肠炎沙门菌LPS合成相关基因rfaQ缺失株的构建及其毒力研究

发布时间：2018-08-26 11:48

【摘要】：肠炎沙门菌(Salmonella enterica serovar Enteritidis, Salmonella Enteritidis, SE)是一类具有广泛宿主适应性的兼性胞内寄生菌。沙门菌在自然感染的情况下,能够在诸如巨噬细胞、树突状细胞等宿主细胞中进行生长繁殖。大量研究证明,在巨噬细胞中的定植是沙门菌引起全身感染的一个重要进程,沙门菌的效应因子能够介导巨噬细胞凋亡,使细菌在细胞与细胞之间传播。因此,沙门菌能够通过受感染的巨噬细胞转移到肠系膜淋巴结,继而进入淋巴循环系统。研究发现,沙门菌的毒力与LPS (lipopolysaccharide)有着密切的关系,LPS是一种高度乙酰化的糖脂,能发挥屏障功能,阻止疏水溶质(如抗生素和洗涤剂)通过被动扩散进入细胞。在大肠杆菌和沙门菌等革兰阴性菌模型的研究中,LPS被认为是合成外膜的重要组成部分,也是细菌发挥毒力的重要因子。本研究通过同源重组法成功地构建了肠炎沙门菌rfaQ基因缺失株和回复株,并通过细胞侵袭、增殖等实验,研究该基因对沙门菌毒力的影响,为进一步探索肠炎沙门菌rfaQ基因的功能奠定基础。1肠炎沙门菌RfaQ蛋白的原核表达及rfaQ缺失株的构建与鉴定本研究利用分子生物学技术成功地构建了原核表达质粒pET30a-rfaQ,并成功将其导入大肠杆菌E. coli BL21 (DE3),通过IPTG的诱导,成功地表达出了肠炎沙门菌C50041LPS的RfaQ蛋白,SDS-PAGE结果显示,重组蛋白His-RfaQ以包涵体的形式表达。并用该His-RfaQ蛋白制备了鼠多克隆抗体。利用λ-Red同源重组系统对肠炎沙门菌rfaQ基因进行了敲除,成功地构建了肠炎沙门菌标准株C50041 rfaQ基因缺失株；通过重组子成功地构建了回复株C50041 ΔrfaQ::rfaQ,该回复株能够体外重组表达rfaQ基因。Realtime-PCR试验结果表明,缺失株的rfaQ基因成功被敲除,而回复株的rfaQ基因的mRNA转录水平则为野生株的20倍以上。对野生株、缺失株及回复株的菌体裂解蛋白进行Western blotting分析,结果显示抗RfaQ多克隆抗体只能从回复株裂解蛋白中检测到大小为44kDa的目的蛋白,表明缺失株已经成功地敲除了rfaQ基因,且回复株能够表达具有免疫学活性的RfaQ蛋白。对rfaQ突变株的生化特性、生长特性、运动性以及LPS结构鉴定等结果显示：与野生株C50041的相比,rfaQ缺失株的生长特性和LPS的整体结构并没有显著差异,但缺失株的运动能力明显减弱,且其生化指标O129R由阳性变成了阴性。肠炎沙门菌标准株C50041rfaQ基因缺失株的成功构建为后期对rfaQ基因功能的研究提供了相应的生物材料。2肠炎沙门菌野生株与rfaQ缺失株毒力测定本研究对野生株和rfaQ缺失株的毒力进行了测定,结果显示：缺失株C50041 ΔrfaQ的LD50(约为1×109CFU)较野生株C50041 LD50(约为1×106CFU)升高了1000倍左右。对鼠源巨噬细胞RAW264.7和禽源巨噬细胞HD-11的细胞黏附摄取实验结果显示：与野生株C50041相比,缺失株对细胞RAW264.7的黏附率有显著下降(P0.05),但摄取率没有明显差异。缺失株对细胞HD-11的黏附率没有显著性差异,但摄取率却有显著下降(P0.05)。细胞内的增殖实验结果显示：在禽源巨噬细胞HD-11中,野生株与缺失株在四个时间点(1 h、5 h、10 h和24 h)的增殖情况没有明显的差异(P0.05)。而在鼠源巨噬细胞RAW264.7中,缺失株在5 h和10 h这两个时间点增殖倍数明显高于野生株(P0.05)。此外,竞争实验结果显示：野生株和缺失株的体外竞争指数为0.117(±0.0002)；在鼠源巨噬细胞RAW264.7中0 h、5 h、10 h的竞争指数分别为0.796(±0.0027)、0.561(±0.013)、0.091(±0.0003)；在禽源巨噬细胞HD-11中0 h、5 h、10 h的竞争指数分别为0.474(±0.055)、0.780(±0.012)、0.531(±0.0056)；在鸡体脾脏内内竞争指数为0.498(±0.015)；在小鼠肝脏和脾脏中竞争指数分别为0.14(±0.0030)和0.32(±0.0025)。所有的竞争实验结果都表明,与野生株C50041相比,rfaQ缺失株竞争力明显弱于野生株。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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