A method for preparing a pyrogen-detectable monoclonal cell line
A monoclonal, cell line technology, applied in botanical equipment and methods, biochemical equipment and methods, measuring devices, etc., can solve the problems of poor stability, low sensitivity, difficult to obtain, etc., and achieve good stability, high sensitivity, Responsive effect
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Embodiment 1
[0051] The construction of embodiment 1 recombinant plasmid and recombinant cell
[0052] 1 Construction of lentiviral recombinant plasmid
[0053] The lentiviral vector pCDH-CMV-MCS-EF1-Puro (abbreviated as pCDH-CMV-Puro) was used as the original vector to construct the reporter gene, such as figure 1 shown. pCDH-CMV-Puro and pNF-κB-Luc or pGL3-IFN-β-Luc ( figure 2 with image 3 (shown) were digested with speI and BamH I restriction endonucleases respectively, rubber-running and rubber tapping were used for recovery, after recovery, a series of operations such as connection, conversion and plating were performed. On the next day, pick 10 clones of each plasmid for colony PCR, run DNA gel, and shake the positive clones out of them until the pCDH-Puro-NF-κB / IFN-β cells grow to 16-18 hours, and the cells are turbid and opaque The plasmid can be extracted, and the operation can be performed according to the instructions of the high-purity mini-extraction kit. After the plas...
Embodiment 2
[0073] Example 2 Verification of Insertion of NF-κB / IFN-β Gene
[0074] 1. RNA Extraction and Inversion of Recombinant Cells
[0075] We mainly use Trizol reagent to extract RNA, taking 6-well plate as an example, the specific steps are as follows:
[0076] (1) Treat cells: After removing the medium, add 1ml Trizol reagent to the 6-well plate with A549 reporter cells, blow down the cells and put them into a 1.5ml sterilized RNA centrifuge tube.
[0077] (2) Layering: Add 200 μl chloroform (chloroform) to every 1 ml Trizol, shake vigorously on a shaker for 15 s, place at room temperature for 3-5 min, centrifuge at 12000 rpm / min, 4 °C for 5 min.
[0078] (3) RNA precipitation: After centrifugation, transfer the upper aqueous phase (about 500 μl) to a new centrifuge tube, rather less than more, add an equal volume of ice-cold isopropanol (put in advance at -20°C), -20 Place at ℃ for 20min, centrifuge at 12000rpm / min at 4℃ for 20min, and discard the supernatant.
[0079] (4) RN...
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