肠炎沙门氏菌ssrAB、hilA、hilD基因缺失菌株的构建及其生物学特性研究

发布时间：2018-03-18 06:30

本文选题：肠炎沙门氏菌　切入点：ssrAB、hilA、hilD基因　出处：《安徽农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：沙门氏菌(Salmonella)能引起人和动物多种不同临床表现的沙门氏菌病,并为人类食物中毒的主要病原之一,对医学、兽医和公共卫生均十分重要。与沙门氏菌致病性相关的基因成簇地分布在基因组上,形成一个个相对独立的单位,称为沙门氏菌毒力岛(Salmonella pathogenicity Island,SPI)。在沙门氏菌毒力岛中,毒力岛1(SPI1)和毒力岛2(SPI2)分别在沙门氏菌侵袭和定殖过程中扮演了重要的角色。研究表明,ssrAB基因调节了沙门氏菌SPI2的表达,hilA基因促进沙门氏菌SPI1的表达,hilD基因则通过对ssrAB基因和hilA基因的调节,实现调控沙门氏菌SPI1和SPI2的作用。然而,ssr AB、hilA、hilD对沙门氏菌致病性作用的研究尚未见报道,ssrAB、hil A、hilD基因缺失菌株的毒力变化也尚不清楚。本研究以肠炎沙门氏菌为研究对象,通过同源重组技术,分别构建ssrAB、hilA、hilD单基因、双基因以及三基因缺失菌株,即ΔssrAB、ΔhilA、ΔhilD、ΔssrABhilA、ΔssrABhilD、ΔhilAhilD、ΔssrABhilAhilD,并将缺失菌株的生长速率、生物膜形成能力、对小鼠的致病性、在小鼠体内的定殖能力、对细胞的粘附性以及刺激机体产生相应细胞因子水平等生物学特性与亲本菌株进行比较。结果显示,缺失菌株的生长速率与亲本菌株无显著差异;除ΔhilD外,其余缺失菌株的生物膜形成能力与亲本菌株之间差异不显著,ΔssrABhilAhilD的生物膜形成能力则显著低于其他缺失菌株和亲本菌株;ΔssrAB、ΔhilA、ΔhilD对小鼠的半数致死量(LD50)显著高于亲本菌株,ΔssrABhilAhilD的LD50在所有构建的缺失菌株中是最高的;所有缺失菌株对小鼠十二指肠的组织损伤程度均明显低于亲本菌株;ΔhilA对HeLa细胞的黏附力显著低于亲本菌株、ΔssrAB以及ΔhilD(P0.05),ΔssrABhilA、ΔssrABhilD、ΔhilAhilD以及ΔssrABhilAhilD的黏附力无显著差异(P0.05),而ΔssrAB的定殖率在所有基因缺失菌株和亲本菌株中最低(P0.05);7个缺失菌株刺激机体产生IL-1、IL-18、IFN-γ的能力与亲本菌株无显著差异(P0.05)。结果表明,本研究通过基因工程技术成功构建了ssrAB、hilA、hilD单基因、双基因以及三基因缺失菌株;所构建的缺失菌株致病力较亲本菌株低,且具有与亲本菌株相似的生长速率,能够刺激机体产生相应的细胞因子,可以作为沙门氏菌减毒活疫苗或活载体的候选菌株。
[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- 纬 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- 纬. 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.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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