肠炎沙门氏菌ssrAB、hilA、hilD基因缺失菌株的构建及其生物学特性

Construction and characterization of ssrAB, hilA, hilD-deficient mutants of Salmonella enteritidis

摘要:

【背景】沙门氏菌(Salmonella)是重要的人畜共患病原菌，由于耐药菌株的不断出现，新型防治方法的研究迫在眉睫。【目的】探究ssrAB、hilA、hilD基因对肠炎沙门氏菌致病作用的影响，筛选安全可靠的沙门氏菌减毒活疫苗或载体用菌株。【方法】采用自杀性质粒介导的同源重组技术，分别构建ssrAB、hilA、hilD单基因缺失株(ΔssrAB、ΔhilA、ΔhilD)、双基因缺失株(ΔssrABhilA、ΔssrABhilD、ΔhilAhilD)和三基因缺失株(ΔssrABhilAhilD)，并比较上述缺失菌株与亲本菌株间生物学特性的差异。【结果】基因缺失后菌株的生长速率无显著变化(P>0.05)；除ΔhilD生物膜形成能力最强外，其他基因缺失菌株生物膜形成能力变化不显著(P>0.05)；ΔssrABhilAhilD和ΔssrABhilA的LD50值最高，ΔssrAB、ΔhilA、ΔhilD次之；亲本菌株引起的小鼠十二指肠的病变程度较所有缺失菌株明显严重；在所有缺失菌株中，ΔssrABhilAhilD对HeLa细胞的黏附力最低，ΔhilD在小鼠体内的定殖率最高；而机体分泌IL-1、IL-18、IFN-γ的能力与沙门氏菌ssrAB、hilA、hilD基因无显著相关性(P>0.05)。【结论】构建的7株基因缺失株中，ΔssrAB、ΔhilA、ΔhilD、ΔssrABhilA和ΔssrABhilAhilD有致病力低、生长速率良好、能有效刺激细胞因子产生等特性，具备作为沙门氏菌减毒活疫苗或载体的潜质。

关键词:肠炎沙门氏菌，ssrAB，hilA，hilD，缺失菌株，构建，生物学特性

Abstract:

[Background] Salmonella is an important zoonotic pathogen, due to the continuous emergence of drug-resistant strains, research on new prevention methods is imminent. [Objective] In order to investigate the effect of ssrAB, hilA, hilD genes on the pathogenicity of Salmonella enteritidis and screen safe and reliable strains for Salmonella live attenuated vaccines or vectors. [Methods] Using suicide plasmid-mediated homologous recombination technology to construct the single (ΔssrAB, ΔhilA, ΔhilD), double (ΔssrABhilA, ΔssrABhilD, ΔhilAhilD) and triple (ΔssrABhilAhilD) gene deletion strains, then comparing the differences in biological characteristics between the above-mentioned deletion strains and the parent strain. [Results] The growth rate of the mutant strains was not significantly different from the parent strains (P>0.05). The biofilm formation ability of ΔhilD was the strongest, and the other gene deletion strains had no significant changes (P>0.05). The LD50 of ΔssrABhilAhilD and ΔssrABhilA were the highest, ΔssrAB, ΔhilA and ΔhilD were the second, and the degree of duodenal lesions in mice caused by the parent strain is significantly more serious than deleted strains. Among all the deleted strains, the adhesion of ΔssrABhilAhilD to HeLa cells was the lowest, while the colonization rate of ΔhilD in mice was the highest. Besides, the body’s ability to secrete IL-1, IL-18 and IFN-γ has no significant correlation with ssrAB, hilA and hilD genes of Salmonella (P>0.05). [Conclusion] Among the 7 gene deletion strains constructed in this study, ΔssrAB, ΔhilA, ΔhilD, ΔssrABhilA and ΔssrABhilAhilD had the characteristics of low pathogenicity, well growth rate, and they could effectively stimulate cytokine production, which had the potential to be Salmonella live attenuated vaccines or vectors.

Key words:Salmonella enteritidis, ssrAB, hilA, hilD, deletions strains, construct, biological characteristic