METHOD FOR CONSTRUCTING CELL MODEL FOR DETECTING PYROGEN, CELL MODEL AND PYROGEN DETECTION KIT

摘要

The present invention provides a construction method of cell models for detecting pyrogens, cell models and pyrogen detection kits. By inducing double-bond breakage in the specific positions of genomes using CRISPR/CAS9, performing directed knock-in of TLR4 and CD14-MD2 on two chromosomes in a cell line based on the principle of homologous recombination and repair, and tracing using green fluorescent protein GFP and red fluorescent protein RFP respectively, we successfully construct the TLR4/CD14/MD2 directed knock-in and fluorescence-traceable cell model; upon the exposure to LPS, the release of IL-6 and TNF-a cytokines in the cell model can be detected by ELISA, Western Blot, mass spectrometry and magnetic bead method; and the cell model has good stability and high sensitivity, and the lowest detection limit can reach 0.005EU/mL, which is far lower than that of the Limulus amebocyte lysate endotoxin test method (0.025EU/mL).