鼠疫耶尔森氏菌与小鼠巨噬细胞蛋白相互作用的研究

发布时间：2018-01-16 19:37

本文关键词：鼠疫耶尔森氏菌与小鼠巨噬细胞蛋白相互作用的研究　出处：《中国人民解放军军事医学科学院》2005年博士论文　论文类型：学位论文

【摘要】：鼠疫耶尔森氏菌是鼠疫的病原体,曾引发三次鼠疫世界大流行。鼠疫菌与宿主巨噬细胞的相互作用是其致病机制的重要环节。本研究试图通过研究鼠疫耶尔森氏菌与巨噬细胞之间的蛋白相互作用进一步深入了解鼠疫菌的致病机制。我们首先建立和优化了蛋白芯片筛选鼠疫菌与巨噬细胞相互作用的方法,用包含149个鼠疫菌毒力相关蛋白的蛋白芯片筛选出17个能与巨噬细胞蛋白相互作用的鼠疫菌蛋白。其中除LcrG、YscW和YscB为鼠疫菌重要毒力因子低钙响应元件的成员、katY为过氧化物歧化酶外,其余蛋白为推测的原噬菌体基因编码蛋白或推测的未知功能蛋白。间接免疫荧光结果显示8个芯片反应中信号最强的蛋白LcrG,YscB,Yscw,YpPCP1.06,YpMT1.25ac,YpMT1.24c,YP02090,YP02108均能与巨噬细胞相互作用,初步证实筛选结果比较可靠性。利用噬菌体重组技术构建相应蛋白表达缺陷的鼠疫菌突变株,分析突变株对巨噬细胞的细胞毒性和在BALB/c小鼠中的LD_(50),发现两者的突变株在小鼠模型中的毒力显著下降,表明鼠疫菌LcrG和YscB蛋白在其致病机制中有非常重要的作用。利用细菌双杂交技术以鼠疫菌LcrG和Yscw蛋白为诱饵蛋白筛选巨噬细胞J774A.1的cDNA文库,发现LcrG在巨噬细胞内作用的靶蛋白为Rp139和Txn2,YscW作用的靶蛋白为Rnf149。Trx-2和Rnf149分别为硫氧还原蛋白和RING结构泛素连接酶相关的蛋白,它们在细胞的正常生理活动中发挥重要的作用,与细胞的免疫响应、信号转导等紧密相关。我们将继续深入研究这些蛋白相互作用,揭示它们在鼠疫菌致病机制中发挥的作用。本研究首次将蛋白芯片用于筛选细菌与宿主之间的蛋白相互作用的研究,并筛选出一些与鼠疫菌毒力相关的靶标蛋白,发现LcrG和YscW在巨噬细胞内作用的靶蛋白,为揭示鼠疫菌与宿主间相互作用的机制奠定基础。
[Abstract]:Yersinia pestis is the pathogen of plague. The interaction between Yersinia pestis and host macrophages is an important part of its pathogenic mechanism. This study attempts to study the protein interaction between Yersinia pestis and macrophages. We first established and optimized a protein chip screening method for the interaction between Yersinia pestis and macrophages. Seventeen Yersinia pestis proteins that interact with macrophage proteins were screened by using a protein chip containing 149 virulence related proteins of Yersinia pestis, except LcrG. YscW and YscB were members of low calcium response element of Yersinia pestis. The other proteins were speculated phage gene encoding proteins or unknown functional proteins. Indirect immunofluorescence results showed that the most signalling protein of the eight chip reactions was LcrGGN YscBU Yscw. YpPCP1. 06 and YpMT1. 25acl. YpMT1. 24cCU YP02090 YP02108 can interact with macrophages. The screening results were confirmed to be more reliable. A mutant strain of Yersinia pestis with protein expression deficiency was constructed by phage recombination technique. By analyzing the cytotoxicity of mutants to macrophages and LDS-50 in BALB/c mice, we found that the virulence of the mutants was significantly decreased in mice. The results indicate that the LcrG and YscB proteins of Yersinia pestis play an important role in the pathogenesis of Yersinia pestis. Using bacterial Two-hybrid technique to screen macrophages J77 with Yersinia pestis LcrG and Yscw proteins as bait proteins. 4A.1 cDNA library. It was found that the target proteins of LcrG in macrophages were Rp139 and Txn2. The target proteins of YscW were Rnf149.Trx-2 and Rnf149, which were thioxy-reducing proteins and RING structure ubiquitin ligase related proteins, respectively. They play an important role in the normal physiological activities of cells and are closely related to the cellular immune response and signal transduction. We will continue to study these protein interactions in depth. In order to reveal their role in the pathogenesis of Yersinia pestis, protein microarray was used to screen the protein interaction between bacteria and host for the first time. Some target proteins related to the virulence of Yersinia pestis were screened, and the target proteins of LcrG and YscW acting on macrophages were found, which laid a foundation for the mechanism of interaction between Yersinia pestis and its host.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R378

【共引文献】