[Deletion-mutant construction, prokaryotic expression and characterization of peptidal-prolyl cis-trans isomerase SlyD from Salmonella enteritidis].

Objective: Salmonella enterica serovar enteritidis is an important food-borne pathogen of human and animal. To further study the function of SlyD associated with virulence and regulation in stress responses of Salmonella Enteritidis, we constructed slyD gene-deletion mutant,, expressed it in E. coli, and characterized the PPIase enzyme obtained. Methods: The slyD gene-deletion mutant of Salmonella enteritidis C50041 was constructed by suicide plasmid mediated homologous recombination. Salmonella enteritidis slyD prokaryotic expression vector was carried out in E. coli, and PPIase activity of recombination SlyD was measured in protease-coupling assay with chymotrypsin. For amino acids conservation studies, functional domain searches and secondary structure predictions, the BLAST, SMART, TMHMM, SignalP, PHD and SWISS MODEL were used. Results: Salmonella enteritidis C50041 ΔslyD mutant strain was successfully constructed. The growth rate of slyD-deleted strain was identified consistent with its parent strain C50041. A soluble recombinant SlyD protein was expressed in Escherichia coli BL21(DE3) cells and confirmed by SDS-PAGE. Catalytic activity confirmed that the SlyD protein was biologically active. Bioinformatic analysis showed that Salmonella Enteritidis SlyD as a multifaceted protein including three separated domains, the FKBP type peptidal-prolyl cis-trans isomerase domain, the IF chaperone domain and the metal-binding domain. Conclusion: Salmonella enteritidis C50041 ΔslyD mutant strain and soluble SlyD protein was obtained, and the present study may provide a basis for further study of the role of SlyD in Salmonella enteritidis.

One-Step PCR Detection of Salmonella Pullorum/Gallinarum Using a Novel Target: The Flagellar Biosynthesis Gene flhB.

Salmonella enterica serovar Pullorum/Gallinarum is an important infectious pathogen that has caused widespread problems for chicken industry. Traditional Salmonella serotyping is an expensive and time-consuming process. In this study, we developed a rapid one-step polymerase chain reaction (PCR) method to identify S. Pullorum/Gallinarum. The PCR-based assay focuses on flhB, which shows a deficient region only in S. Pullorum/Gallinarum, compared with that of other serovars. The specificity and sensitivity of the PCR system were evaluated. The developed PCR method could identify S. Pullorum/Gallinarum from 27 different Salmonella serovars and eight non-Salmonella pathogens. The minimum limit of DNA and the lowest number of cells of S. Pullorum for the PCR detection were no less than 5.85 pg/µL and 10 CFU, respectively. The method was applied to the analysis of Salmonella strains isolated from the chicken farm. The PCR-based testing results of the farm isolates were in concordance with those obtained using traditional serotyping method. This newly developed PCR-based system could be used to accurately screen for the presence of S. Pullorum/Gallinarum, and support traditional serotyping methods, especially in high-throughput screening situations.

[Prokaryotic expression and application of SpiC protein of Salmonella pullorum].

OBJECTIVE: To express the SpiC protein of Salmonella pullorum and establish an indirect ELISA method with SpiC protein as antigen. METHODS: The 384 bp spiC gene of Salmonella pullorum was amplified by PCR from the genomic DNA and cloned into pET30a vector. The recombinant plasmid pET30a-spiC was transformed into competent E.coli BL21(DE3) cells and induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Indirect ELISA based on purified SpiC protein was applied to detect 144 clinical serum samples. RESULTS: SDS-PAGE and Western blotting confirmed that a soluble recombinant His-SpiC protein of 19.2 ku was expressed in BL21(DE3) cells. SPF chicken antibodies against GST-SpiC could recognize His-SpiC, indicating that His-SpiC had a good immunogenicity. The indirect ELISA that we established using His-SpiC protein as coating antigen for detecting antibodies against SpiC could differentiate infected from vaccinated animals (DIVA). CONCLUSION: The recombinant His-SpiC was successfully expressed and the indirect ELISA with it as coating antigen could be used as DIVA method for the related vaccine of pullorum disease.