本文選題：鼠傷寒沙門菌 + cpxR基因��； 參考：《河南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：沙門菌病是引起養(yǎng)殖業(yè)的主要細(xì)菌病之一,禽畜感染沙門氏菌可引起相應(yīng)的傳染病,如豬霍亂、雞白痢等,而且該菌污染肉類食品后,可進(jìn)入人類食物鏈而引發(fā)人類食物中毒,對人類的健康有著巨大的威脅。本試驗(yàn)?zāi)康脑谟谘芯侩p組份信號轉(zhuǎn)導(dǎo)系統(tǒng)cpx AR對鼠傷寒沙門菌致病性的調(diào)控作用,為控制其嚴(yán)重的致病性尋找出路,也為減毒活疫苗的研究奠定基礎(chǔ)。1.本研究通過ERIC-PCR、MLST分型技術(shù)篩選流行克隆株JS_分,應(yīng)用噬菌體轉(zhuǎn)導(dǎo)技術(shù)構(gòu)建基因缺失突變株JS_分△cpx R,并進(jìn)行Js標(biāo)、JS_標(biāo)△cpx R、JS_分、JS_分△cpx R小鼠致病性試驗(yàn)。LD50結(jié)果顯示:JS_標(biāo)△cpx R突變株與親本株Js標(biāo)相比,LD50升高了1.36倍,JS_分△cpx R突變株與親本株Js分相比。LD50升高了17.54倍,基因缺失突變株LD50均升高。上述試驗(yàn)結(jié)果表明:cpx R基因與細(xì)菌毒力具有緊密的相關(guān)性,影響著細(xì)菌的致病特性。2.測定標(biāo)準(zhǔn)菌JS_標(biāo)、JS_分及其缺失株JS_標(biāo)△cpx R、JS_分△cpx R黏附、侵襲PK—15細(xì)胞的能力。結(jié)果表明:流行克隆株JS_分的黏附、侵襲力很強(qiáng),其黏附力、侵襲力均高于標(biāo)準(zhǔn)菌株。缺失cpx R后,受試菌黏附、侵襲能力均顯著下降,本試驗(yàn)中標(biāo)準(zhǔn)菌株、流行克隆cpx R缺失后的對PK-15細(xì)胞的黏附量分別較缺失前的親本菌株分別降低了1.35、2倍,侵襲量分別降低了3.4、3倍,表明cpx R基因與鼠傷寒沙門菌黏附侵襲能力密切相關(guān)。3.通過熒光定量PCR技術(shù),對7株不同來源的菌株及其cpx R基因缺失株的11個相關(guān)毒力基因的m RNA表達(dá)水平進(jìn)行的檢測,結(jié)果表明:鼠傷寒沙門菌中與毒力相關(guān)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_標(biāo)△cpx R的m RNA表達(dá)量與JS_標(biāo)相比總體呈下降趨勢,然而在臨床分離菌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號菌株中的相對表達(dá)量與6、18、25′、35、57、69、110號菌株相比較,fur、spv C在臨床菌株cpx R缺失株中呈總體下降趨勢,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中呈小幅下降趨勢,在57△cpx R、69△cpx R、110△cpx R號菌株中表達(dá)量卻上升及其顯著,高至354.6倍,變化呈現(xiàn)多樣性,提示cpx R基因與鼠傷寒沙門菌的致病性有密切關(guān)系,但在臨床分離菌中,其毒力或致病力差異較大,調(diào)控鼠傷寒沙門菌毒力基因表達(dá)的不僅僅是cpx R,還可能存在與cpx R的調(diào)控機(jī)制密切相關(guān)的復(fù)雜的調(diào)控網(wǎng)絡(luò)。
[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.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.61

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