Construction And Characterization Of Attenuated Salmonella Typhimurium ΔcrpsL1344 And Δcyasl1344

Salmonella typhimurium, belongs to Salmonella group B, is a group of enteric pathogenic bacteria with a broad host spectrum and pathogenic. It causes paratyphoid fever in a variety of animal and human, and causes great economic losses for breeding industry. Food poisoning and pediatric nosocomial infection caused by S. typhimurium, which has greatly threatened human health. The drug resistant spectrum of S. typhimurium been broadened in recent years, which make the prevention and control of this disease becomes more difficult. Therefore, it has vital significance for veterinary, medical and public health.Due to drug resistance and food safety issues, vaccination was an ideal choice for the prevention and control of this disease. S. typhimurium live vaccine has put into use in some countries, and achieved a successful effect. However, the genetic background and the mechanism of immune protection of these live vaccine strain attenuated by chemical mutagenesis remain still unknown. In addition, there was a strong risk of back mutation, its safety and quality was not guaranteed. Therefore, how to get low virulence or no virulence, good immunogenicity live vaccine to be efficiently against S. typhimurium infection has become a pressing issue.The genes encoding the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) were an important virulence regulation gene for Salmonella. It has been reported that the virulence of S. typhimurium cya crp mutant was decreased significantly, but was immunogenicity. Based on crp, cya gene sequence, theΔcrpSL1344 orΔcyaSL1344 mutant was constructed by the allelic exchange introduced by the transduction of the recombinat suicide plasmids. Then, comparison study of the two mutant of biological characteristics were carried out. The results were summarized as follows:1. Cloning and sequential analysis of S. typhimurium SL1344 crp and cya geneThe crp and cya gene were cloned from genomic DNA of S. typhimurium SL1344 strain according to the corresponding sequences of S. typhimurium LT2 strain (AE006468.1) from GenBank. The results of sequence alignment analysis showed that the SL1344 crp and cya gene were highly conserved compared with other three Salmonella, S. typhimurium LT2, S. typhi Ty2 and S. enteritidis p125109, whose complete genome sequences were published.2. Construction of recombinant suicide plasmids pREΔcyaTo construct of the recombinant suicide plasmids were designated as pREΔcya which contained 1182 bp-deleted cya fragement. The upstream and downstream fragements of cya gene were amplified from genomic DNA of S. typhimurium SL1344, then successfully subcloned into the suicide plasmid pRE112, and transformed into E. coliχ7213.3. Transconjugation and identification of theΔcrpSL1344 orΔcyaSL1344 mutantThe E. coil donor strainχ7213 transformed with recombination suicide plasmid pREΔcrp or pREΔcya was conjugated with the recipient S. typhimurium SL1344 strain. TheΔcrpSL1344 orΔcyaSL1344 mutant was constructed by the allelic exchange introduced by the transduction of the recombinat suicide plasmids. Then, theΔcrpSL1344 orΔcyaSL1344 mutant was determined by PCR and sequencing analysis.4. Comparison analysis for biological characteristics of theΔcrpSL1344 andΔcyaSL1344 mutantThe phenotypes, growth properties, drug resistance, genetic stability and virulence in chick of theΔcrpSL1344 andΔcyaSL1344 mutant were characterized. The results of the phenotypes, growth properties, drug resistance and genetic stability showed that there were no significantly difference between theΔcrpSL1344 andΔcyaSL1344 mutant. The serotype of the two mutant did not change, compared with the parent SL1344 strain, was still 1, 4, 5, 12: i: 1, 2. But the two mutant were not able to ferment maltose, glycerol, sorbitol, while the parental strain SL1344 did. Other biochemical characteristics and drug resistance of the two mutant were consistent with the parent strain SL1344. The two mutant were stable with the recombinant ?crp gene and ?cya gene in vitro. However, the growth rate of the two mutant was slower than the parent SL1344 strain obviously. The chicks lethal test showed that the virulence of theΔcrpSL1344 orΔcyaSL1344 mutant was more lower 21574 times or 16706 times than SL1344.In the current study, theΔcrpSL1344 orΔcyaSL1344 mutant was constructed successfully by the allelic exchange introduced by the transduction of the recombinat suicide plasmids. All these data provided a new method for make further study on relationship among genes crp, cya and virulence. It was also lay the foundation for exploit a more safer and effective attenuated S. typhimurium vaccine and the further application in an oral live vector vaccine for mucosal immunity.