【摘要】：腸炎沙門菌(Salmonella enterica serovar Enteritidis, Salmonella Enteritidis, SE)是一類具有廣泛宿主適應性的兼性胞內(nèi)寄生菌。沙門菌在自然感染的情況下,能夠在諸如巨噬細胞、樹突狀細胞等宿主細胞中進行生長繁殖。大量研究證明,在巨噬細胞中的定植是沙門菌引起全身感染的一個重要進程,沙門菌的效應因子能夠介導巨噬細胞凋亡,使細菌在細胞與細胞之間傳播。因此,沙門菌能夠通過受感染的巨噬細胞轉(zhuǎn)移到腸系膜淋巴結(jié),繼而進入淋巴循環(huán)系統(tǒng)。研究發(fā)現(xiàn),沙門菌的毒力與LPS (lipopolysaccharide)有著密切的關(guān)系,LPS是一種高度乙�；奶侵�,能發(fā)揮屏障功能,阻止疏水溶質(zhì)(如抗生素和洗滌劑)通過被動擴散進入細胞。在大腸桿菌和沙門菌等革蘭陰性菌模型的研究中,LPS被認為是合成外膜的重要組成部分,也是細菌發(fā)揮毒力的重要因子。本研究通過同源重組法成功地構(gòu)建了腸炎沙門菌rfaQ基因缺失株和回復株,并通過細胞侵襲、增殖等實驗,研究該基因?qū)ι抽T菌毒力的影響,為進一步探索腸炎沙門菌rfaQ基因的功能奠定基礎。1腸炎沙門菌RfaQ蛋白的原核表達及rfaQ缺失株的構(gòu)建與鑒定本研究利用分子生物學技術(shù)成功地構(gòu)建了原核表達質(zhì)粒pET30a-rfaQ,并成功將其導入大腸桿菌E. coli BL21 (DE3),通過IPTG的誘導,成功地表達出了腸炎沙門菌C50041LPS的RfaQ蛋白,SDS-PAGE結(jié)果顯示,重組蛋白His-RfaQ以包涵體的形式表達。并用該His-RfaQ蛋白制備了鼠多克隆抗體。利用λ-Red同源重組系統(tǒng)對腸炎沙門菌rfaQ基因進行了敲除,成功地構(gòu)建了腸炎沙門菌標準株C50041 rfaQ基因缺失株；通過重組子成功地構(gòu)建了回復株C50041 ΔrfaQ::rfaQ,該回復株能夠體外重組表達rfaQ基因。Realtime-PCR試驗結(jié)果表明,缺失株的rfaQ基因成功被敲除,而回復株的rfaQ基因的mRNA轉(zhuǎn)錄水平則為野生株的20倍以上。對野生株、缺失株及回復株的菌體裂解蛋白進行Western blotting分析,結(jié)果顯示抗RfaQ多克隆抗體只能從回復株裂解蛋白中檢測到大小為44kDa的目的蛋白,表明缺失株已經(jīng)成功地敲除了rfaQ基因,且回復株能夠表達具有免疫學活性的RfaQ蛋白。對rfaQ突變株的生化特性、生長特性、運動性以及LPS結(jié)構(gòu)鑒定等結(jié)果顯示：與野生株C50041的相比,rfaQ缺失株的生長特性和LPS的整體結(jié)構(gòu)并沒有顯著差異,但缺失株的運動能力明顯減弱,且其生化指標O129R由陽性變成了陰性。腸炎沙門菌標準株C50041rfaQ基因缺失株的成功構(gòu)建為后期對rfaQ基因功能的研究提供了相應的生物材料。2腸炎沙門菌野生株與rfaQ缺失株毒力測定本研究對野生株和rfaQ缺失株的毒力進行了測定,結(jié)果顯示：缺失株C50041 ΔrfaQ的LD50(約為1×109CFU)較野生株C50041 LD50(約為1×106CFU)升高了1000倍左右。對鼠源巨噬細胞RAW264.7和禽源巨噬細胞HD-11的細胞黏附攝取實驗結(jié)果顯示：與野生株C50041相比,缺失株對細胞RAW264.7的黏附率有顯著下降(P0.05),但攝取率沒有明顯差異。缺失株對細胞HD-11的黏附率沒有顯著性差異,但攝取率卻有顯著下降(P0.05)。細胞內(nèi)的增殖實驗結(jié)果顯示：在禽源巨噬細胞HD-11中,野生株與缺失株在四個時間點(1 h、5 h、10 h和24 h)的增殖情況沒有明顯的差異(P0.05)。而在鼠源巨噬細胞RAW264.7中,缺失株在5 h和10 h這兩個時間點增殖倍數(shù)明顯高于野生株(P0.05)。此外,競爭實驗結(jié)果顯示：野生株和缺失株的體外競爭指數(shù)為0.117(±0.0002)；在鼠源巨噬細胞RAW264.7中0 h、5 h、10 h的競爭指數(shù)分別為0.796(±0.0027)、0.561(±0.013)、0.091(±0.0003)；在禽源巨噬細胞HD-11中0 h、5 h、10 h的競爭指數(shù)分別為0.474(±0.055)、0.780(±0.012)、0.531(±0.0056)；在雞體脾臟內(nèi)內(nèi)競爭指數(shù)為0.498(±0.015)；在小鼠肝臟和脾臟中競爭指數(shù)分別為0.14(±0.0030)和0.32(±0.0025)。所有的競爭實驗結(jié)果都表明,與野生株C50041相比,rfaQ缺失株競爭力明顯弱于野生株。
[Abstract]:Salmonella enterica serovar Enteritidis (SE) is a facultative intracellular parasite with wide host adaptability. Salmonella can grow and reproduce in host cells such as macrophages and dendritic cells when naturally infected. Salmonella colonization is an important process of systemic infection caused by Salmonella. The effector factors of Salmonella can mediate the apoptosis of macrophages and the transmission of bacteria between cells. LPS is a highly acetylated glycolipid that acts as a barrier against the passive diffusion of hydrophobic solutes (such as antibiotics and detergents) into cells. In the study of gram-negative bacterial models such as Escherichia coli and Salmonella, LPS is considered to be an important component of the synthetic outer membrane. This study successfully constructed the deleted and restored strains of Salmonella enteritidis rfaQ gene by homologous recombination method, and studied the effect of this gene on the virulence of Salmonella enteritidis by cell invasion and proliferation experiments, laying a foundation for further exploring the function of the rfaQ gene of Salmonella enteritidis. Prokaryotic expression of the protein and construction and identification of rfaQ-deleted strain in E. coli BL21 (DE3) were successfully constructed by molecular biology technique. The recombinant plasmid pET30a-rfaQ was successfully transfected into E. coli BL21 (DE3). The RfaQ protein of Salmonella enteritidis C50041LPS was successfully expressed by induction of IPTG. SDS-PAGE results showed that the recombinant plasmid was successfully expressed. The murine polyclonal antibody was prepared with his-RfaQ protein. The rfaQ gene of Salmonella enteritidis was knocked out by a lambda-Red homologous recombinant system, and the C50041 rfaQ gene deletion strain of Salmonella enteritidis standard strain was successfully constructed. The results of Realtime-PCR showed that the rfaQ gene of the deleted strain was successfully knocked out, and the transcription level of rfaQ gene of the restored strain was more than 20 times that of the wild strain. Western blotting analysis of lysis proteins of the wild strain, the deleted strain and the restored strain showed more resistance to RfaQ. The clonal antibody could only detect the target protein of 44 kDa from the cleavage protein of the recombinant strain, indicating that the deleted strain had successfully knocked out the rfaQ gene and the recombinant strain could express the immunocompetent rfaQ protein. Compared with 50041, the growth characteristics of rfaQ-deficient strain and the overall structure of LPS were not significantly different, but the motility of the deleted strain was significantly weakened, and its biochemical index O129R changed from positive to negative. MATERIALS.2 Virulence test of Salmonella enteritidis wild strain and rfaQ-deleted strain The virulence of wild strain and rfaQ-deleted strain was determined. The results showed that the LD50 of C50041 rfaQ-deleted strain (about 1 x 109 CFU) was about 1000 times higher than that of wild strain C50041 LD50 (about 1 x 106 CFU). Compared with the wild strain C50041, the cell adhesion rate of the deleted strain to RAW264.7 was significantly decreased (P 0.05), but the uptake rate was not significantly different. The cell adhesion rate of the deleted strain to HD-11 was not significantly different, but the uptake rate was significantly decreased (P 0.05). In avian macrophage HD-11, there was no significant difference in the proliferation between wild and absent strains at four time points (1 h, 5 h, 10 h and 24 h) (P 0.05). In mouse macrophage RAW 264.7, the multiplication of the absent strain at 5 h and 10 h was significantly higher than that of the wild strain (P 0.05). The in vitro competitive index of the murine macrophage RAW 264.7 was 0.117 (+0.0002), 0.796 (+0.0027), 0.561 (+0.013), 0.091 (+0.0003), 0.474 (+0.055), 0.780 (+0.012), 0.531 (+0.0056) in the spleen of chicken, and 0.474 (+0.055) in the avian macrophage HD-11, 5 h and 10 h, respectively. The competition index was 0.498 (+0.015), 0.14 (+0.0030) and 0.32 (+0.0025) in the liver and spleen of mice, respectively.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.61

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