沙門(mén)菌密度感應(yīng)系統(tǒng)對(duì)六型分泌系統(tǒng)調(diào)控的相關(guān)研究
發(fā)布時(shí)間：2018-10-13 18:59
【摘要】：沙門(mén)菌是一種重要的人畜共患病原菌,在自然界分布廣泛。絕大多數(shù)沙門(mén)菌對(duì)人和動(dòng)物都有致病性,能引起人和動(dòng)物多種不同臨床表現(xiàn)的沙門(mén)菌病。沙門(mén)菌分為腸道沙門(mén)菌和邦戈沙門(mén)菌兩個(gè)種。沙門(mén)菌血清型眾多,大約擁有2500多種血清型,其中腸炎沙門(mén)菌和鼠傷寒沙門(mén)菌在人類(lèi)食源性沙門(mén)菌病中尤為重要。六型分泌系統(tǒng)(Type 6 secretion system,T6SS)是近年來(lái)發(fā)現(xiàn)的革蘭陰性菌中一種新的蛋白轉(zhuǎn)運(yùn)系統(tǒng),它可將蛋白質(zhì)運(yùn)送到胞外。T6SS廣泛存在于細(xì)菌中,其結(jié)構(gòu)組成包括Icm F家族、ATP酶-Clp V、Fha調(diào)控蛋白以及分泌相關(guān)的Vgr G和Hcp蛋白。目前發(fā)現(xiàn)T6SS在細(xì)菌對(duì)外界環(huán)境的適應(yīng)性、對(duì)宿主細(xì)胞的致病力、生物被膜形成等方面發(fā)揮作用。與其他分泌系統(tǒng)一樣,T6SS的轉(zhuǎn)錄和表達(dá)水平也受到嚴(yán)格調(diào)控的。有研究證實(shí),在霍亂弧菌中密度感應(yīng)(Quorum sensing,QS)系統(tǒng)lux S基因能顯著影響T6SS主要元件的轉(zhuǎn)錄水平。因此,本課題通過(guò)構(gòu)建沙門(mén)菌lux S基因缺失株,研究沙門(mén)菌中QS對(duì)T6SS的影響。1.沙門(mén)菌毒力因子多重PCR檢測(cè)方法的建立及應(yīng)用通過(guò)建立沙門(mén)菌不同毒力島基因的多重PCR方法,對(duì)沙門(mén)菌毒力基因進(jìn)行快速、靈敏的檢測(cè)。根據(jù)Gen Bank序列設(shè)計(jì)合成16對(duì)引物,優(yōu)化反應(yīng)條件,建立四組多重PCR方法。利用建立的多重PCR方法對(duì)124株沙門(mén)菌進(jìn)行毒力基因檢測(cè),結(jié)果顯示除了sop E基因分布率為15%外,其它15個(gè)毒力基因分布率可達(dá)90%以上。選取代表性菌株,測(cè)定其對(duì)小鼠的致病力,結(jié)果顯示沙門(mén)菌均可導(dǎo)致小鼠發(fā)病死亡。藥敏試驗(yàn)結(jié)果顯示沙門(mén)菌對(duì)紅霉素、阿奇霉素、克林霉素、新霉素、大觀霉素、四環(huán)素、利福平等抗生素的耐藥率可達(dá)90%以上,且存在多重耐藥性。2.沙門(mén)菌lux S基因缺失株的構(gòu)建及生物學(xué)特性研究利用Red同源重組系統(tǒng)構(gòu)建沙門(mén)菌lux S基因缺失株,并對(duì)其生長(zhǎng)速度、運(yùn)動(dòng)性、生物被膜形成、致病力以及對(duì)鼠巨噬細(xì)胞RAW264.7黏附入侵等生物學(xué)特性進(jìn)行研究。結(jié)果顯示,Lux S對(duì)沙門(mén)菌的生長(zhǎng)速度、生物被膜形成能力、致病力及細(xì)胞黏附侵襲能力均無(wú)明顯影響。但lux S缺失株的運(yùn)動(dòng)性顯著高于野生株。3.密度感應(yīng)系統(tǒng)Lux S蛋白對(duì)T6SS影響的分析克隆表達(dá)并純化T6SS核心組分Hcp1、Hcp2和Clp V蛋白,制備相應(yīng)的免疫血清。應(yīng)用熒光定量PCR以及免疫印跡技術(shù)對(duì)lux S基因缺失株中hcp1、hcp2和clp V的基因轉(zhuǎn)錄水平和蛋白表達(dá)水平進(jìn)行檢測(cè)。結(jié)果顯示,無(wú)論在對(duì)數(shù)生長(zhǎng)期和平臺(tái)期,lux S基因缺失株與野生株中T6SS核心組分的轉(zhuǎn)錄水平和表達(dá)水平均無(wú)明顯差異。本研究表明QS系統(tǒng)Lux S蛋白并不影響或直接調(diào)控沙門(mén)菌T6SS核心組分的轉(zhuǎn)錄及表達(dá)。沙門(mén)菌中T6SS系統(tǒng)的調(diào)控有待進(jìn)一步研究。
[Abstract]:Salmonella is an important zoonotic pathogen, which is widely distributed in nature. Most Salmonella have pathogenicity to human and animal, and can cause many different clinical manifestations of Salmonella. Salmonella is divided into intestinal Salmonella and Bongo Salmonella species. Salmonella serotype has more than 2500 serotypes, among which Salmonella enteritidis and Salmonella typhimurium are particularly important in human foodborne salmonella. Type 6 secretion system,T6SS is a new protein transporter system found in Gram-negative bacteria in recent years. It can transport protein to extracellular. T6SS is widely found in bacteria. Its structure consists of Icm F family, ATP enzyme-Clp VN Fha regulatory protein, and secretory Vgr G and Hcp proteins. At present, it has been found that T6SS plays an important role in the adaptability of bacteria to the environment, the pathogenicity of host cells and the formation of biofilm. Like other secretory systems, T6SS transcription and expression levels are tightly regulated. In Vibrio cholerae, the density sensing (Quorum sensing,QS) system lux S gene can significantly affect the transcription level of the main components of T6SS in Vibrio cholerae. Therefore, the effect of QS on T6SS in Salmonella was studied by constructing Salmonella lux S gene deletion strain. 1. Establishment and Application of multiple PCR Assay for the Detection of Salmonella virulence Factor the rapid and sensitive detection of Salmonella virulence gene was carried out by establishing multiple PCR method for different virulence island genes of Salmonella. According to the Gen Bank sequence, 16 pairs of primers were designed and synthesized, the reaction conditions were optimized, and four groups of multiplex PCR methods were established. The virulence genes of 124 Salmonella strains were detected by using the multiplex PCR method. The results showed that the distribution rate of the other 15 virulence genes was more than 90%, except that the sop E gene distribution rate was 15%. The pathogenicity of the representative strains to mice was determined. The results showed that salmonella could lead to morbidity and death of mice. The results of drug sensitivity test showed that the resistance rate of Salmonella to erythromycin, azithromycin, clindamycin, neomycin, spectinomycin, tetracycline and rifampicin was more than 90%. Construction and Biological characteristics of Salmonella lux S Gene deletion strain; Red homologous recombination system was used to construct Salmonella lux S gene deletion strain and its growth rate, motility and biofilm formation were studied. The pathogenicity and RAW264.7 adhesion invasion of mouse macrophages were studied. The results showed that, Lux S had no significant effect on the growth rate, biofilm formation, pathogenicity and cell adhesion and invasion of Salmonella. But the motility of lux S deletion strain was significantly higher than that of wild strain. Analysis of the effect of Lux S protein on T6SS by density Induction system; cloning and purification of Hcp1,Hcp2 and Clp V proteins, the core components of T6SS, to prepare the corresponding immune serum. The transcription and protein expression of hcp1,hcp2 and clp V in lux S gene deletion strains were detected by fluorescence quantitative PCR and Western blotting. The results showed that there was no significant difference in the transcription and expression levels of the core components of T6SS between the deletion strains of, lux S gene and wild strains in logarithmic growth period and platform stage. This study showed that QS Lux S protein did not affect or directly regulate the transcription and expression of the core components of Salmonella T6SS. The regulation of T6SS system in Salmonella needs further study.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.61
本文編號(hào)：2269609
[Abstract]:Salmonella is an important zoonotic pathogen, which is widely distributed in nature. Most Salmonella have pathogenicity to human and animal, and can cause many different clinical manifestations of Salmonella. Salmonella is divided into intestinal Salmonella and Bongo Salmonella species. Salmonella serotype has more than 2500 serotypes, among which Salmonella enteritidis and Salmonella typhimurium are particularly important in human foodborne salmonella. Type 6 secretion system,T6SS is a new protein transporter system found in Gram-negative bacteria in recent years. It can transport protein to extracellular. T6SS is widely found in bacteria. Its structure consists of Icm F family, ATP enzyme-Clp VN Fha regulatory protein, and secretory Vgr G and Hcp proteins. At present, it has been found that T6SS plays an important role in the adaptability of bacteria to the environment, the pathogenicity of host cells and the formation of biofilm. Like other secretory systems, T6SS transcription and expression levels are tightly regulated. In Vibrio cholerae, the density sensing (Quorum sensing,QS) system lux S gene can significantly affect the transcription level of the main components of T6SS in Vibrio cholerae. Therefore, the effect of QS on T6SS in Salmonella was studied by constructing Salmonella lux S gene deletion strain. 1. Establishment and Application of multiple PCR Assay for the Detection of Salmonella virulence Factor the rapid and sensitive detection of Salmonella virulence gene was carried out by establishing multiple PCR method for different virulence island genes of Salmonella. According to the Gen Bank sequence, 16 pairs of primers were designed and synthesized, the reaction conditions were optimized, and four groups of multiplex PCR methods were established. The virulence genes of 124 Salmonella strains were detected by using the multiplex PCR method. The results showed that the distribution rate of the other 15 virulence genes was more than 90%, except that the sop E gene distribution rate was 15%. The pathogenicity of the representative strains to mice was determined. The results showed that salmonella could lead to morbidity and death of mice. The results of drug sensitivity test showed that the resistance rate of Salmonella to erythromycin, azithromycin, clindamycin, neomycin, spectinomycin, tetracycline and rifampicin was more than 90%. Construction and Biological characteristics of Salmonella lux S Gene deletion strain; Red homologous recombination system was used to construct Salmonella lux S gene deletion strain and its growth rate, motility and biofilm formation were studied. The pathogenicity and RAW264.7 adhesion invasion of mouse macrophages were studied. The results showed that, Lux S had no significant effect on the growth rate, biofilm formation, pathogenicity and cell adhesion and invasion of Salmonella. But the motility of lux S deletion strain was significantly higher than that of wild strain. Analysis of the effect of Lux S protein on T6SS by density Induction system; cloning and purification of Hcp1,Hcp2 and Clp V proteins, the core components of T6SS, to prepare the corresponding immune serum. The transcription and protein expression of hcp1,hcp2 and clp V in lux S gene deletion strains were detected by fluorescence quantitative PCR and Western blotting. The results showed that there was no significant difference in the transcription and expression levels of the core components of T6SS between the deletion strains of, lux S gene and wild strains in logarithmic growth period and platform stage. This study showed that QS Lux S protein did not affect or directly regulate the transcription and expression of the core components of Salmonella T6SS. The regulation of T6SS system in Salmonella needs further study.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.61
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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，本文編號(hào)：2269609
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