伤寒沙门菌三基因敲除株的构建及其减毒效果分析

Construction of three-gene-deletion mutant of Salmonella Typhi and preliminary safety assessment

目的通过敲除伤寒沙门菌与体内生存相关的基因构建减毒株。方法采用同源重组方法进行基因敲除,将涉及铁摄取的外膜转运蛋白编码基因iroN、fepA、cirA全部敲除。采用小鼠感染模型测定野生株和突变株的毒力水平,采用概率单位加权回归法计算半数致死量(LD50)。结果采用同源重组方法将3个基因一一敲除,成功构建三基因敲除株。贫铁培养基中的生长曲线显示突变株生长较野生株明显缓慢;以动物模型检测的毒力水平显示,野生株和突变株的LD50分别为3.64×10CFU和3.56×10^6CFU。结论伤寒沙门菌外膜转运蛋白编码基因iroN、fepA、cirA敲除后病原菌毒力显著减低,为减毒疫苗的后续评价奠定了基础。

Objective Global incidence of typhoid fever remains high.Vaccine is one of the protective means against this infectious disease.Among various vaccines,attenuated vaccine has advantages of comprehensive immune protection,low cost,convenient use and high compliance.The present study aims to construct an attenuated strain from Salmonella Typhi Ty2 via deletion of three outer-membrane-transporter-encoding genes,iron,fepA and cirA. Methods The target genes,iron,fepA and cirA,were deleted from the chromosome of wild type one by one via homologous recombination.LD 50 of the mutant and the wild type was determined by using mouse infection model. Results In iron-limited medium,the growth rate of the mutant was lower than that of the wild type.The LD 50 of the wild type and the mutant was 3.64×10 CFU and 3.56×10^6 CFU,respectively. Conclusion The deletion of outer membrane transporter encoding genes,iron,fepA and cirA,led to significant attenuation.The resultant strain is suitable for subsequent assessment of immune protective efficacy.