鼠伤寒沙门菌sseK2基因缺失株的构建及其生物学特性研究

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

目的检测sseK2基因的缺失对鼠伤寒沙门菌生物学特性的影响,为探究该基因的功能及开发安全有效的鼠伤寒沙门菌活疫苗提供新思路。方法首先通过重组自杀性质粒介导,两步筛选法敲除1 047 bp sseK2基因并成功构建缺失株及其回复株,进而检测其生物学和免疫学特性。结果PCR、双酶切及测序结果分析,证实构建了SL1344△sseK2缺失株及其回复株。生物学特性研究表明,缺失株SL1344△sseK2保留了亲本株SL1344的血清型1,4,[5],12:i:1,2,且能够稳定遗传缺失后的sseK2基因,生化特性和生长速度并未发生明显改变,但与亲本株SL1344相比,其毒力发生明显改变。缺失株SL1344△sseK2口服感染6周龄BALB/c小鼠的半数致死量(median lethal dose,LD50)为3.44×10^8菌落形成单位(colony-forming units,CFU),毒力较亲本株SL1344降低了1 620倍。免疫保护效力试验显示,缺失株免疫17 d后对鼠伤寒沙门菌野生株攻毒小鼠的保护率为62.5%,小鼠免疫14 d后血清抗体IgG达到最高。回复株与亲本株生物学特性无显著性差异,基本回复到野生型水平。结论成功构建了SL1344△sseK2基因缺失株,其遗传稳定,毒力明显降低,且具有较好的免疫原性,推测sseK2对SL1344的毒力具有重要的调节作用,为探究该基因的功能及评估其作为用于减毒活疫苗的候选株提供参考。

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 3.44×10^8 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.