Abstract: In order to investigate the role of SopB effector secreted by Salmonella typhimurium LT2, the sopB gene deletion mutant with λ Red recombination system was constructed. To amplify the homologous fragment for sopB deletion, a pair of knockout primer was designed according to the full-length sequence of sopB gene of Salmonella typhimurium LT2. With pKD4 plasmid as template, the homologous fragments including homologous regions which are similar to sopB gene upstream sequence and downstream sequence respectively and kanamycin cassette with two FRT sites were amplified. Then, the homologous fragments were electrotransformed into the Red-induced S.Typhimurium LT2 competent cells. Under the pressure of antibiotic and with the work of λ Red system, homologous recombination occurred between the fragments and genome of host strain, and the recombinants were selected on kanamycin agar plate. After the resultant recombinants were verified with PCR, the positive recombinants were cultured at 43℃overnight to eliminate pKD46. To remove the kanamycin resistant relevant DNA fragment, FLP recombinase-encoding plasmid pCP20 was introduced into the recombinants, resulting in a single FRT site within the targeted genomic segment. The markerless mutant strains were detected by genome PCR. The removal of sopB was further verified by the secretion of SopB protein and the induction of pAkt activation in HeLa cells upon S. typhimurium LT2 infection. Considering the results including genome PCR, SopB secretion and pAkt activation in HeLa cells upon infection, it was confirmed that the sopB gene was successfully knocked out from S. typhimurium LT2 genome with λ Red recombination system. In summary, it is concluded that the sopB gene of S. typhimurium LT2 could be successfully knocked out. Moreover, this paper provides a powerful tool for the functional study of SopB effectors during the interaction between Salmonella and host. Also, it supplies the clue for the gene knock-out of other types of bacteria.

Key words: Salmonella typhimurium LT2, λ Red recombination system, sopB gene, Gene knock-out, Homologous recombination