本文選題：腸炎沙門氏菌　切入點：ssrAB、hilA、hilD基因　出處：《安徽農(nóng)業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：沙門氏菌(Salmonella)能引起人和動物多種不同臨床表現(xiàn)的沙門氏菌病,并為人類食物中毒的主要病原之一,對醫(yī)學、獸醫(yī)和公共衛(wèi)生均十分重要。與沙門氏菌致病性相關(guān)的基因成簇地分布在基因組上,形成一個個相對獨立的單位,稱為沙門氏菌毒力島(Salmonella pathogenicity Island,SPI)。在沙門氏菌毒力島中,毒力島1(SPI1)和毒力島2(SPI2)分別在沙門氏菌侵襲和定殖過程中扮演了重要的角色。研究表明,ssrAB基因調(diào)節(jié)了沙門氏菌SPI2的表達,hilA基因促進沙門氏菌SPI1的表達,hilD基因則通過對ssrAB基因和hilA基因的調(diào)節(jié),實現(xiàn)調(diào)控沙門氏菌SPI1和SPI2的作用。然而,ssr AB、hilA、hilD對沙門氏菌致病性作用的研究尚未見報道,ssrAB、hil A、hilD基因缺失菌株的毒力變化也尚不清楚。本研究以腸炎沙門氏菌為研究對象,通過同源重組技術(shù),分別構(gòu)建ssrAB、hilA、hilD單基因、雙基因以及三基因缺失菌株,即ΔssrAB、ΔhilA、ΔhilD、ΔssrABhilA、ΔssrABhilD、ΔhilAhilD、ΔssrABhilAhilD,并將缺失菌株的生長速率、生物膜形成能力、對小鼠的致病性、在小鼠體內(nèi)的定殖能力、對細胞的粘附性以及刺激機體產(chǎn)生相應(yīng)細胞因子水平等生物學特性與親本菌株進行比較。結(jié)果顯示,缺失菌株的生長速率與親本菌株無顯著差異;除ΔhilD外,其余缺失菌株的生物膜形成能力與親本菌株之間差異不顯著,ΔssrABhilAhilD的生物膜形成能力則顯著低于其他缺失菌株和親本菌株;ΔssrAB、ΔhilA、ΔhilD對小鼠的半數(shù)致死量(LD50)顯著高于親本菌株,ΔssrABhilAhilD的LD50在所有構(gòu)建的缺失菌株中是最高的;所有缺失菌株對小鼠十二指腸的組織損傷程度均明顯低于親本菌株;ΔhilA對HeLa細胞的黏附力顯著低于親本菌株、ΔssrAB以及ΔhilD(P0.05),ΔssrABhilA、ΔssrABhilD、ΔhilAhilD以及ΔssrABhilAhilD的黏附力無顯著差異(P0.05),而ΔssrAB的定殖率在所有基因缺失菌株和親本菌株中最低(P0.05);7個缺失菌株刺激機體產(chǎn)生IL-1、IL-18、IFN-γ的能力與親本菌株無顯著差異(P0.05)。結(jié)果表明,本研究通過基因工程技術(shù)成功構(gòu)建了ssrAB、hilA、hilD單基因、雙基因以及三基因缺失菌株;所構(gòu)建的缺失菌株致病力較親本菌株低,且具有與親本菌株相似的生長速率,能夠刺激機體產(chǎn)生相應(yīng)的細胞因子,可以作為沙門氏菌減毒活疫苗或活載體的候選菌株。
[Abstract]:Salmonella-salmonella, a disease that causes many different clinical manifestations in humans and animals, and is one of the main pathogens of food poisoning in humans. Veterinary and public health are important. Genes associated with the pathogenicity of salmonella are clustered around the genome to form a relatively independent unit called Salmonella pathogenicity Islandland spider.In Salmonella virulence Island, Virulence island 1 (SPI1) and virulence island 2 (SPI2) play an important role in the invasion and colonization of salmonella respectively. Studies have shown that the SSRAB gene regulates the expression of SPI2 of Salmonella, and the HILA gene promotes the expression of SPI1 of Salmonella by means of the HILD gene. Regulation of ssrAB gene and hilA gene, However, the study on the pathogenicity of Salmonella sp. SPI1 and hilAhilD has not been reported. The virulence of Salmonella enteritidis (Salmonella enteritidis) is not clear. By homologous recombination technique, single gene, double gene and three gene deletion strains of ssrABhilA, 螖 hilA, 螖 hilD, 螖 ssrABhilA, 螖 ssrABhilA, 螖 ssrABhilD, 螖 AhhililD, 螖 ssrABhilAhild were constructed, and the growth rate, biofilm forming ability, pathogenicity, pathogenicity, colonization ability in mice were studied. The biological characteristics of cell adhesion and stimulation of cytokine production were compared with parent strain. The results showed that there was no significant difference in growth rate between deletion strain and parent strain, except for 螖 hilD. There was no significant difference between the biofilm forming ability of the other deletion strains and their parents, the biofilm forming ability of 螖 ssrABhilAhilD was significantly lower than that of other deleted strains and parent strains, and the half lethal dose of 螖 ssrAB, 螖 hilA, 螖 hilD to mice was significantly higher than that of parent strains. The LD50 of 螖 ssrABhilAhilD was the highest among all the strains with deletion. The adhesion of 螖 hilA to HeLa cells was significantly lower than that of parent strains, and the adhesion of 螖 ssrAB to HeLa cells was significantly lower than that of parent strains. There was no significant difference in the adhesion of 螖 ssrAB and 螖 hilDH P0.05, 螖 ssrABhilA, 螖 hilAhilD and 螖 ssrABhilAhilD. However, the colonization rate of 螖 ssrAB was higher than that of 螖 ssrAB. There was no significant difference in the ability of 7 deletion strains to stimulate the production of IL-1 IL-18 and IFN- 緯 from their parent strains, and the results showed that there was no significant difference between the seven deletion strains and their parent strains in the production of IL-18 and IFN- 緯. The results showed that there was no significant difference between the seven deletion strains and their parent strains. In this study, we successfully constructed the single gene, double gene and three gene deletion strains of ssrAbhilhilA hilD by genetic engineering technology, the pathogenicity of the deleted strain was lower than that of the parent strain, and the growth rate was similar to that of the parent strain. It can stimulate the production of corresponding cytokines and can be used as a candidate for live attenuated Salmonella vaccine or live vector.
【學位授予單位】：安徽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.61

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