Recombinant vectors and cells for biological detection of dioxin-like substances
A recombinant cell and biological detection technology, applied in the direction of cells modified by the introduction of foreign genetic material, biochemical equipment and methods, microbial measurement/testing, etc., to achieve the effect of reducing the requirements for detection parameters and reducing detection costs
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Embodiment 1
[0052] 1. Construction of pCL-FL plasmid
[0053] Mouse genomic DNA was obtained from hepatocarcinoma Hepa1 cells and extracted according to the standard method of the kit. And construct pCL-FL by standard molecular cloning means ( figure 1 ).
[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 Xho I and Hind III, and the vector plasmid pGL3-basic was subjected to the same double digestion, and finally the fragment was constructed into pGL3-basic. Finally, after identification by enzyme digestion and sequencing, th...
Embodiment 2
[0079] 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
[0081]Next, Hepa 1 cells were transiently transfected with pCL-FL or pCL-CR1. 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 pCL-FL or pCL-CR1 plus 1 / 30 amount of pRL-SV40 (control reporter gene, expressing Renilla luciferase) were co-transfected into 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 control (ie Renilla luciferase) were detected respectively ...
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