Construction And Characterization Of A Salmonella Choleraesuis C78-1 â–³crp Deletion Mutant

Salmonella choleraesuis is one of the main pathogens of piglet's paratyphoid, and it affect mainly weaned piglets. A large number of weaned piglets can be dead when secondary infection with other pathgens, or not treated in time. In addition, this pathogen play a significant role in public health for causing human food poisoning through the infected animals or the contaminated products. Vaccination against S. choleraesuis is an appropriate strategy for control and prevention of this disease. In China, the most effective attenuated paratyphoid fever vaccine strain was C500 strain. It was attenuated from highly virulent S. choleraesuis C78-1 strain by maintaining in the thallium acetate's culture medium for hundreds of generations, and had stronger immunogenicity. However, the genetic background and the mechanism of immune protection of C500 strain attenuated by chemical methods remain still unknown. In addition, the vaccine strain remains some residual virulence and risk of back mutation.In the current study, a safer attuanted S. choleraesuis vaccine was developed through deleting the crp gene, which coding a cAMP receptor protein. The ?crpC78-1 mutant was constructed through the two-step method introduced by the transduction of recombinant suicide plasmid. The biological characteristics of the ?crpC78-1 was further analysed. The main research was described as follows:1. Cloning and sequential analysis of crp geneThe crp gene were cloned from genomic DNA of C78-1 strain according to the corresponding sequences of S. typhimurium LT2 strain (AE008859) from GenBank. The results of sequence alignment analysis showed that the C78-1 crp gene was completely same as C500 strain's and highly conserved compared with other three salmonella whose whole genome sequences were published.2. Construction of recombination suicide plasmids pREΔcrpThe upstream and downstream fragements of crp gene were amplified from genomic DNA of S. choleraesuis C78-1, then successfully subcloned into suicide plasmid pRE112, transformed into E. coliχ7213. The recombination suicide plasmid were designated as pREΔcrp which contained 320bp-deleted crp fragement.3. Transconjugation and identification of theΔcrpC78-1 mutantThe E. coil donor strainχ7213 transformed with recombination suicide plasmid pREΔcrp was conjugated with the recipient S. choleraesuis C78-1 strain. The ?crp mutant of S. choleraesuis C78-1 was constructed by the allelic exchange introduced by the transduction of the recombination suicide plasmid. Then, theΔcrpC78-1 mutant was determined by PCR and sequencing analysis.4. Analysis for Biological Characteristics ofΔcrpC78-1 mutantThe phenotypes, genetic stability, growth properties and virulence in mice ofΔcrpC78-1 mutant were characterized. The O and H antigens of the mutant was 6,7:C:1,5, identical to the parent C78-1 strain. TheΔcrpC78-1 mutant was able to ferment glucose, not maltose, mannose and xylose, while the parental strain C78-1 did. The mutant was stable with the recombinant ?crp gene in vitro. However, fermentation patterns and growth rate of the mutant were differed from the parent C78-1 strain obviously. The mouse lethal test showed that the virulence of the muntant was lower 750 times than C78-1.