Construction and characterization of △sseK2 mutant strain of Salmonella typhimurium SL1344

Objective To analyze the biological characteristics of a mutant strain of Salmonella typhimurium SL1344 with sseK2-deletion (SL1344△sseK2) in order to provide reference for further study of safe and effective live vaccines. Methods The mutant strain SL1344△sseK2 with a deletion of 1 047 bp in sseK2 gene was constructed through a two-step allelic exchange using recombinant suicide plasmid. Its complemented strain, SL1344C△sseK2, was also constructed. Biological and immunological characteristics of the mutant strain were detected. Results PCR, double-enzyme digestion and sequencing analysis showed that the mutant strain SL1344△sseK2 and the complemented strain SL1344C△sseK2 were successfully constructed. The serotype of the mutant strain was 1, 4, [5], 12: i: 1, 2, identical to the parent strain SL1344. In addition, the mutant strain showed no significant change in biochemical characteristics or growth rate and was genetically stable in vitro. Compared with the parent strain SL1344, the virulence of SL1344△sseK2 was attenuated in BALB/c mice. The median lethal dose of SL1344△sseK2 for 6-week-old BALB/c mice was 3.44×108 colony-forming units (CFU), which was 1 620 times lower than that of SL1344. Oral immunization with SL1344△sseK2 protected 62.5% of the mice against challenge with wild Salmonella typhimurium strains on 17 d after vaccination. The levels of serum IgG antibody peaked on 14 d after immunization. No significant difference in biological characteristics was observed between the complemented and the parent strains, indicating that the mutant strain was basically complemented to the wild-type strain. Conclusions The mutant strain SL1344△sseK2 was constructed successfully and genetically stable with significantly attenuated virulence and good immunogenicity. This study suggested that sseK2 gene played an important role in regulating the virulence of SL1344, which might provide reference for further study of its function and for assessing its potential as a candidate live attenuated vaccine. Key words: Salmonella typhimurium SL1344; Recombinant suicide vector; Mutant strain SL1344△sseK2; Complemented strain SL1344C△sseK2; Biological characteristics