Abstract:

λRed recombination system and pBAD prokaryotic expression vector were used to construct spvBC gene editing strains of Salmonella typhimurium,which may provide tools for further study on the pathogenic mechanisms and functions of spv as well as its role in host anti-infection immunity. Plasmid pKD4 was used as template,and kanamycin resistance gene containing spvBC homologous arm was amplified to construct homologous linear DNA fragment via PCR. Then the linear fragment was electrically transferred to a recombinant strain of S. typhimurium with pKD46. After the recombination,plasmid pCP20 was electrically transferred to positive colonies to delete the kanamycin resistance gene,and gene deletion was identified by PCR. spvBC gene fragment containing enzyme loci was amplified by PCR. Both the amplified product and the prokaryotic expression vector pBAD/gⅢ were double digested and then ligated to construct recombinant plasmid pBAD-spvBC. Positive colonies were screened by PCR and identified by sequencing. Finally,the constructed pBAD-spvBC recombinant plasmid was electrically transferred to spvBC deletion strain. The expression of SpvB and SpvC induced by L-arabinose at different concentrations was determined by Western blot. PCR results indicated that the knockout of spvBC in S. typhimurium was successful. PCR and sequencing results demonstrated the successful construction of the recombinant plasmid pBAD-spvBC,Western blot results showed 13 mmol/L L-arabinose induced normal expression of SpvB and SpvC. λRed recombination system can be used to knock out large gene fragments on Salmonella plasmid,as well as pBAD prokaryotic expression vector can be used to complement large fragments on Salmonella plasmid,which enriches the gene modification and editing strategies of bacterial plasmids.

Key words: Salmonella typhimurium, spvBC gene, λRed recombination system, gene editing