单核细胞增多性李斯特菌溶血素LLO的功能关键位点鉴定及表面展示系统的构建

发布时间：2018-04-25 05:42

本文选题：单核细胞增多性李斯特菌 + 溶血素LLO　；参考：《浙江农林大学》2015年硕士论文

【摘要】：单核细胞增多性李斯特菌(Listeria monocytogenes,LM),简称单增李斯特菌,是一种革兰氏阳性、食源性胞内病原菌。LM主要通过分选酶(Sortase)系统将含有LPXTG基序的蛋白锚定至胞壁并发挥相应功能。溶血素Listeriolysin O(LLO)是属于依赖于胆固醇的膜穿孔蛋白家族(CDCs),在游离状态下可以形成直径35 nm的孔状结构,插入到宿主细胞膜内,在李斯特菌逃离到细胞质中扮演着重要的角色。本研究分别以单增李斯特菌参考菌株EGD-e和弱毒株M7溶血素蛋白(Listeriolysin O,LLO)为研究对象,通过同源重组及氨基酸突变技术将编码LLO蛋白的基因hly在基因组水平上进行如下修饰:(1)构建LLO缺失突变株(ΔLLO);(2)将野生株LLO蛋白第472-483位氨基酸缺失,使其形成携带LPXTG基序的重组LLO菌株(LLOLPXTG)。Western blot试验结果显示EGD-e和M7菌株缺失LLO后均未检测到LLO蛋白的表达,而LLOLPXTG缺失株检测到重组LLO蛋白的表达,证实ΔLLO缺失株和LLOLPXTG缺失株均构建成功;鼠源巨噬细胞RAW264.7胞内增殖试验表明,EGD-e的ΔLLO缺失株和LLOLPXTG缺失株的胞内增殖力显著减弱;溶血试验表明,M7的ΔLLO缺失株和LLOLPXTG缺失株的溶血活性完全丧失。体外原核表达纯化LLO及其第472-483位氨基酸突变体蛋白,并研究其体外溶血活性。结果显示只有LLO第478和479位氨基酸影响LLO的溶血活性。体内外试验表明单增李斯特菌LLO蛋白与细菌感染宿主细胞后的增殖能力密切相关。本研究首次发现并证实LLO第472-483位氨基酸为其关键位点,决定了LLO的活性及功能。LLO缺失株的构建可以为细菌体内表面展示技术在宿主细胞内进行抗原展示和递呈的研究奠定重要基础。综上所述,本研究首次发现并证实单增李斯特菌LLO第472-483位氨基酸为其关键位点或功能域,决定了LLO的活性及功能;单增李斯特菌LLOLPXTG表面展示重组菌株为将来胞内菌的抗原展示技术开发提供了一种工具,可用于病毒病的预防和治疗。
[Abstract]:Listeria monocytogenes monocytogenes monocytogenes (Listeria monocytogenesus) is a Gram-positive bacteria. The protein-containing LPXTG motifs are anchored to the cell wall and play a corresponding function by the food-borne intracellular pathogen. Hemolysin (Listeriolysin) is a cholesterol dependent membrane perforated protein family, which can form a 35 nm diameter pore structure in the free state and insert into the host cell membrane, which plays an important role in the escape of Listeria to the cytoplasm. In this study, the reference strain of Listeria monocytogenes EGD-e and the attenuated strain M7 hemolysin Olol were studied. By homologous recombination and amino acid mutation technique, the gene hly encoding LLO protein was modified as follows at genomic level to construct LLO deletion mutant (螖 Llosian2), which deleted the 472-483 amino acid of LLO protein of wild strain. The expression of LLO protein was not detected in the EGD-e and M7 strains without LLO, while the expression of the recombinant LLO protein was detected in the LLOLPXTG deletion strain. It was confirmed that both 螖 LLO deletion and LLOLPXTG deletion were successfully constructed, and the intracellular proliferation of murine macrophage macrophage RAW264.7 was significantly decreased by RAW264.7 deletion of EGD-e and LLOLPXTG deletion. Hemolysis test showed that the hemolytic activity of 螖 LLO deletion strain and LLOLPXTG deletion strain of M7 was completely lost. LLO and its 472-483 amino acid mutant protein were expressed and purified in vitro and their hemolytic activity was studied. The results showed that only the 478th and 479-amino acids of LLO affected the hemolytic activity of LLO. In vitro and in vivo tests showed that the LLO protein of Listeria monocytogenes was closely related to the proliferation of host cells. In this study, we first found and confirmed that the 472-483 amino acid of LLO is the key site. It is determined that the activity and function of LLO. The construction of LLO deletion strain can lay an important foundation for the study of antigen display and presentation in host cells by the technique of bacterial surface display in vivo. To sum up, the amino acid at position 472-483 of Listeria monocytogenes LLO is the key site or functional domain, which determines the activity and function of LLO. The recombinant strain of Listeria monocytogenes LLOLPXTG surface display provides a tool for the development of antigen display technology of intracellular bacteria in the future and can be used in the prevention and treatment of virus diseases.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【相似文献】