摘要：

为探究鼠伤寒沙门菌sRNA STnc1220的分子特征、生物学功能及对细菌毒力的影响,通过PCR扩增了鼠伤寒沙门菌sRNA STnc1220基因,分析了其分子特征,利用TargetRNA2网站预测STnc1220基因可能调控的靶基因;利用λ-Red 同源重组技术构建沙门菌(STM)-基因缺失突变株,并与亲本SL1344比较,对其生长特性、生化特性、遗传稳定性、生物被膜(BF)形成能力、在不同应激环境中(pH值、H₂O₂、高盐和乙醇胁迫环境)的适应性、通过黏附侵染巨噬细胞和小鼠感染试验对致病力进行研究。结果显示,STnc1220 基因全长73 bp、其上游5'-UTR存在-10和-35启动子结合位点,预测其潜在的靶基因为barA基因;试验成功构建缺失株STM-ΔSTnc1220,且具有良好的遗传稳定性;与SL1344亲本株相比,缺失株的生长速度、生化特性及生物被膜形成能力没有发生改变;在pH=4.8,pH=9和H₂O₂条件下生长速率差异不显著,但在4% NaCl环境下,缺失株在7 h后生长速率明显高于亲本株,在3.8%乙醇环境中缺失株在对数期的生长速率显著升高(P<0.05);其对巨噬细胞中黏附和侵袭能力极显著降低(P<0.01),小鼠LD₅₀显著升高,对小鼠的肝脏和脾脏的病理损伤致病力减弱。表明STnc1220基因在环境适应性和毒力调控中发挥了重要的作用。为阐明鼠伤寒沙门菌的致病机制提供理论依据。

关键词: 鼠伤寒沙门菌, sRNA, STnc1220, 生物学特性, 毒力

Abstract:

In order to explore the molecular characteristics,biological functions and effects of sRNA STnc1220 of Salmonella typhimurium on bacterial virulence,the sRNA STnc1220 gene of Salmonella typhimurium was amplified by PCR,and its molecular characteristics were analyzed.Use the TargetRNA2 website to predicted the target genes that might be regulated by the STnc1220 gene;the λ-Red homologous recombination technology was used to construct STM(Salmonella)-The gene deletion mutant strain,and compared with the parent SL1344,its growth characteristics,biochemical characteristics,genetic stability,biofilm(BF)formation ability,and its effects in different stress environments(pH,H₂O₂,high salt and ethanol stress)adaptability,through adhesion and infection of macrophages and mouse infection tests to study pathogenicity.The results showed that the full length of STnc1220 gene was 73 bp,and its upstream 5'-UTR had-10 and-35 promoter binding sites.It was predicted that its potential target gene was barA gene;The test successfully constructed the deletion strain STM-ΔSTnc1220,and it had good performance genetic stability;compared with the SL-1344 parent strain,the growth rate,biochemical characteristics and biofilm formation ability of the missing strain did not change;the growth rate was not significantly different under the conditions of pH=4.8,pH=9 and H₂O₂.However, in the 4% NaCl environment, the growth rate of the deletion strain was significantly higher than that of the parent strain after 7 h, and the growth rate of the deletion strain in the logarithmic phase was significantly increased in the 3.8% ethanol environment(P<0.05);It significantly reduces the ability of adhesion and invasion in macrophages(P<0.01), and the LD₅₀ of mice was significantly elevated,the pathological damage to the liver and spleen of mice was weakened.It showed that the STnc1220 gene played an important role in environmental adaptation and virulence regulation.

Key words: Salmonella typhimurium, sRNA, STnc1220, Biological characteristics, Virulence