鼠疫耶尔森氏菌蛋白YopN与人蛋白相互作用的研究

The protein-protein interactions between Yersinia pestis and host cells are the foundation of pathogenic mechanism of Pestis. In this study, the interaction between the protein YopN of Yersinia pestis and human protein TGFB1I1 and LPXN which had been screened with the yeast two-hybrid system will be investigated deeply to find the evidence on molecular level to reveal the nosogenesis of Yersinia pestis. Many technologies including GST-Pull down, Co-IP, Confocal microscopy will be used to ascertain the interaction between YopN and TGFB1I1 and/or LPXN even the binding domain. Then the secretion and the translocation of YopN into HeLa cells will be analyzed after having been infected by Yersinia pestis bacillus. Many biologic effect such as focal adhesion, cytoskeleton, apoptosis, immune regulation and actin polymerization will be researched based on the binding between YopN and two other proteins. Subsequently, the flow cytometry technology will be used to evaluate the adhesion ability of Yersinia pestis to the HeLa cells and the phagocytosis of this bacillus by RAW264.7 cells after being infected by Yersinia pestis with fluorescence tag. Finally, mutants of YopN by replacing critical amino acids will be constructed to inhibit its binding to target proteins to investigate the changes of phenotype of the infected cells. Based on our research, we want to reveal preliminarily the function of the interaction between YopN and two human proteins in pathogenic procedure to clarify the pathogenic mechanism of Yersinia pestis, thereby to profit for the prophylaxis and therapy of the Pestis.

鼠疫菌和宿主细胞之间的蛋白相互作用是其致病性的重要基础。本研究以酵母双杂交技术筛选到的鼠疫菌蛋白YopN与人蛋白TGFB1I1、LPXN作为研究靶标，深入研究YopN与两种蛋白间的相互作用，以期为深入揭示鼠疫菌的致病机制提供分子水平的证据。本研究拟首先通过GST-pull down、Co-IP等技术验证和分析YopN与两种蛋白间的相互作用和作用的结构域；鼠疫菌感染宿主细胞后分析YopN的细胞定位；细胞内过表达YopN研究其对宿主细胞粘着斑、细胞骨架、细胞凋亡、细胞免疫调节能力等生物学效应的改变；鼠疫菌经荧光标记后感染过表达LPXN、TGFB1I1的细胞，流式细胞仪分析细胞粘附或吞噬能力的改变；最后，通过关键氨基酸点突变的方法使YopN不能与靶蛋白相互结合情况下，观察鼠疫菌感染细胞后的表型改变。初步阐明该相互作用在鼠疫菌致病过程中发挥的作用，从而为鼠疫菌致病机制、预防和治疗的研究奠定基础。

鼠疫菌和宿主细胞之间的蛋白相互作用是其致病性的重要基础。本研究以酵母双杂交技术筛选到的鼠疫菌蛋白 YopN 与人蛋白 TGFB1I1、LPXN作为研究靶标，本研究首先通过 GST-pull down技术验证和分析 YopN 与两种蛋白间的相互作用；鼠疫菌感染宿主细胞后分析 YopN的细胞定位；细胞内过表达 YopN 研究其对宿主细胞细胞骨架的改变；鼠疫菌经荧光标记后感染过表达 LPXN、TGFB1I1 的细胞，流式细胞仪分析细胞粘附能力的改变；但由于不同方法测定鼠疫菌感染宿主细胞后YopN的细胞定位的结果不一致，导致后期部分研究未继续进行。初步阐明YopN对鼠疫菌感染宿主细胞的粘附能力有影响，但YopN能否进入宿主细胞、YopN 与TGFB1I1、LPXN相互作用在鼠疫菌致病过程中发挥的作用有待于进一步研究。