介导金葡菌侵袭细胞的FnBPA蛋白功能肽段的初步研究

发布时间：2018-03-24 02:25

本文选题：金黄色葡萄球菌　切入点：FnBPA　出处：《中国人民解放军军事医学科学院》2007年硕士论文

【摘要】： 金黄色葡萄球菌(以下简称金葡菌)是革兰染色阳性病原菌，，能引起多种疾病。过去研究认为金葡菌属于胞外寄生菌。但近来的研究表明，金葡菌可以侵袭多种非专职吞噬细胞，FnBP蛋白是介导金葡菌侵袭宿主细胞的关键表面蛋白。研究认为FnBP蛋白通过与纤连蛋白结合从而介导细菌与细胞的结合，其中FnBPA与纤连蛋白结合的关键位点位于蛋白D结构域，以重复序列为单位，包括3个连续的完整的重复序列D1、D2、D3和一个截短的D_4序列。由于空间位阻的关系，如果FnBP仅仅以重复序列为单位结合Fn分子，势必造成实际结合位点的减少和D4截短序列功能的冗余。为保证最大限度的生物利用和结合，我们推测FnBPA D domain中的Fn结合位点可能并不是以重复序列为单位的，某些截短的重复序列或具有结合功能，或和下一个重复序列的一部分组合产生新的结合位点。本实验的目的是对以上推测进行初步验证。根据文献报道，我们建立了细菌侵袭293细胞的实验模型，并通过此模型找到了金葡菌有效侵袭株04018作为实验用菌株。利用基因工程的方法，克隆了04018菌株的FnBPA蛋白D_1区C端和D_2区N端肽段的基因片段，在大肠杆菌中表达了GST与该肽(FnBPA-D1c/D2n)的融合蛋白。ELISA实验证实融合蛋白中的目的肽能够结合Fn，细菌侵袭细胞的实验证实融合蛋白的目的肽能够抑制金葡菌侵袭293细胞，蛋白的50％抑制剂量约为20μg/ml，相当于2.5μg/ml肽。通过本课题的研究，我们找到了金葡菌FnBPA蛋白中与纤连蛋白结合的新的位点，提示FnBPA蛋白的D结构域中或者具有更小的结合单位，或者存在更多的Fn结合位点，而不是预测的3-4个，并为抑制金葡菌侵袭细胞的应用研究提供了一定的基础。
[Abstract]:Staphylococcus aureus (S. aureus) is a Gram-positive pathogen, can cause a variety of diseases. The past research that Staphylococcus aureus belongs to extracellular bacteria. But recent research showed that Staphylococcus aureus can invade a variety of non professional phagocytic cells, FnBP protein is a key surface protein mediated Staphylococcus aureus the invasion of the host cell.
Studies suggest that FnBP protein by binding protein and fiber combination and even mediated by bacteria and cells, one of the key sites of FnBPA and fibronectin binding protein in the D domain, to repeat units, including 3 consecutive complete repeat sequences of D1, D2, D_4 sequence D3 and a truncated. Because the relationship between the steric, if only to FnBP repeat units with Fn molecules, will inevitably result in redundant actual binding sites decreased and D4 truncated sequence function. In order to ensure the maximum bioavailability and the combination of FnBPA D domain, we speculate that Fn binding sites may not repeat units, repeat some sequence truncated or binding function, and a repeat or part of a combination of new binding sites. The purpose of this experiment is to verify the above speculation.
According to the literatures, we established the experimental model of bacterial invasion of 293 cells, and through this model to find the effective strains of Staphylococcus aureus invasion 04018 as experimental strains. Using the method of genetic engineering, cloning gene fragment of FnBPA protein D_1 C 04018 strain and D_2 N end peptide in Escherichia coli. The expression of GST and the peptide (FnBPA-D1c/D2n) fusion protein.ELISA fusion protein in the experiment confirmed that the target peptide is capable of binding to Fn cells, bacterial invasion experiments confirmed that the target peptide fusion protein can inhibit Staphylococcus aureus invasion of 293 cells, 50% protein inhibitor was about 20 g/ml, equivalent to 2.5 g/ml peptide.
Through this study, we found FnBPA protein of Staphylococcus aureus and fibronectin binding new sites, suggesting that FnBPA protein D domain or in combination with smaller units, or there are more Fn binding sites, 3-4 rather than forecast, and provide a basis for the application of research inhibition of Staphylococcus aureus invasion.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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