleotide sequences "cacgc" and "gcgtg" which may be unique to dioxin-responsive fragments. Background technique [0002] Due to the excessive development of civilization, many tangible or intangible pollution factors have repeatedly invaded the human society living in the civilized world, and among the various pollution factors, dioxins are considered to be the most toxic chemicals to humans and animals today. Products (chemicals), and therefore known as "the poison of the century." In the past 20 years of research, it has been found that dioxins not only affect human fertility, cause developmental disorders, damage the im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12N15/63C12N15/52C12N5/10C12Q1/00C12Q1/68
Inventor 李伟山李心予张简国平黄怡玮卢冠妤
Owner CHENG SHIU UNIVERSITY
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