本文選題：鞭毛蛋白 + 減毒鼠傷寒沙門菌��； 參考：《揚(yáng)州大學(xué)》2011年碩士論文

【摘要】：細(xì)菌鞭毛蛋白可誘導(dǎo)宿主固有免疫應(yīng)答的產(chǎn)生,但是鞭毛蛋白在重組減毒沙門菌固有免疫應(yīng)答中作用的研究鮮見。本研究旨在探討鞭毛蛋白在減毒鼠傷寒沙門菌對家禽和小鼠固有免疫誘導(dǎo)中的作用,進(jìn)而為篩選合適的減毒沙門菌作為疫苗載體提供理論依據(jù)。 根據(jù)雞IL-6、IL-1β、IFN-γ、TGF-β4、β-actin mRNA的保守序列設(shè)計(jì)特異性引物,提取經(jīng)鞭毛蛋白刺激的雞巨噬細(xì)胞HD11總RNA,并逆轉(zhuǎn)錄成cDNA,建立實(shí)時(shí)熒光定量PCR(real-time fluorescence quantitative PCR, qRT-PCR)方法。通過熔解曲線及擴(kuò)增曲線的分析表明,本研究所建立的qRT-PCR方法特異性強(qiáng)、重復(fù)性好。HD11細(xì)胞經(jīng)鞭毛蛋白刺激后,前炎性細(xì)胞因子IL-6和IL-1β的相對表達(dá)量與對照組相比顯著升高(P0.05)；而IFN-γ與抗炎性細(xì)胞因子TGF-β4的相對表達(dá)量無顯著變化(P0.05)。接著分離1周齡非免疫雞血液中的異嗜性白細(xì)胞,用鞭毛蛋白、減毒鼠傷寒沙門菌X4550(pYA3334-F)及其鞭毛蛋白缺失株X4550△FlhD (pYA3334-F)進(jìn)行體外刺激,應(yīng)用qRT-PCR方法檢測刺激不同時(shí)間后這些細(xì)胞因子的表達(dá)水平。結(jié)果顯示,鞭毛蛋白能顯著誘導(dǎo)異嗜白細(xì)胞中前炎性細(xì)胞因子IL-6和IL-1β的產(chǎn)生,TGF-β4的相對表達(dá)量無明顯變化(P0.05)。兩株細(xì)菌刺激2、3、4h后異嗜白細(xì)胞均能上調(diào)IL-6和IL-1β的表達(dá),TGF-β4在刺激4h后表達(dá)顯著上調(diào),IFN-γ在檢測的時(shí)間點(diǎn)均未出現(xiàn)擴(kuò)增。鼠傷寒沙門菌X4550(pYA3334-F)與異嗜白細(xì)胞作用3h和4h后對IL-6、IL-1β和TGF-β4的轉(zhuǎn)錄誘導(dǎo)能力要顯著高于X4550△FlhD(pYA3334-F)。 以5×109劑量的X4550(pYA3334-F)和X4550△FlhD(pYA3334-F)分別口服感染1周齡非免疫雞,在感染后不同時(shí)間點(diǎn)對兩組雞的盲腸、肝臟、脾臟進(jìn)行細(xì)菌涂板計(jì)數(shù),并以qRT-PCR方法比較兩株細(xì)菌早期感染(1、3、5d)后血液異嗜白細(xì)胞、盲腸、脾臟中CXCLi2、IL-1β、TGF-β4以及TLR5的轉(zhuǎn)錄表達(dá)差異。結(jié)果表明感染早期X4550△FlhD (pYA3334-F)在雞體內(nèi)定植能力要高于X4550(pYA3334-F),但是其體內(nèi)定植時(shí)間要短于X4550(pYA3334-F)。與X4550△FlhD(pYA3334-F)組相比X4550(pYA3334-F)在感染3d后,誘導(dǎo)雞體內(nèi)異嗜白細(xì)胞的炎癥反應(yīng),產(chǎn)生較多的CXCLi2、ⅠL-1β、TGF-β4和TLR-5以控制X4550(pYA3334-F)的早期感染數(shù)量。X4550(pYA3334-F)在感染早期能誘導(dǎo)雞盲腸和脾臟產(chǎn)生CXCLi2(1d、3d),IL-1β(3d,5d),TGF-β4(1d)以及TLR5(1d、3d),且與X4550△FlhD(pYA3334-F)組存在顯著差異(P0.05)。 以10。劑量的X4550(pYA3334-F)和X4550△FlhD(pYA3334-F)分別口服感染6-8周齡BALB/c小鼠,在感染不同時(shí)間點(diǎn)對兩組小鼠的派伊爾氏結(jié)、腸系膜淋巴結(jié)、肝臟、脾臟進(jìn)行細(xì)菌涂板計(jì)數(shù),并比較兩株細(xì)菌早期感染(8h、1d、3d)后小腸、脾臟中MIP-2,MCP-1,IL-1β,IFN-γ,TNF-α,TGF-β,IL-4等細(xì)胞因子的轉(zhuǎn)錄表達(dá)差異。結(jié)果表明X4550△FlhD(pYA3334-F)在感染早期的體內(nèi)定植能力高于X4550(pYA3334-F)。 X4550(pYA3334-F)在感染早期(8h、1d)誘導(dǎo)產(chǎn)生的MIP-2、MCP-1及IL-1p的表達(dá)水平要顯著高于X4550△FlhD(pYA3334-F)組(P0.05),其中X4550(pYA3334-F)感染組脾臟中TNF-a的轉(zhuǎn)錄表達(dá)水平顯著高于X4550△FlhD(pYA3334-F)組,但在小腸中均未出現(xiàn)顯著上調(diào)。IFN-γ在兩株細(xì)菌感染1d小腸中的轉(zhuǎn)錄表達(dá)水平出現(xiàn)顯著差異,但在脾臟中未出現(xiàn)顯著上調(diào)。早期感染后小腸和脾臟中IL-4均處于較低的轉(zhuǎn)錄表達(dá)水平。 體內(nèi)感染實(shí)驗(yàn)結(jié)果表明,與X4550△FlhD(pYA3334-F)相比,C4550(pYA3334-F)能誘導(dǎo)雞和小鼠產(chǎn)生較強(qiáng)的細(xì)胞因子應(yīng)答,減少早期階段的感染,說明鞭毛蛋白的存在會(huì)增強(qiáng)減毒株的固有免疫應(yīng)答水平,這為減毒沙門菌疫苗載體的研究提供了理論依據(jù)。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1

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相關(guān)期刊論文 前10條

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本文編號：2062005