Expression of Chicken NLRP3 Protein and Preparation and Application of Its Polyclonal Antibodies
In order to explore the molecular mechanism of inflammation triggered by avian disease pathogens,chicken-origin NLRP3 gene was cloned and analyzed,NLRP3 protein was prokaryotically expressed and purified,and then polyclonal antibodies against NLRP3 was prepared by immunizing mice with the purified NLRP3 protein,followed by titer determination as well as preliminary testing of its effectiveness in indirect immunofluorescence assay(IFA)and Western blot test.The results showed that the recombinant protein NLRP3 was successfully expressed with a molecular weight of about 87 kDa,which could specifically bind to His-tag antibodies;the polyclonal antibodies against chicken NLRP3 protein was successfully prepared,with the ELISA titer up to 1:32 000;according to the results of Western blot and IFA,the NLRP3 polyclonal antibodies could specifically bind to both the prokaryotic and eukaryotic expressed proteins of NLRP3,and up-regulated expression of endogenous NLRP3 protein in macrophage HD11 induced by IBDV or stimulated by LPS could be detected.In conclusion,the prepared polyclonal antibodies had good immunogenicity and high ELISA titer,acting as an important tool for further researches on the molecular mechanism of inflammation induced by avian pathogens.
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维普
