双组份信号转导系统CpxAR对鼠伤寒沙门菌致病性的调控作用

发布时间：2018-04-17 12:23

本文选题：鼠伤寒沙门菌 + cpxR基因　；参考：《河南农业大学》2016年硕士论文

【摘要】：沙门菌病是引起养殖业的主要细菌病之一,禽畜感染沙门氏菌可引起相应的传染病,如猪霍乱、鸡白痢等,而且该菌污染肉类食品后,可进入人类食物链而引发人类食物中毒,对人类的健康有着巨大的威胁。本试验目的在于研究双组份信号转导系统cpx AR对鼠伤寒沙门菌致病性的调控作用,为控制其严重的致病性寻找出路,也为减毒活疫苗的研究奠定基础。1.本研究通过ERIC-PCR、MLST分型技术筛选流行克隆株JS_分,应用噬菌体转导技术构建基因缺失突变株JS_分△cpx R,并进行Js标、JS_标△cpx R、JS_分、JS_分△cpx R小鼠致病性试验。LD50结果显示:JS_标△cpx R突变株与亲本株Js标相比,LD50升高了1.36倍,JS_分△cpx R突变株与亲本株Js分相比。LD50升高了17.54倍,基因缺失突变株LD50均升高。上述试验结果表明:cpx R基因与细菌毒力具有紧密的相关性,影响着细菌的致病特性。2.测定标准菌JS_标、JS_分及其缺失株JS_标△cpx R、JS_分△cpx R黏附、侵袭PK—15细胞的能力。结果表明:流行克隆株JS_分的黏附、侵袭力很强,其黏附力、侵袭力均高于标准菌株。缺失cpx R后,受试菌黏附、侵袭能力均显著下降,本试验中标准菌株、流行克隆cpx R缺失后的对PK-15细胞的黏附量分别较缺失前的亲本菌株分别降低了1.35、2倍,侵袭量分别降低了3.4、3倍,表明cpx R基因与鼠伤寒沙门菌黏附侵袭能力密切相关。3.通过荧光定量PCR技术,对7株不同来源的菌株及其cpx R基因缺失株的11个相关毒力基因的m RNA表达水平进行的检测,结果表明:鼠伤寒沙门菌中与毒力相关的基因hil D、sop D、fur、inv A、sop E2、ssr A、spv C、hil A、inv F、sop B、sip A在JS_标△cpx R的m RNA表达量与JS_标相比总体呈下降趋势,然而在临床分离菌cpx R基因缺失株6△cpx R、18△cpx R、25′△cpx R、35△cpx R、57△cpx R、69△cpx R、110△cpx R号菌株中的相对表达量与6、18、25′、35、57、69、110号菌株相比较,fur、spv C在临床菌株cpx R缺失株中呈总体下降趋势,而sip A、hil A、inv F、sop B、hil D、sop D、inv A、sop E2、ssr A在6△cpx R、18△cpx R、25′△cpx R、35△cpx R中呈小幅下降趋势,在57△cpx R、69△cpx R、110△cpx R号菌株中表达量却上升及其显著,高至354.6倍,变化呈现多样性,提示cpx R基因与鼠伤寒沙门菌的致病性有密切关系,但在临床分离菌中,其毒力或致病力差异较大,调控鼠伤寒沙门菌毒力基因表达的不仅仅是cpx R,还可能存在与cpx R的调控机制密切相关的复杂的调控网络。
[Abstract]:Salmonellosis is one of the major bacterial diseases in the aquaculture industry. The infection of salmonella in livestock can cause corresponding infectious diseases, such as pig cholera, white dysentery in chickens and so on. After the bacteria pollutes meat food, it can enter the human food chain and lead to human food poisoning.There is a great threat to human health.The aim of this study was to study the role of cpx AR in regulating the pathogenicity of Salmonella typhimurium, so as to find a way to control its serious pathogenicity and lay a foundation for the study of live attenuated vaccine.In this study, EIC-PCRN MLST typing technique was used to screen the JS _ score of the epidemic clone strain.Phage transduction technique was used to construct the mutant JS_ subunit cpx R, and the pathogenicity test. LD50 of Js-labeled cpx cpx cpx R strain. LD50 showed that the LD50 of cpx R mutant strain was higher than that of parent strain Js.Compared with parent strain Js, the 1.36-fold JS-score of cpx R mutant was 17.54 times higher than that of parent strain Js.The LD50 of the deletion mutant was increased.The results showed that the proportion of CPX R gene was closely related to the virulence of bacteria and affected the pathogenicity of bacteria.The ability of the standard strain JS-labeled JS _ 1 and its missing strain JS-labeled cpx RGS-R to adhere to and invade PK-15 cells was determined.The results showed that the adhesion and invasiveness of the strains were higher than those of the standard strains.After the deletion of cpx R, the adhesion and invasion ability of the tested strains were significantly decreased. In this experiment, the adhesion of the standard strains to PK-15 cells after the deletion of the popular clone cpx R was 1.35 times lower than that of the parent strains before deletion, respectively.The amount of invasion decreased by 3.4 times, indicating that the cpx R gene was closely related to the adhesion and invasion ability of Salmonella typhimurium.The m RNA expression levels of 11 virulence genes in 7 strains from different sources and their cpx R gene deletion strains were detected by fluorescence quantitative PCR.The results showed that the m RNA expression of the virulence related gene of Salmonella typhimurium, hil Dendrosop, furinv Ajsop E2Ossr Aspv Aspv Caphil, in JS _ standard cpx R, showed a general downward trend compared with that of JS _ standard, and the results showed that the expression level of m RNA of Salmonella typhimurium was significantly lower than that of JS _ 2, and the expression of m RNA in JS _ standard cpx R was decreased.However, the relative expression levels of 6 cpx Rn18 cpx Rn25' cpx Rn35 cpx Rn57 cpx Rn69 cpx Rn110 cpx R strains in clinical isolates were compared with those of 6 cpx 1825FN 357, 69110 strains in clinical cpx R deletion strains.There is a general downward trend,In contrast, the expression of sip Acarinhil in 6 cpx RN18 cpx RG25 'cpx Rn35 cpx R was slightly decreased, while in 57 cpx Rn69 cpx RG110 cpx R, the expression level increased significantly, reaching 354.6 times, indicating a diversity of changes, while the expression level of Acarinhil was significantly increased to 354.6 times in 57 cpx Rn69 cpx RM110 cpx R, and increased significantly to 354.6 times, and showed a slight decrease in 6 cpx RV 18 cpx RN 25' cpx Rn35 cpx R, but increased significantly in 57 cpx Rn69 cpx RM110 cpx R.The results suggest that the cpx R gene is closely related to the pathogenicity of Salmonella typhimurium, but the virulence or pathogenicity of the isolates is different.The expression of virulence gene of Salmonella typhimurium is not only regulated by cpx R, but also by complex regulatory network which is closely related to the regulation mechanism of cpx R.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61

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