Expression, Purification And Polyclonal Antibody Preparation Of FliC In Salmonella Typhimurium

Salmonella was one kind of person and livestock pathogens.It could cause food poisoning and pose the very big threat to the humanity.The main aim of the study was to express FliC fusion protein in.E.coli in high effieiene by cloning the.fliC gene of Salmone lla into an expression plasmid to generate pET-fliC recombinant and expressed. Then the recombinant FliC protein was purified by Ni-NTA affinity chromatography and the polyclonal antibodies were obtained via imunning BALA/c mice with the purified protein. The characteristics of the polyclonal antibodies such as titer and the specificity were detected. This research would be helpful for the further study on establishing immunologic detection of Salmonella.The paper included three parts:1Cloning and expression of flagellar gene fliC of Salmonella Typhimurium in Escherichia colithe flagellar gene fliC of Salmonella Typhimurium was amplified by PCR from the Salmonella genome, and cloned into the prokaryotic expression vector pET28a(+) to construct the recombinants pET28a-fliC,then the recombinants were transformed to the strain BL21(DE3)pLysS,after enzyme digested and sequenced, The recombinant strains were induced by IPTG to express the protein.The results showed that the PCR product was about1509bp as expected, the enzyme digestion and sequencing identification showed the prokaryotic expression system was constructed successfully. The molecular weight of the protein which was induced by IPTG was about54.6kD as expected.2Purification and identification of Phase I flagellar of Salmonella TyphimuriumThe fusion protein was purified by Ni-NTA affinity chromatography,then the purity and weight were detected by SDS-PAGE.The concentration and specificity were determined by BCA and Western-blot.The results showed that the purity of the protein purified by Ni-NTA was high, and the weight was as expected. Western-blot analysis showed that the expressed protein had a specific reaction with the monoclonal antibody anti-His-tag at55KD,indicating that the expressed protein had His-tag and was the target protein.3The production of anti-FliC Polyclonal antibodiesThe polyclonal antibodies were obtained by immunning BALA/c mice with the purified protein, and the titer and specificity were detected by indirect ELISA.The results showed that the purified protein could stimulate the mice to produce specific polyclonal antibodies, the antibody titers reached1:32000and had good specificity and broad spectrum in Salmonella. This reearch would be helpful for the further study on establishing immunologic detection of Salmonella.