Preparation and Application of Polyclonal Antibodies Against the Virulence Protein SptP in Salmonella Typhimurium
[Objective]The aim of this study was to prepare the polyclonal antibodies against SptP of Salmonella Typhimurium Sal-14028,and apply them in the detection of Salmonella SptP protein.[Method]Using the Sal-14028 genome DNA as a template,the SptP gene fragment was amplified by PCR and a prokaryotic expression plasmid pET28a-SptP was constructed.The recombinant plasmid was transformed into Escherichia coli BL21(DE3)competent cells,the protein expression was induced by IPTG,and the recombinant SptP protein was identified by SDS-PAGE and Western blotting.The expression product was purified by nickel column and then was used to immunize the rabbits to prepare polyclonal antibodies.The titer and specificity of the antibodies were detected by indirect ELISA and Western blotting.Cell immunofluorescence was used to detect the distribution of SptP in Raw264.7 cells infected with Salmonella,and the total protein of the infected cells was immunoprecipitated.A 25 ku band was enzymatically hydrolyzed and identified by mass spectrometry.[Result]The results showed that a SptP gene fragment of 1 173 bp was cloned,and enzyme digestion and sequencing showed that the recombinant plasmid pET28a-SptP was successfully constructed.The recombinant SptP protein with the size of about 45 ku was obtained by inducing expression and purification.Polyclonal antibody with titer of 1∶25 600 was obtained by immunizing rabbits with recombinant SptP protein.The antibody could both recognize recombinant SptP protein and Salmonella SptP whole protein,and also could be used in tracking the distribution of SptP within Salmonella-infected cells.The immunoprecipitation test showed that the antibody could bind to multiple proteins,and the 25 ku protein that the antibody binded was identified as mouse Rac1 protein using peptide mass fingerprint.[Conclusion]This study successfully prepared polyclonal antibodies with high titer,which could recognize recombinant SptP and SptP whole protein,and could be used in Western blotting,cellular immunofluorescence and immunoprecipitation experiments.The results of this study laid a foundation for the detection of Salmonella SptP protein and the exploration of target molecules of Salmonella SptP.
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