To create a stable cell line expressing equine NOD-like receptor pyrilin domain-associated protein 3(NLRP3)and to study the activation of the eqNLRP3 inflammasome,we cloned PCR-amplified eqNLRP3 and GFP genes into pLPCX by homologous recombination.The recombinant expression plasmid pLPCX-Flag-eqNLRP3-GFP was constructed and correctly identified by PCR and sequencing,and co-transfected with pCGP and pVSV-G into HEK293T cells.Lentivirus carrying eqNLRP3 was obtained 48 hours later.The lentivirus was infected with HEK293T cells,and the green fluorescent(GFP+)monoclonal cells were screened by purinomycin screening.The expression,reactivity and genetic stability of eqNLRP3 protein were determined by western blot.Western blot results showed that specific bands were present at 146.1ku,and the bands were present in all the cells transmitted to the 1st,4th,7th and 10th generations,indicating that HEK293T cell line HEK293TeqNLRP3(2G1 cells),which statically expressed NLRP3 protein,had been obtained.2G1 cells were treated with the NLRP3 activator Nigeriectin(Nig)for 1 hour.The expression plasmid pCDNA3.1-eqASC-HA was transfected into 2G1 cells and treated with Nig for 1 hour.The aggregation state of eqNLRP3 in the above cells was observed by confocal laser microscopy,and whether ASC spots(eqNLRP3-eqASC complex,which is typical of NLRP3 inflammasome activation)could be formed after expression of eqASC in 2G1 cells.The results showed that eqNLRP3 with green fluorescence in normal 2G1 cells was dispersed in the cytoplasm,and after the addition of Nig,the dispersed eqNLRP3 formed a typical green fluorescent dot aggregation in the cytoplasm.The red fluorescence(eqASC)in 2G1 cells transfected with eqASC was mainly distributed in the cytoplasm.2G1 cells transfected with eqASC were treated with Nig to form 2.5μm typical ASC spots with green fluorescence in the middle and red fluorescence around.These results indicate that HEK293TeqNLRP3 can respond to specific activator stimulation and induce the aggregation of NLRP3,recruiting ASC to form ASC spots.HEK293T cells were transfected with four kinds of inflammasome-expressing protein granules in the iGLuc reporting system constructed in our laboratory and corresponding plasmids of EIV proteins,respectively.The luciferase activity in each group of cells was detected by the reporting system 24 hours later.Western blot was used to detect the effect of the expression of EIV proteins in the cotransfected cells on the expression of four inflammasome proteins in the system,and the EIV proteins that can activate the eqNLRP3 inflammasome were screened through the above two tests.The results of iGLuc reporting system showed that the luciferase activity in the supernub of cells expressing only EIV M2 protein granules was higher than that in positive control group(P<0.05).Western blot results showed that the expression of EIV M2 protein had no significant effect on the expression of the four inflammasome proteins,indicating that EIV M2 protein activated the eqNLRP3 inflammasome.Plasmid expressing EIV M2 protein and eqASC were co-transfected into 2G1 cells and observed by confocal laser microscope 24 hours later to further verify the above results.The results showed that eqNLRP3 with green fluorescence appeared spot-like aggregation in this group of cells,and eqASC with red fluorescence in the cytoplasm was recruited to form ASC spots,which was consistent with the screening results of iGLuc reporting system.These results suggest that EIV infection has a aggregative effect on NLRP3 in HEK293TeqNLRP3 cell line and recruits ASCs to form ASC spots.In this study,HEK293T cell line with stable expression of eqNLRP3 protein was established,which laid a material basis for the evaluation of NLRP3 inflammasome activated by pathogen infection in equine animals.
NLRP3 inflammasomeequine NLRP3stable cell lineactivation of inflammation
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