大口黑鲈NLRP3蛋白多克隆抗体的制备及初步应用OA北大核心CSTPCD

NLRP3,a classic pattern recognition receptor,combines with ASC and pro-caspase 1 to form the NLRP3 inflammasome,which is responsible for the activation of pyroptosis in teleost fish.To further investigate the role of NLRP3 protein in largemouth bass(Micropterus salmoides),this study amplified an Msnlrp3 segment using largemouth bass cDNA as the template and constructed the pET-32a-MsNLRP3 prokaryotic recombinant plasmid.The recombinant plasmid was transformed into BL21(DE3)cells,and a soluble fusion protein was expressed after induction with 0.8 mmol/L isopropyl β-D-1-thiogalactopyranoside overnight at 16℃.The obtained recombinant protein was purified using affinity chromatography,and then identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and mass spectrometry analysis to confirm the successful construction of the prokaryotic expression system for MsNLRP3.Subsequently,the purified MsNLRP3 protein was used to immunize Japanese long-eared rabbits and Balb/C mice to obtain polyclonal antibodies,respectively.The titers and specificity of the obtained antibodies were determined using enzyme-linked immunosorbent assay(ELISA)and western blotting.Results showed that the obtained antisera,after immunization,could specifically recognize the recombinant and endogenous NLRP3 protein in largemouth bass tissues,as indicated by a single,clear target band consistent with the expected molecular weight.Meanwhile,the titers of rabbit and mouse antisera were 1∶10240 and 1∶1024,respectively.To test the application potential,a Flavobacterium columnare infection model via immersion was constructed in largemouth bass,in which the gills exhibited significant histopathological changes.Western blotting analysis with the polyclonal antibody obtained from this study indicated that NLRP3 protein levels were significantly increased in gills after bacterial infection.Therefore,the polyclonal antibody against MsNLRP3 prepared in this study provides an important material basis for the functional research of NLRP3 protein in largemouth bass.