Construction Of SpvB And SpvC Gene-deleted Mutants In Salmonella Typhimurium And Culture Characterization Of The Mutants

Objective：To investigate the influence of the Salmonella plasmid virulence gene B/C(spvB/spvC) on biological characteristics, we construct spvB/spvC defect strains.Therefore provide theoretical and experimental basis for discussing the pathogenicmechanism of the spvB/spvC, and provide tools and means for further study on thefunction of the gene using cell and mouse infection model.Methods：The spvB/spvC deleted mutants of Salmonella typhimurium was prepared byhomologous recombination mediated by suicide plasmid pCVD442. According to theSalmonella spvB/spvC gene sequence in the GeneBank sequence database, two pair’sspecific PCR primers were designed using Primer Premier5.0, upper-anddown-stream of the spvB/spvC gene to amplify two homologous DNAfragments. Thehomologous recombinant DNA fragment of the defective target gene was cloned intothe suicide plasmid pCVD442，which was then transduced into the target cell of thewidetype strain of Salmonella typhimurium for homologous recombination．Themutant spvB/spvC was selected by screening on the LB sucrose plate and selected byspecific PCR and finally verified by the relative DNA sequencing. The growthproperties, genetic stability and survival ability in acid condition in chick of thespvB/spvC gene-deleted mutants and the widetype strain were characterized. Thestrains were cultured on the LB medium12hours, inoculated LB agar plate andcalculated the flat on the colony-forming units every one hour. The strains werecultured on the LB agar plate for fifty generations, pick the single colony and PCRamplification to study the genetic stability of the gene deletion strains every fivegenerations. The spvB/spvC deleted mutant and the widetype strain were developedwith2mL PH4.0fresh LB medium and shaked two hours at37℃, properly dilutedand inoculated LB agar plate every hour starting at time0h until2h, calculate the flaton the colony-forming units, and caculate the survival rates. Results：pCR and sequecing analysis showed that1749bp in the encoding region of thespvB gene was deleted,711bp in the encoding region of the spvC gene was deleted,and demonstrated that the spvB/spvC gene deleted mutant strains without polarmutation was constructed successfully. The results of the growth properties andgenetic stability showed that there were no significantly difference between the spvB,spvC gene-deleted mutant and the widetype strain. Under acid condition, the growthof the spvB,spvC gene deleted mutants and the widetype strain were significantlylower than the growth at non-acid condition, and the survival rates of the three strainsat two hour was siginficantly lower than at one hour, the differences were statisticallysignificant(P<0.01). At1h and2h, the growth(P<0.01) and the survival rates(P<0.05)of the spvB/spvC gene deleted mutants were lower than the widetype strain. Thedifferences of the growth and the survival rates between the two mutant strains at onehour were not statistically significant (P>0.05); but at two hour the growth of thespvB-deleted mutant strain was lower than the spvC-deleted mutant srain(P<0.05),and the differences of the survival rates were not statistically significant(P>0.05).Conclusion：The spvB gene-deleted mutant of S. enterica serovar Typhi was generatedsuccessfully. The acid condition has a selective effect on the growth of Salmonella,and the effect increased gradually over time within the first two hours. The spvB andspvC genes may play an important role for Salmonella typhimurium to overcome theacid stress and there has no difference between the spvB and the spvC gene.