鞭毛蛋白在重组减毒鼠伤寒沙门菌诱导固有免疫应答中的作用研究

发布时间：2018-06-24 15:35

本文选题：鞭毛蛋白 + 减毒鼠伤寒沙门菌　；参考：《扬州大学》2011年硕士论文

【摘要】：细菌鞭毛蛋白可诱导宿主固有免疫应答的产生,但是鞭毛蛋白在重组减毒沙门菌固有免疫应答中作用的研究鲜见。本研究旨在探讨鞭毛蛋白在减毒鼠伤寒沙门菌对家禽和小鼠固有免疫诱导中的作用,进而为筛选合适的减毒沙门菌作为疫苗载体提供理论依据。根据鸡IL-6、IL-1β、IFN-γ、TGF-β4、β-actin mRNA的保守序列设计特异性引物,提取经鞭毛蛋白刺激的鸡巨噬细胞HD11总RNA,并逆转录成cDNA,建立实时荧光定量PCR(real-time fluorescence quantitative PCR, qRT-PCR)方法。通过熔解曲线及扩增曲线的分析表明,本研究所建立的qRT-PCR方法特异性强、重复性好。HD11细胞经鞭毛蛋白刺激后,前炎性细胞因子IL-6和IL-1β的相对表达量与对照组相比显著升高(P0.05)；而IFN-γ与抗炎性细胞因子TGF-β4的相对表达量无显著变化(P0.05)。接着分离1周龄非免疫鸡血液中的异嗜性白细胞,用鞭毛蛋白、减毒鼠伤寒沙门菌X4550(pYA3334-F)及其鞭毛蛋白缺失株X4550△FlhD (pYA3334-F)进行体外刺激,应用qRT-PCR方法检测刺激不同时间后这些细胞因子的表达水平。结果显示,鞭毛蛋白能显著诱导异嗜白细胞中前炎性细胞因子IL-6和IL-1β的产生,TGF-β4的相对表达量无明显变化(P0.05)。两株细菌刺激2、3、4h后异嗜白细胞均能上调IL-6和IL-1β的表达,TGF-β4在刺激4h后表达显著上调,IFN-γ在检测的时间点均未出现扩增。鼠伤寒沙门菌X4550(pYA3334-F)与异嗜白细胞作用3h和4h后对IL-6、IL-1β和TGF-β4的转录诱导能力要显著高于X4550△FlhD(pYA3334-F)。以5×109剂量的X4550(pYA3334-F)和X4550△FlhD(pYA3334-F)分别口服感染1周龄非免疫鸡,在感染后不同时间点对两组鸡的盲肠、肝脏、脾脏进行细菌涂板计数,并以qRT-PCR方法比较两株细菌早期感染(1、3、5d)后血液异嗜白细胞、盲肠、脾脏中CXCLi2、IL-1β、TGF-β4以及TLR5的转录表达差异。结果表明感染早期X4550△FlhD (pYA3334-F)在鸡体内定植能力要高于X4550(pYA3334-F),但是其体内定植时间要短于X4550(pYA3334-F)。与X4550△FlhD(pYA3334-F)组相比X4550(pYA3334-F)在感染3d后,诱导鸡体内异嗜白细胞的炎症反应,产生较多的CXCLi2、ⅠL-1β、TGF-β4和TLR-5以控制X4550(pYA3334-F)的早期感染数量。X4550(pYA3334-F)在感染早期能诱导鸡盲肠和脾脏产生CXCLi2(1d、3d),IL-1β(3d,5d),TGF-β4(1d)以及TLR5(1d、3d),且与X4550△FlhD(pYA3334-F)组存在显著差异(P0.05)。以10。剂量的X4550(pYA3334-F)和X4550△FlhD(pYA3334-F)分别口服感染6-8周龄BALB/c小鼠,在感染不同时间点对两组小鼠的派伊尔氏结、肠系膜淋巴结、肝脏、脾脏进行细菌涂板计数,并比较两株细菌早期感染(8h、1d、3d)后小肠、脾脏中MIP-2,MCP-1,IL-1β,IFN-γ,TNF-α,TGF-β,IL-4等细胞因子的转录表达差异。结果表明X4550△FlhD(pYA3334-F)在感染早期的体内定植能力高于X4550(pYA3334-F)。 X4550(pYA3334-F)在感染早期(8h、1d)诱导产生的MIP-2、MCP-1及IL-1p的表达水平要显著高于X4550△FlhD(pYA3334-F)组(P0.05),其中X4550(pYA3334-F)感染组脾脏中TNF-a的转录表达水平显著高于X4550△FlhD(pYA3334-F)组,但在小肠中均未出现显著上调。IFN-γ在两株细菌感染1d小肠中的转录表达水平出现显著差异,但在脾脏中未出现显著上调。早期感染后小肠和脾脏中IL-4均处于较低的转录表达水平。体内感染实验结果表明,与X4550△FlhD(pYA3334-F)相比,C4550(pYA3334-F)能诱导鸡和小鼠产生较强的细胞因子应答,减少早期阶段的感染,说明鞭毛蛋白的存在会增强减毒株的固有免疫应答水平,这为减毒沙门菌疫苗载体的研究提供了理论依据。
[Abstract]:Bacterial flagellin can induce the inherent immune response of the host, but the role of flagellin in recombinant immune response to Salmonella is rare. The purpose of this study is to explore the use of flagellin in the innate immunity induction of Salmonella typhimurium to poultry and mice, and then to screen suitable attenuated Salmonella. It provides a theoretical basis for the vaccine carrier.
Based on the conservative sequence of chicken IL-6, IL-1 beta, IFN- gamma, TGF- beta 4 and beta -actin mRNA, specific primers were designed to extract HD11 total RNA of chicken macrophages stimulated by flagellin and reverse transcriptase to cDNA. The qRT-PCR method established by the Institute was strong, and the relative expression of proinflammatory cytokines IL-6 and IL-1 beta was significantly higher than that of the control group (P0.05) after the stimulation of flagellin, and the relative expression of IFN- gamma and anti inflammatory cytokine TGF- beta 4 was not significantly changed (P0.05). Then, 1 weeks of non immunized chicken blood were separated. The heterophilic leukocytes in the liquid were stimulated with flagellin, attenuated Salmonella typhimurium X4550 (pYA3334-F) and the flagellin deletion strain X4550 Delta FlhD (pYA3334-F) in vitro. The expression level of these cytokines was detected by qRT-PCR method. The results showed that flagellin could significantly induce proinflammatory in heterophils. The relative expression of TGF- beta 4 was not significantly changed (P0.05). The expression of IL-6 and IL-1 beta was up-regulated by two strains of bacteria stimulated by 2,3,4h. The expression of TGF- beta 4 was up significantly up after the stimulation of 4h, and IFN- gamma was not expanded at the time point of detection. X4550 (pYA3334-F) and isosophil of Salmonella typhimurium was found. The transcriptional induction ability of 3H and 4H to IL-6, IL-1 beta and TGF- beta 4 was significantly higher than that of X4550 Delta FlhD (pYA3334-F).
5 x 109 doses of X4550 (pYA3334-F) and X4550 Delta FlhD (pYA3334-F) were taken orally for 1 weeks of non immune chickens. The cecum, liver and spleen of two groups of chickens were counted at different time points after infection, and the blood isophils, cecum, spleen, IL-1 beta and TG were compared with two strains of early infection (1,3,5d) by qRT-PCR. The transcriptional expression difference between F- beta 4 and TLR5 showed that the colonization of X4550 Delta FlhD (pYA3334-F) in the early stage of infection was higher than X4550 (pYA3334-F), but the time of colonization in vivo was shorter than X4550 (pYA3334-F). Compared with X4550 Delta FlhD (pYA3334-F) group, it induced the inflammatory reaction of leukocytes in chicken after infection. Producing more CXCLi2, I L-1 beta, TGF- beta 4 and TLR-5 to control the early infection number of X4550 (pYA3334-F).X4550 (pYA3334-F) in the early infection can induce chicken cecum and spleen to produce CXCLi2 (1D, 3D), IL-1 beta 4, etc.
10. doses of X4550 (pYA3334-F) and X4550 Delta FlhD (pYA3334-F) were used to infect 6-8 weeks old BALB/c mice respectively. The bacterial smear plates were counted on the PY's knot, mesenteric lymph nodes, liver and spleen at different time points of infection in two groups, and two strains of early infection (8h, 1D, 3D) were compared, and MIP-2, MCP-1, IL-1 beta, IL-1 beta in the spleen. The transcriptional expression difference of TNF- alpha, TGF- beta, IL-4 and other cytokines. The results showed that the colonization ability of X4550 Delta FlhD (pYA3334-F) in early infection was higher than that of X4550 (pYA3334-F). X4550 (pYA3334-F) in the early infection (8h, 1D) was significantly higher than that of X4550 (pYA3334-F). PYA3334-F) the transcriptional expression level of TNF-a in the spleen of the infected group was significantly higher than that of the X4550 Delta FlhD (pYA3334-F) group, but no significant up regulation of.IFN- gamma in the small intestine was found in the small intestine, but there was a significant difference in the level of the transcriptional expression in the small intestine of the two strains of bacterial infection, but there was no significant change in the spleen. The IL-4 in the small intestine and spleen in the early infection was in the same position after early infection. Low level of transcriptional expression.
The results of the infection in vivo showed that compared with X4550 Delta FlhD (pYA3334-F), C4550 (pYA3334-F) could induce a stronger cytokine response in chickens and mice and reduce the infection in the early stage. It shows that the presence of flagellin will enhance the inherent immune response of the attenuated strain, which provides a theoretical basis for the research of the attenuated Salmonella vaccine carrier.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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