Recombinant vectors and cells for biological detection of dioxin-like substances

A technology of recombining cells and dioxins, applied in the direction of cells modified by introducing foreign genetic material, microorganism-based methods, biochemical equipment and methods, etc., to reduce requirements, improve detection sensitivity, and reduce detection costs.

Active Publication Date: 2016-11-30
拓感(深圳)科技创意有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current problem is that in the future, the purity of the sample can be improved by optimizing the pretreatment method of the environmental sample, and the specificity of the CALUX system can be further improved

Method used

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  • Recombinant vectors and cells for biological detection of dioxin-like substances
  • Recombinant vectors and cells for biological detection of dioxin-like substances
  • Recombinant vectors and cells for biological detection of dioxin-like substances

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Experimental program
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Effect test

Embodiment 1

[0057] Mouse genomic DNA was obtained from hepatocarcinoma Hepa 1 cells and extracted according to the standard method of the kit.

[0058] 1. Construction of pCL-FL plasmid

[0059] Mouse genomic DNA was obtained from Hepa1 cells and extracted according to the standard method of the kit. And pCL-FL was constructed separately by standard molecular cloning means.

[0060] The purpose of this experiment is to construct a dioxin reporter gene detection plasmid including the original sequence of the mouse CYP1A1 promoter (-1400 ~ +3), which contains 6 DRE sequences. Using mouse genomic DNA as a template, a PCR reaction was performed with the following primers.

[0061] The 5' primer is: 5'-CACGCTCGAGAACAGGTTGAGTTAGAC-3' (SEQ ID NO: 1)

[0062] The 3' primer is: 5'-CACGAAGCTTCAGGGTTAGGGTGAAG-3' (SEQ ID NO: 2)

[0063] After the PCR fragment was obtained, it was digested with XhoI and HindIII, and the vector plasmid pGL3-basic was subjected to the same double digestion, and fina...

Embodiment 2

[0101] Next, Hepa 1 cells were transiently transfected with pCL-FL. Hepa 1 was introduced into 24-well plates in equal amounts one day in advance, and the plasmid was transiently transfected with liposomes (LTX) when the cell density reached 50-80% the next day. That is, 0.5 μg of PCL-FL plus 1 / 30 of the amount of pRL-SV40 (control reporter gene, expressing Renilla luciferase, purchased from Promega) co-transfected cells for 18-24 hours (refer to Promega dual luciferase reporter Instructions for genetic testing kit). Then, according to the specific requirements of the experiment, the cells were stimulated with 1 nM concentration of TCDD for 4 hours, and the cells were treated with the same conditions at the final concentration of 0.1% DMSO as a negative control. The cells were lysed, and the activities of firefly luciferase and Renilla luciferase were detected by a microplate reader with a double reporter method. Finally, the reaction value of Renilla luciferase was used as ...

Embodiment 3

[0103] The obtained plasmids pCL-FL, pCL-CR1 and pCL-CR2 were respectively transiently transfected into Hepa 1 cells. The same amount of Hepa 1 was introduced into 24 empty plates one day in advance, and when the cell density reached 50-80% the next day, the plasmid was transiently transfected with liposomes (LTX). That is, 0.5 μg of pCL-CR1 or pCL-CR2 or pCL-FL plus 1 / 30 of the amount of pRL-SV40 (control reporter gene, expressing Renilla luciferase) co-transfected the cells for 18-24 hours. Cells were then treated with α-MEM containing a final concentration of 0.1% DMSO or 1 nM TCDD (also at a final concentration of DMSO of 0.1%) for 24 hours. Aspirate the medium and wash the cells once with PBS, then lyse the cells with 150 μl Promega lysis buffer for 15 minutes. Then pipette 20 μl of cell lysate into a 96-well white opaque microtiter plate, and repeat 3 times for each sample. Then, the reaction value of firefly luciferase induced by TCDD and the reaction value of system ...

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Abstract

The invention relates to a recombinant vector and a recombinant cell, in particular to a recombinant vector for biological detection of dioxins and a recombinant cell containing the recombinant vector. The present invention also relates to the use of the recombinant vector and cells in the biological detection of dioxins.

Description

technical field [0001] The invention relates to a recombinant vector and a recombinant cell, in particular to a recombinant vector for biological detection of dioxins and a recombinant cell containing the recombinant vector. The present invention also relates to the use of the recombinant vector and cells in the biological detection of dioxins. Background technique [0002] With the development of human society, the problem of environmental pollution has become increasingly complex and diverse, which makes the negative impact of environmental pollution on human health increasingly serious. The World Health Organization (WHO) pointed out in its "2004 World Health Report" that an estimated 24% of the disease burden (years of healthy life lost) and 23% of all deaths (premature death) worldwide can be attributed to environmental factors, And one of the most important factors is the increasing chemical pollutants. Among many chemical pollutants, persistent organic pollutants (P...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C12Q1/68C12Q1/66C12Q1/02C12R1/91
Inventor 赵斌李帅章谢群慧郑明辉裴新辉周志广
Owner 拓感(深圳)科技创意有限公司
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