分析表达SspH2-EscI融合蛋白的重组沙门菌感染早期小鼠炎症因子分泌及细菌定殖的变化

发布时间：2018-04-24 07:57

本文选题：鼠伤寒沙门菌 + 小鼠　；参考：《扬州大学》2017年硕士论文

【摘要】：沙门菌(Salmonella)是引起人食物中毒的主要病源菌之一。其感染后,能够通过调节宿主细胞(如巨噬细胞)功能逃避机体免疫系统的杀伤,从而在细胞内存活。而在感染早期,机体也能够启动天然免疫反应(如分泌炎症因子),以发挥清除作用。因此,分析沙门菌感染早期炎症因子的分泌与沙门菌定殖的关系具有重要意义。本研究首先对流式蛋白定量分析过程进行优化,然后选取了野生型鼠伤寒沙门菌和表达融合蛋白SspH2-EscI的重组鼠伤寒沙门菌为研究对象,探讨其感染早期炎症因子的分泌与细菌定殖的相关性。1鼠源炎症因子流式蛋白定量试剂盒的优化利用流式蛋白定量(cytometric bead array,CBA)技术分析细胞因子含量,方法简便、易操作且灵敏度高,但试剂成本较高。本实验以鼠源炎症因子检测试剂盒为参考,分别从降低试剂用量和缩短孵育时间两个方面进行优化,结果表明,试剂用量由50μL降至20μL、孵育时间由2 h缩短至30 min仍然可以得到较好的检测效果。以上分析为CBA技术的推广应用提供了重要数据。2利用CBA技术分析小鼠对沙门菌感染的应答沙门菌感染后,机体产生应答并分泌细胞因子。本实验利用CBA技术动态分析小鼠对鼠伤寒沙门菌感染(腹腔注射或口服)的应答。结果表明,与未感染组相比,腹腔注射(1×104CFU/只)或口服(2×107CFU/只)感染鼠伤寒沙门菌后,小鼠血液凝固加重;血清中炎症因子(IFN-γ、IL-6、TNF)的分泌呈现先升后降的趋势,该变化与脾脏重量、细菌的体内定殖的变化趋势一致。腹腔注射和口服途径感染具有类似的变化趋势。这说明炎症因子的分泌与细菌的体内定殖呈正相关。3表达融合蛋白SspH2-EscI的重组沙门菌体外感染巨噬细胞分析前期研究表明,融合蛋白SspH2-EscI的表达能够增强沙门菌诱导的巨噬细胞内炎性体途径的活化。本实验以含有空载体的重组菌X4550(pYA3334)和表达SspH2的重组菌X4550(pYA3334-P-SspH2)为对照,分析表达融合蛋白SspH2-EscI的重组沙门菌X4550(pYA3334-P-SspH2-EscI)体外感染RAW264.7细胞后的细胞应答情况。结果表明,在感染期间,两对照组的变化趋势相似。与两对照组相比,体外感染(MOI=100)5 h后,X4550(pYA3334-P-SspH2-EscI)感染组细胞膜受损严重,caspase-1的活化和乳酸脱氢酶的释放明显增加,说明细胞内炎性体途径被激活;另外,细胞培养上清中炎症因子的含量在感染期间均呈现下降趋势,而对照组明显不同升高。在细胞代谢方面,各组在细胞内线粒体膜电位、活性氧、NO和钙离子浓度等方面的变化趋势一致,但在pH方面,X4550(pYA3334-P-SspH2-EscI)组在感染5h后细胞内pH值升高,而对照组下降。说明炎性体途径的活化影响了炎症因子的分泌和细胞内pH值的变化。4表达融合蛋白SspH2-EscI的重组沙门菌感染小鼠分析据推测,细胞内炎性体途径的活化有利于沙门菌的清除。本研究以含有空载体的重组沙门菌X4550(pYA3334)和表达SspH2蛋白的重组沙门菌X4550(pYA3334-P-SspH2)为对照,通过感染小鼠(腹腔注射和口服),分析表达融合蛋白SspH2-EscI的重组沙门菌X4550(pYA3334-P-SspH2-EscI)在小鼠体内的定殖能力和机体炎症因子分泌情况。结果表明,在感染期间,两对照组呈现相似的变化趋势,小鼠血液凝固加重,血清中炎症因子的分泌增加,脾脏重量升高,细菌定殖严重。而X4550(pYA3334-P-SspH2-EscI)感染组小鼠血液凝固状态、脾脏重量、细菌定殖和炎症因子分泌均明显低于对照组。口服与腹腔注射感染呈现类似的现象。表明,Ssph2-EscI的表达降低了沙门菌的体内定殖和小鼠血清中炎症因子的分泌。
[Abstract]:Salmonella (Salmonella) is one of the main pathogenic bacteria causing human food poisoning. After infection, it can escape the killing of the immune system by regulating the function of host cells (such as macrophages) to survive in the cell. In the early stage of infection, the body can also start the natural immune response (such as secreting inflammatory factors) to play a scavenging effect. Therefore, it is of great significance to analyze the relationship between the early inflammatory factors of Salmonella infection and the colonization of Salmonella. Firstly, the quantitative analysis process of convective protein was optimized. Then the wild Salmonella typhimurium and the recombinant Salmonella typhimurium expressing the fusion protein SspH2-EscI were selected as the research object, and the early infection was discussed. The relationship between the secretion of inflammatory factors and bacterial colonization.1 mouse source of inflammatory factor flow protein quantitative reagent box optimization using flow protein quantitative (cytometric bead array, CBA) technology to analyze the cytokine content. The method is simple, easy to operate and highly sensitive, but the reagent cost is high. This experiment took the mouse source inflammatory factor detection kit as the reference. The test was optimized from two aspects: reducing the dosage of reagents and shortening the incubation time. The results showed that the dosage of reagents was reduced from 50 L to 20 mu, and the incubation time was shortened from 2 h to 30 min. The above analysis provided a heavy requirement for the popularization and application of CBA Technology,.2 using CBA technology to analyze the sense of Salmonella in mice. After infected with Salmonella infection, the body produced responses and secreted cytokine. The CBA technique was used to dynamically analyze the response to Salmonella typhimurium infection (intraperitoneal injection or oral) in mice. The results showed that the blood of mice infected with Salmonella typhimurium (1 x 104CFU/) or (2 x 107CFU/) infected with Salmonella typhimurium from the uninfected group The secretion of inflammatory factors (IFN- gamma, IL-6, TNF) in the serum showed a tendency to rise first and then descend, which was in accordance with the tendency of the spleen weight and the colonization of bacteria in the body. Intraperitoneal injection and oral infection showed a similar trend of change. This shows that the secretion of inflammatory factors is positively related to the colonization of bacteria in the body of.3 expression. The preliminary study of recombinant Salmonella infected by recombinant Salmonella protein SspH2-EscI in vitro showed that the expression of fusion protein SspH2-EscI enhanced the activation of Salmonella induced inflammatory pathway in macrophages. The experiment was compared with the recombinant bacteria X4550 (pYA3334) containing empty carriers and the recombinant strain X4550 (pYA3334-P-SspH2) expressing SspH2. The cell response of the recombinant Salmonella X4550 (pYA3334-P-SspH2-EscI) expressing the fusion protein SspH2-EscI in RAW264.7 cells in vitro was analyzed. The results showed that the change trend of the two control groups was similar during the infection period. Compared with the two control group, the cell membrane of the X4550 (pYA3334-P-SspH2-EscI) infection group was seriously damaged after the infection (MOI=100) 5 h. C The activation of aspase-1 and the release of lactate dehydrogenase showed an obvious increase, indicating that the intracellular inflammatory pathway was activated. In addition, the content of inflammatory factors in the cell culture supernatant decreased during the infection, and the control group was significantly different. In cell metabolism, the mitochondrial membrane potential, reactive oxygen species, NO and calcium ions in cells were in cell metabolism. The changes in concentration were consistent, but in pH, X4550 (pYA3334-P-SspH2-EscI) group increased the pH value in the cells after infection with 5h, while the control group decreased. It was suggested that the activation of the inflammatory pathway affects the secretion of inflammatory factors and the changes in the intracellular pH value of the recombinant Salmonella infected mice with the.4 expression of the fusion protein SspH2-EscI. The activation of the intracellular inflammatory pathway is beneficial to the removal of Salmonella. This study uses recombinant Salmonella X4550 (pYA3334) containing empty carriers and recombinant Salmonella X4550 (pYA3334-P-SspH2) expressing SspH2 protein as the control. By infecting mice (intraperitoneal injection and oral), the recombinant Salmonella X4550 (pYA3334-P) expressing the fusion protein SspH2-EscI is analyzed. -SspH2-EscI) the colonization of the mice and the secretion of inflammatory factors in the mice. The results showed that during the infection, the two control groups showed a similar trend, the blood coagulation in the mice increased, the secretion of inflammatory factors in the serum increased, the weight of the spleen increased and the bacterial colonization was severe. The blood coagulation of mice in the X4550 (pYA3334-P-SspH2-EscI) infection group Solid state, spleen weight, bacterial colonization and inflammatory factors were significantly lower than those in the control group. The oral and intraperitoneal infection showed a similar phenomenon. The expression of Ssph2-EscI decreased the colonization of Salmonella and the secretion of inflammatory factors in the serum of mice.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R516.3

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