[Show abstract][Hide abstract] ABSTRACT: A small plasmid with 4,080bp long, designated pSPI12, was purified from Salmonella enterica serovar Pullorum using a gene knock-in method by inserting a kanamycine resistance cassette in the plasmid. The G+C content of the plasmid was 51.8%, which is in the range of Salmonella genomic DNA. A sequence analysis revealed that pSPI12 had 99.1% homology to pSFD10, which was first reported in the vaccine strain Salmonella enterica Serovar Chloreaesuis C500, but not prevalent among other strains of S. Chloreaesuis. The plasmid has seven open reading frames (ORFs), with one ORF containing a putative virulence-related protein, which had 49% homology with invasion plasmid antigen J protein (IpaJ) secreted by type III secretion system of Shigella flexneri. The putative IpaJ protein was expressed and purified as a His-tagged fusion protein reacted with convalescent sera against S. Pullorum, confirming its identification as an immunogen of the pathogen. In addition, the gene was upregulated for 1 hour post-infection of HD-11 cells with the pathogen by a quantitative real-time reverse transcription PCR assay. The results suggest that IpaJ may be a virulent protein involved in the early stage of infection by S. Pullorum.
[Show abstract][Hide abstract] ABSTRACT: Cloning of ipaJ gene from Salmonella pullorum C79-13, and identification of expressed IpaJ protein as an immunogen of the pathogen.
With suppression subtractive hybridization (SSH) between Salmonella pullorum strain C79-13 (tester) and Salmonella enteritidis strain 50041 (driver), three subtracted fragments PEA3, PE31 and PE44 showed high homology with ipaJ in plasmid pSFD10 of Salmonella choleraesuis C500. The three subtracted sequences were spliced together into the whole sequence of ipaJ in Salmonella pullorum. Then the ipaJ gene was amplified from Salmonella pullorum by polymerase chain reaction (PCR) and cloned into prokaryotic expressive vector pET-30a(+). Western-blot was used to identify it as an immunogen. The distribution of the gene was also detected in Salmonella pullorum isolates.
The ipaJ gene cloned from Salmonella pullorum was 840 bp, and the expressed fusion protein was 37 kDa. Specific reaction was found between Salmonella pullorum positive serum and expressed protein by Western-blot assay, confirming its identification as an immunogen of Salmonella pullorum. The PCR results showed that the gene exists in all Salmonella pullorum strains.
The ipaJ gene from Salmonella pullorum was first reported and cloned, and the expressed IpaJ protein was confirmed as an immunogen of Salmonella pullorum.
[Show abstract][Hide abstract] ABSTRACT: Pullorum disease affecting poultry is caused by Salmonella enterica serovar Pullorum and results in severe economic loss every year, especially in countries with a developing poultry industry. The pathogenesis of S. Pullorum is not yet well defined, as the specific virulence factors still need to be identified. Thus, to isolate specific DNA fragments belonging to S. Pullorum, this study used suppression subtractive hybridization. As such, the genome of the S. Pullorum C79-13 strain was subtracted from the genome of Salmonella enterica serovar Gallinarum 9 and Salmonella enterica serovar Enteritidis CMCC(B) 50041, respectively, resulting in the identification of 20 subtracted fragments. A sequence homology analysis then revealed three types of fragment: phage sequences, plasmid sequences, and sequences with an unknown function. As a result, several important virulence-related genes encoding the IpaJ protein, colicinY, tailspike protein, excisionase, and Rhs protein were identified that may play a role in the pathogenesis of S. Pullorum.
Journal of Microbiology and Biotechnology 09/2009; 19(9):898-903. DOI:10.4014/jmb.0812.694 · 1.53 Impact Factor