Construction Of Porcine NLRP6 Gene Expression Vector And Preparation Of Its Polyclonal Antibody

NOD-like receptors are one of the pathogen recognition receptors in congenital immunity and play an important role in the body's defense.NLRP6 is a class of pro teins with a special mechanism of action in the NOD-like family,and its role in im munity is related to its ability to regulate caspase-1 and NF-?B activity.This experiment used the porcine NLRP6 gene as the research object.Specific primers were designed based on the porcine NLRP6 full-length nucleotide sequence(Gen Bank accession number: XM-003124236.4),and the NLRP6 gene was amplified by PCR from the pMD-19T-NLRP6 recombinant vector.The NLRP6 gene fragment was ligated into the prokaryotic expression vector pET-32a(+)to obtain the recombin ant expression plasmid pET-32a-NLRP6,and the recombinant plasmid was identified by resistance screening,restriction enzyme digestion and PCR reaction.The recombinant plasmid pET-32a-NLRP6 was transformed into E.coli Rosetta(DE3)competent cells,and IPTG induced its high expression to obtain NLRP6 reco mbinant fusion protein with molecular weight of about 34 KDa and inclusion in the form of inclusion bodies.Western blot results showed that it was positive with mo use anti-6 × His monoclonal antibody.After purification,denaturation and renaturation of NLRP6 recombinant fusion protein by nickel ion column,soluble NLRP6 reco mbinant fusion protein was obtained.Using purified fusion protein as antigen,BALB/c female mice were immunized according to reasonable immunization procedure to prepare polyclonal antibody.The polyclonal antibody was identified by Western blot.The results showed that the mou se anti-NLRP6 polyclonal antibody can specifically recognize NLRP6 protein.In this experiment,NLRP6 protein was successfully prepared by prokaryotic exp ression system,and the mouse anti-NLRP6 polyclonal antibody with good immunolo gical activity and specificity was prepared,which provided the basic material for the biological function of NLRP6 protein and the pathogenesis of related diseases.