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Recombinant vector used for biologically detecting dioxin substances

A recombinant vector, dioxin technology, applied in the determination/inspection of microorganisms, the introduction of foreign genetic material using a vector, and the cells modified by the introduction of foreign genetic material, etc., can solve the problem of increasing the uncertainty of biological detection systems, MMTV promoter Area length and other problems, to achieve the effect of reducing detection cost, improving detection sensitivity, and reducing requirements

Active Publication Date: 2014-05-28
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] CALUX uses MMTV as the backbone of the recombinant promoter, which has both advantages and disadvantages. The advantage is that MMTV itself is a strong promoter, so it can amplify the signal of dioxin reaction to a certain extent. The disadvantage is that the MMTV promoter region is very long and may also Regulated by unknown other transcription factors, adding uncertainty to this bioassay system

Method used

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  • Recombinant vector used for biologically detecting dioxin substances
  • Recombinant vector used for biologically detecting dioxin substances
  • Recombinant vector used for biologically detecting dioxin substances

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Embodiment 1

[0051] Mouse genomic DNA was obtained from hepatocarcinoma Hepa1 cells and extracted according to the standard method of the kit.

[0052] 1. Construction of pCL-FL plasmid

[0053] 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.

[0054] 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.

[0055] The 5' primer is: 5'-CACGCTCGAGAACAGGTTGAGTTAGAC-3' (SEQ ID NO: 1)

[0056] The 3' primer is: 5'-CACGAAGCTTCAGGGTTAGGGTGAAG-3' (SEQ ID NO: 2)

[0057] 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 final...

Embodiment 2

[0095] Next Hepa1 cells were transiently transfected with pCL-FL or pGL6.1. Hepa1 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 or pGL6.1 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 (for the experimental method, refer to Promega double Luciferase reporter gene detection kit instructions). Then, according to the specific requirements of the experiment, the cells were stimulated with TCDD at a concentration of 1 nM for 4 h, and the cells were treated with the same conditions at a 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 reactio...

Embodiment 3

[0098] The obtained plasmids pCL-FL, pCL-CR1 and pCL-CR2 were respectively transiently transfected into Hepa1 cells. Hepa1 was introduced into 24 empty 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-CR1 or pCL-CR2 or pCL-FL plus 1 / 30 of the amount of pRL-SV40 (control reporter gene, expressing Renilla luciferase) co-transfected 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 0.1% DMSO) 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 the system control ...

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Abstract

The invention relates to a recombinant vector and in particular relates to a recombinant vector used for biologically detecting dioxin substances and a cell containing the recombinant vector. The invention also relates to an application of the recombinant vector and the cell to biological detection of the dioxin substances.

Description

technical field [0001] The invention relates to a recombinant vector, in particular to a recombinant vector used for biological detection of dioxins and cells 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] Dioxin is a kind of persistent organic pollutants (POPs), known as the "poison of the century" because of its strong toxicity and great harm to organisms. [0003] As far as the current detection methods of dioxins are concerned, there are generally two types of methods, namely instrumental analysis and biological analysis. The standard methods US EPA Method 1613 and 1618 for the detection of dioxins announced by the US Environmental Protection Agency in the 1990s defined the online detection of dioxins by high-resolution gas chromatography-high-resolution mass spectrometry (HRGC-HRMS), which is also recognized as The "gold standard"...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12Q1/68C12Q1/66C12Q1/02
Inventor 赵斌裴新辉谢群慧
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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