Effect Of Regulator PhoP On The Expression Of FljB Encoding Z66 Flagellar Antigen Of Salmonella Enterica Serovar Typhi

Object:To study the effect of the regulator PhoP on the expression of flagellin gene fljB:z66 in Salmonella enterica serovar Typhi.Methods:1)Constraction of the phoP deleted mutant of S.enterica serovar Typhi by homologious recombination mediated by suicide plasmid. As the genomic information,two pair's primers were designed at upper-and downstreams of the phoP gene of S.enterica serovar Typhi to amplify two homologous DNA fragments.A BglⅡsite was added in 5'-termini of the reverse primer of the upper stream fragment and the forward primer of the down stream fragment. After amplification and purification,two fragments were digested by BglⅡand ligated as the homologous recombinant DNA fragment,which contains the defective target phoP gene.The homologous recombinant DNA fragment was inserted into the BamH I site of the suicide plasmid pGMB 151.The positive vector was then transferred into the target cell of S.enterica serovar Typhi wild strain by using of electroporation.The recombination was visualized by PCR,and the complete recombinant strain was selected as the phoP deleted mutant strain and confirmed by the corresponding sequencing analysis.2)The fljB::lacZ inserted mutant was prepared by homologous recombination mediated by suicide plasmid.The positive vector with the recombinant fragment fljB::lacZ was transformed into the wild and phoP~-mutant strains of S.enterica serovar Typhi by electroporation.Inserted mutants were selected on the LB sucrose plate containing X-Gal and verified by relative PCR and sequencing analysis.3)Strains of fljB::lacZ and phoP~-fljB::lacZ mutant were incubated LB broth to log-phase and submitted to environmental stresses, including hyperosmotic stress,weak acidic stress,bile stress and oxidative stress.Whether the regulatory protein PhoP regulates the expression of flagella antigen gene fljB:z66 could be investigated by comparing the activities ofβ-galactosidae of strains under environmental stresses.Results:1)A deletion of 297bp from the phoP gene was confirmed by PCR and sequencing analysis suggested that the phoP deleted mutant was constructed successfully.2)A 763 bp of fljB gene was substituted by 3550bp of lacZ gene in S. enterica serovar Typhi wild strain and phoP~-mutant strain in sequencing analysis suggested that the fljB::lacZ mutant and phoP~-fljB::lacZ mutant of S.enterica serovar Typhi were generated successfully.3)There was obvious difference of fljB:z66 expression between the fljB::lacZ strain and the phoP~-fljB::lacZ mutant strain in the osmotic stress condition,while no obvious expressional difference in other stress conditions including of oxidative,bile and acidic stress.Conclusions:1)The phoP gene deleted mutant of S.enterica serovar Typhi was constructed successfully. 2)The lacZ gene was successfully inserted in the fljB in wild and phoP~-mutant strain of S.enterica serovar Typhi.3)Regulator PhoP could serve as a possible factor to fljB:z66 gene expression in the osmotic stress condition.PhoP did not obviously regulate the expression of z66 flagella antigen in conditions of oxidative,bile and weak acidic stresses.