Construction And Biological Characteristics Of Attuanted Salmonella Typhimurium ΔcrpΔcyaΔasdSL1344Mutant Strain

Salmonella was a kind of aggressive intracellular bacteriumand which could be used as a vaccine carrier carrying the prokaryotic expression plasmid and eukaryotic expression plasmid. Attenuated recombinant salmonella vaccine was preparation simplely and immunity conveniently. It also has the strong immunogenicity and could induce strong and lasting immune response.As Salmonella carrier Salmonella typhimurium was of more extensive applicated in research,but with as the carrier to carry exogenous antigen expression of exogenous gene,it may not stable because of the pressure to survive.Recent studies of a host carrier without resistance balanced lethal system may provide a solution to efficiently express foreign genes without resistance marker. Asd (aspartic semialdehyde dehydrogenase) gene which was encoded aspartic acid beta galactose dehydrogenase, the enzymes were diaminopimelic acid (DAP) essential enzymes in the biosynthesis pathway and DAP was a major component of gram-negative bacteria cell walls. Strains lacking asd gene was deathed in the absence of exogenous lysis. Carrier contains asd gene which was complementary to the host asd gene,the transformation was growed in the absence of exogenou DAP. The main contents were included.1. Construction and identification of the Δcrp cya asdSL1344E.coli χ7213(pREΔasd) which was contained with recombinant plasmid suicid as the donor strain was conjugated with ΔcrpΔcyaSL1344which was as the recipient strain. Through of the allelic exchange introduced by the transduction of the recombination suicide plasmid,the ΔcrpΔcya asdSL1344mutant was constructed by two steps. Then,the strain with resistance of sucrose (SUCr) and relying DAP on colony wasdetermined by PCR and sequencing analysis.2. Comparison analysis for biological characteristics of theΔcrpΔcya asdSL1344and other strains The serotype of ΔcrpΔcya asdSL1344strain was1,4,5,12:i:1,2identically to the parent ΔcrpΔcyaSL1344strain and wild SL1344strain. The mutant was stabile with the recombinant asd gene in vitro. The growth rate of the mutant was almost same as the parent ΔcrpΔcyaSL1344strain, but more slowly than virulent SL1344strain. The ΔcrpΔcya asdSL1344mutant was not able to ferment maltose, lactose, and sorbitol. It also had no ability to decompose H2S, galactose and rat lee sugar. The one-day chicken lethal test was showed that ΔcrpΔcya asdSL1344(pYA3493) mutant was only10times lower than ΔcrpΔcyaSL1344strain, but at least105times lower than wild SL1344strain. It showed that the ΔcrpΔcya asdSL1344was greatly safe as a live vector.