Construction Of A Salmonella Typhimurium SL1344ΔcrpΔasd Mutant Strain And Its Application As A Vehicle For The ND-HN Gene

Salmonella typhimurium, which is a kind of very important zoonotic bacteria,belongs to intestinal and intracellular bacterial species and has a strong addict tomucous membrane. It can be attenuated without loss of invasiveness by varioustechniques in order to be used for delivering immunizing antigen. Recent studiesshowed that the attenuated Salmonella typhimurium is one of the focuses of vaccinedevelopment for its usage as vehicle of orally vaccine.In our previous studies, attenuated Salmonella typhimurium SL1344Δcrp mutantstrain was obtained through the allelic exchange introduced by the transduction of therecombinant suicide plasmids. Its growth properties, drug tolerance, genetic stabilityand safety has been evaluated. The results showed that it had good stability and safety.Asd（aspartic beta-semialdehyde）gene was one of the auxotrophic genes in Salmonellawhich could lead the lysis and death of bacteria when it deleted. The normal ability ofasd-mutant strain could be restored if the host-vector balanced lethal system wasconstructed. As a result, this study was aimed to develop a safer attenuated Salmonellatyphimurium vaccine and exploit it as a live vaccine vector. TheΔasd gene was deletedfrom SL1344Δcrp strain to construct host-vector balanced lethal system for stablyexpressing heterologous genes. Then the biological characteristics of these strains werecompared and further more, the SL1344ΔcrpΔasd mutant strain was used as a vehiclefor the ND-HN gene. It’s likely that this SL1344ΔcrpΔasd mutant strain could be apotential oral vaccine vector to express foreign antigen or act as a eukaruotic expressionplasmid carrier.1. Construction and identification of the SL1344ΔcrpΔasdE.coli donor strain χ⁷²¹³（pREΔasd） was conjugated with SL1344Δcrp, therecipient. The SL1344ΔcrpΔasd mutant was constructed through the allelic exchangeintroduced by the transduction of the recombination suicide plasmid. Then, theSL1344ΔcrpΔasd mutant strain was determined by PCR and sequencing analysis. 2. Analysis for biological characteristics of the SL1344ΔcrpΔasdThe SL1344ΔcrpΔasd mutant strain can grow normally when foreign DAP wassupplied. The study showed that there had some significantly differences between theSL1344ΔcrpΔasd and SL1344. SL1344ΔcrpΔasd was not able to ferment maltose,glycerol, sorbitol and H₂S. However, it could also ferment glucose, galactose, rhamnoseand mannitol what was consistent with the parent strain SL1344Δcrp. The growth ratesof the SL1344ΔcrpΔasd and the parent strain SL1344Δcrp were slower than SL1344strain, obviously. The serotype of the SL1344ΔcrpΔasd mutant did not change, and itwas stable with the recombinant Δasd gene in vitro. The virulence of SL1344ΔcrpΔasd（pYA3493）strain was10⁵times lower than SL1344strain.3. Construction and identification of the attenuated Salmonella typhimuriumrecombined with NDV-HN geneThe HN gene was cloned from the plasmid pMD18-T-HN by PCR and insertedinto the vector pYA3493. Electricity transformation method was used in thisrecombinant plasmid pYA3493-HN transformating to recepted state Salmonellatyphimurium SL1344ΔcrpΔasd. The recombinant plasmid pYA3493-HN was identifiedby digestion of endonuclease, PCR and sequencing. The results confirmed thatrecombinant attenuated Salmonella typhimurium vaccine SL1344ΔcrpΔasd（pYA3493-HN）was constructed succeed. The gene of HN can be transmited stablely in the liquidnutrient medium of Luria Bertani after60generation. It established foundation ofresearch and development in ND-HN orally gene engineering live vaccine.