鼠伤寒沙门菌SL1344株侵袭性蛋白B缺失株的构建及生物学特性

Construction and characterization of invasion protein B gene deleted mutant of Salmonella typhimurium SL1344 strain

目的:为了研制更加安全的鼠伤寒沙门菌弱毒株,本研究构建了鼠伤寒沙门菌SL1344Δsip B基因缺失突变株。方法:首先构建含缺失585 bp sipB基因的重组自杀性质粒PREΔsipB,利用重组自杀性质粒介导的等位基因交换技术,两步法筛选出SL1344的sip B缺失株。结果:PCR及测序结果表明SL1344ΔsipB构建成功。进一步生物学特性研究表明,缺失株SL1344ΔsipB保留了亲本菌株SL1344的血清型1,4,5,12:i:1,2,且能够稳定遗传缺失585 bp的sipB基因,生长速度没有发生明显改变;但是,与亲本株SL1344相比,其毒力发生明显改变,缺失株SL1344ΔsipB口服感染6周龄BALB/c小鼠的LD50为1.70×108CFU,毒力较亲本株SL1344降低至1.4%,免疫保护效力实验显示,对6周龄BALB/c小鼠免疫后第17天,鼠伤寒沙门菌野生株攻毒有50%的保护率。在给小鼠免疫后第14天血清抗体IgG达到最高。结论:结果表明,SL1344ΔsipB基因缺失株构建成功,遗传稳定,毒力明显降低,且具有较好的免疫原性,为研究鼠伤寒沙门菌sipB基因与其致病力之间的关系提供了方法,并为研制安全有效的沙门菌疫苗弱毒株奠定了基础。

Objective： In order to develop a safer vaccine strain exploit Salmonella typhimurium vaccine strain . A AsipB mutant of Salmonella typhimurium SL1344 strain was constructed. Methods： Firstly, the recombinant suicide plasmid containing the missing 585 bp sipB （PREAsipB） was built by homologous recombination, and screenned by two-step method. Results： PCR and sequencing results showed that SL1344AsipB was successfully constructed. It was no significant changes compared with SL1344. But compared with the parent strains SL1344, the mutant strain had obvious change in its virulence, oral challenge of bacteria in mice revealed that LDs0 of the mutant strain was l. 70 x l0s CFU,the toxicity reduced about 1.4%. The protection rate induced by the sipB mutant was 50% ,and the serum antibody peaked 14 d post-immunization. Conclusion： The SL1344AsipB mutant was constructed suc- cessfnlly, and genetic stability, significantly reduced virulence. The study provides a new approach for further study of the relationship between the gene and pathogenicity of Salmonella typhimurium. It is likely that the AsipB mutant could be adapted to develope attenuated Salmonella vaccine.