| Salmonella Choleraesuis(S.Choleraesuis) is the main pathogen of Piglet’s paratyphoid for2-4-month-old piglets. Paratyphoid can occur throughout the year and cause mixed infections with swine plague. S.Choleraesuis could cause high morbidity and mortality resulting in significant losses. It is also highly pathogenic to humans, usually causing fever, septicemic disease and other systemic infections. In addition, the multidrug resistance of S.Choleraesuis has been a serious problem, and it has a seriously potential threat to human’s health. Till now, S. Choleraesuis has been paid more and more attention.In this study, the S. Choleraesuis subtractive library was constructed and analyzed by comparison of genomic differences between S.Choleraesuis virulent strain C78-2and attenuated strain C500by suppression subtractive hybridization (SSH).2-dim ensional electrophoresis (2-DE) and real-time fluorescent quantitative PCR were used to analyze the expression difference of proteins between the two strains.1Construction of suppression subtractive library between S.Choleraesuis virulent and attenuated strains and identification of the different genesUsing suppression subtractive hybridization (SSH) technique, the S.Choleraesuis subtractive library was constructed by comparison of genomic differences between S.Choleraesuis virulent strain C78-2(tester) and attenuated strain C500(driver). The subtracted fragments were sequenced and searched homologically in GenBank after identification by Dot-blot. The results showed five different fragments were identified, including asr, ydgF, rpoS, ptsG and a sequence with unknown function. In addition, a specific deletion region was found in the genome of C500. It’s size is3,290bp which contains four genes:asr, ydgD, ydgE, ydgF. The results suggested that the subtracted library was very useful for determining the attenuated mechanism of C500.2Comparison of the Proteome between S. Choleraesuis virulent and attenuated strainsTo compare the whole cell proteins between the two S.Choleraesuis strains,2-DE technique was used to get a set of reproducible gel maps. After using PDQuest software to analyze the maps, five specific protein points were chosed in C78-2gel for Mass Spectrometry ananlysis. Four points were successfully identified. The identifiable rate was80%. The four proteins respectively were the phosphatase of CheY, Bacterioferritin, OsmY and Ribose-5-phosphate isomerase A. It was shown that CheZ is the phosphatase of CheY, the response regulator in bacterial chemotaxis. Bacterioferritin is an iron-storage hemoprotein which store the superfluous iron and prevent the problem of cytotoxicity. OsmY is a periplasmic protein, and it could have the function of resisting hyperosmotic stress. Ribose-5-phosphate isomerase A (RpiA) is an enzyme which is essential for the pentose phosphate pathway and Calvin cycle. These results of the study provided a theoretical basis for understanding of the attenuation mechanism of S.Choleraesuis, and laying the foundation for the demonstration of genetic evolution.3Differential expression of S.Choleraesuis related proteins detected by real-time fluorescent quantitative PCR in in vitro conditionAccording to the results of the comparison between the two S.Choleraesuis strains. Eight pairs of primers which were specific to rpoS, spvA, spvB, spvC, bfr, cheZ, osmY, rpiA were synthesized to analyze their differential expression in the two strains, and the gene of S.Choleraesuis gmk was used as reference. The total RNA was extracted from the two strains cultured in LB, and reverse transcribed into cDNA and used as templates for assessment of their differential expression by real-time quantitative PCR.The results showed that rpoS gene didn’t express in C500. It could be a further evidence for the deletion of rpoS in C500. The expression level of the rpoS-regulated genes (spvA, spvB, spvC, osmY), bfr, cheZ and rpiA in C78-2was also significantly higher than C500. And the results were in accordance with expectation. |
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