禽病原性大肠杆菌O2分离株外膜蛋白OmpT纯化与结晶条件的初步研究

发布时间：2018-10-12 10:11

【摘要】：禽大肠杆菌病是由禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)引起禽类的一种复杂的多系统综合征的总称。禽致病性大肠杆菌病目前已成为鸡群中非常严重的一种细菌病,大肠杆菌败血症、心包炎、肝周炎、气囊炎等是大肠杆菌病在临床上的主要表现特征,并常常伴随不同程度的死亡率,给养殖业造成严重的经济损失,同时越来越多的证据表明APEC具有人兽共患病的潜能。外膜蛋白T(Outer membrane protein T,OmpT)定位于大肠杆菌外膜,是具有高度底物特异性的蛋白水解酶,属于革兰氏阴性菌外膜蛋白家族。APEC中的OmpT蛋白由位于染色质的ompT基因(c-ompT)和位于质粒中的ompT基因(p-ompT)分别编码,但在行使功能方面存在一定差异。通过基因的序列比对可以发现位于质粒pAPEC-O2-ColV中的ompT基因(p-ompT)与位于染色质的ompT基因(c-ompT)同源性为79%。本研究旨在探讨p-OmpT的结构和功能之间的关系,了解c-OmpT和p-OmpT的结构差异,从而阐明c-OmpT和p-OmpT之间功能上的差异问题,为了解APEC的致病机理和探求特异性防治措施奠定基础。通过同时构建含有c-ompT或p-ompT基因的重组表达载体,并分别转化到工程宿主菌BL21(DE3)中进行诱导表达。鱼精蛋白抑制试验显示,含有p-ompT基因的细菌生长受到抑制,而含有c-ompT的细菌则生长不受影响。为了进一步研究p-OmpT的结构和功能之间的关系。本实验以禽致病性大肠杆菌E058的基因组为DNA模板,PCR扩增出p-ompT基因片段,并且连接在表达载体上,构建成重组表达质粒。然后在大肠杆菌BL21(DE3)中进行了异源表达。由于诱导表达过程中形成无蛋白活性的包涵体,需要经过变性和复性,以恢复蛋白生物活性,然后利用离子交换和凝胶过滤层析进行蛋白纯化,得到高纯度且聚合状态单一的p-OmpT蛋白样品。将纯化出来的蛋白利用气相扩散坐滴法进行蛋白结晶条件筛选,并对结晶条件进行优化。我们获得了 p-OmpT的蛋白晶体,并对其进行X-射线的衍射,收集数据分析。为p-OmpT晶体的进一步优化和获得优质蛋白晶体打下基础。根据大肠杆菌OmpT能够降解抗菌肽的特性来筛选和改造新型抗菌肽,为开发抗OmpT蛋白酶水解新抗菌肽序列提供新的思路。
[Abstract]:Avian Escherichia coli (E. coli) is a complex multisystem syndrome caused by avian pathogenic Escherichia coli (avian pathogenic Escherichia coli,APEC). Avian pathogenic Escherichia coli disease has become a very serious bacterial disease in chickens. E. coli septicemia, pericarditis, pericarditis and balloon inflammation are the main clinical manifestations of Escherichia coli. It is often accompanied by different degrees of mortality, causing serious economic losses to the aquaculture industry, and more evidence shows that APEC has the potential of zoonosis. The outer membrane protein (T (Outer membrane protein Tory OmpT) is located in the outer membrane of Escherichia coli and is a highly substrate-specific proteolytic enzyme. The OmpT protein in APEC is encoded by ompT gene (c-ompT) located in chromatin and ompT gene (p-ompT) in plasmid respectively. The homology of ompT gene (p-ompT) in plasmid pAPEC-O2-ColV and ompT gene (c-ompT) in chromatin was found to be 79% by sequence alignment. The purpose of this study was to explore the relationship between the structure and function of p-OmpT, to understand the structural differences between c-OmpT and p-OmpT, to clarify the functional differences between c-OmpT and p-OmpT, and to lay a foundation for understanding the pathogenesis of APEC and exploring specific preventive and therapeutic measures. The recombinant expression vector containing c-ompT or p-ompT gene was constructed at the same time and transformed into BL21 (DE3) to induce expression. Protamine inhibition test showed that the growth of bacteria containing p-ompT gene was inhibited, but the growth of bacteria containing c-ompT was not affected. In order to further study the relationship between the structure and function of p-OmpT. In this experiment, the genome of avian pathogenic Escherichia coli E058 was used as the DNA template. The p-ompT gene fragment was amplified by PCR and ligated into the expression vector to construct the recombinant expression plasmid. Then heterologous expression was carried out in Escherichia coli BL21 (DE3). In order to recover the biological activity of the protein, the inclusion bodies with no protein activity should be denatured and renatured during the induction and expression, and then purified by ion exchange and gel filtration chromatography. P-OmpT protein samples with high purity and polymerization state were obtained. The purified protein was selected by gas phase diffusion drop method and the crystallization conditions were optimized. The protein crystals of p-OmpT were obtained and analyzed by X-ray diffraction. It lays the foundation for the further optimization of p-OmpT crystal and the obtaining of high quality protein crystal. According to the characteristic that Escherichia coli OmpT can degrade antimicrobial peptides, new antimicrobial peptides can be selected and modified, which provides a new idea for the development of new antimicrobial peptides hydrolyzed by anti OmpT protease.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.612

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