百日咳杆菌气管定植因子（TcfA）的功能分析

发布时间：2018-11-12 15:19

【摘要】：百日咳是由百日咳鲍特氏菌（以下简称百日咳杆菌）引起的一种呼吸道传染病，在婴幼儿当中较为常见。百日咳杆菌致病机理包括粘附、侵袭、产毒等一系列的过程。丝状血凝素（Filamentous hemagglutmin）、粘附素（Pertactin)、百日咳气管定植因子（Tracheal colonization factor; TcfA）等诸多膜表面蛋白参与到细菌的粘附过程。百日咳气管定植因子是百日咳杆菌膜表面蛋白，相关文献报道，推测该蛋白可能是百日咳杆菌的粘附分子，但对其粘附功能尚未进行系统的验证。因此本研究采用DNA重组技术，在大肠杆菌（BL21；DE3）内表达TcfA蛋白，并纯化重组TcfA蛋白。然后通过重组表达菌的体外细胞粘附实验、重组蛋白免疫荧光实验、竞争抑制实验证明TcfA蛋白的黏附作用。并在此基础上，就TcfA蛋白第317-319位RGD三联肽的功能进行验证。同时通过构建FHA-PRN-基因缺失株，探究TcfA蛋白在粘附过程中的重要性，并尝试找出TcfA蛋白与经典模型细胞HeLa相互作用的受体。 通过基因重组技术成功的表达了TcfA蛋白，并纯化出纯度达85%的重组蛋白，免疫印迹实验结果显示，重组蛋白能与疫苗生产菌株（CS菌）全菌体血清发生抗原抗体反应，说明重组蛋白具有良好的免疫反应性和抗原特异性；玻片凝集实验和V管凝集实验证明TcfA蛋白表达于大肠杆菌膜表面；荧光粘附试验证实表达有TcfA的大肠杆菌与含有空载体的大肠杆菌对经典模式细胞的粘附有明显的差异，CFU实验定量证实两者之间具有统计学差异。粘附ELISA实验从另一技术方法反映了两者之间具有统计学差异。重组蛋白的免疫荧光实验间接反映了TcfA蛋白与细胞受体的相互作用，重组蛋白的竞争抑制实验表明TcfA蛋白能竞争抑制重组TcfA大肠杆菌的粘附，并表现出蛋白浓度的依赖性。TcfA小鼠多抗封闭实验结果显示，封闭了重组大肠杆菌膜表面的TcfA蛋白，细菌的粘附率出现明显的下降，说明了TcfA蛋白的粘附的特异性（依赖性）。含RGD的短肽与含RGE的短肽的竞争抑制试验表明，TcfA蛋白可能通过其氨基酸位点上的RGD模序发挥作用。庆大霉素胞外杀伤实验是检测病原菌是否具有入侵宿主细胞能力的有效方法之一，该方法主要是检测粘附分子是否介导了病原菌的入侵。实验结果显示，TcfA蛋白并不介导重组大肠杆菌入侵HeLa细胞。FHA、PRN基因缺失株与抗TcfA小鼠多克隆抗体作用后，粘附力相比于FHA、PRN基因缺失株和CS野生株有较明显的下降，说明TcfA蛋白作为百日咳杆菌膜表面的一个粘附因子，，在细菌的粘附过程中发挥着重要的作用。 GST pull down实验初步证明Myosin-9蛋白可能是TcfA蛋白与HeLa细胞相互作用的受体，但需要进一步验证。
[Abstract]:Pertussis is a respiratory infectious disease caused by Bordetella pertussis (hereinafter referred to as pertussis bacillus). The pathogenic mechanism of pertussis bacillus includes a series of processes, such as adhesion, invasion, toxin production and so on. Many membrane surface proteins such as fibroin (Filamentous hemagglutmin), adhesin (Pertactin), pertussis tracheobronchial colonization factor (Tracheal colonization factor; TcfA) and so on are involved in the bacterial adhesion process. Pertussis trachea colonization factor is a pertussis bacillus membrane surface protein. It is speculated that this protein may be the adhesion molecule of pertussis bacillus, but its adhesion function has not been systematically verified. Therefore, TcfA protein was expressed in Escherichia coli (BL21;DE3) by DNA recombination technique, and the recombinant TcfA protein was purified. Then the adhesion of TcfA protein was proved by cell adhesion assay in vitro, immunofluorescence assay and competitive inhibition test. On this basis, the function of RGD tripeptide at position 317-319 of TcfA protein was verified. At the same time, by constructing the FHA-PRN- gene deletion strain, the importance of TcfA protein in the adhesion process was explored, and the receptor of HeLa interaction between TcfA protein and classical model cells was found. TcfA protein was successfully expressed by gene recombination technique, and the recombinant protein was purified with 85% purity. The results of Western blot showed that the recombinant protein could react with the whole cell serum of vaccine producing strain (CS). The results showed that the recombinant protein had good immunoreactivity and antigen specificity. Glass slide agglutination assay and V tube agglutination assay showed that TcfA protein was expressed on the surface of Escherichia coli membrane. Fluorescence adhesion test confirmed that there was significant difference between E. coli expressing TcfA and Escherichia coli containing empty vector on classical model cell adhesion. Quantitative CFU experiment confirmed that there was a statistical difference between them. Adhesion to ELISA test from another technical method reflects a statistical difference between the two. The immunofluorescence assay of the recombinant protein indirectly reflected the interaction between the TcfA protein and the cell receptor. The competitive inhibition test of the recombinant protein showed that the TcfA protein could compete to inhibit the adhesion of the recombinant TcfA Escherichia coli. The results of TcfA mouse polyclonal blocking test showed that the adhesion rate of the bacteria to TcfA protein on the surface of recombinant Escherichia coli was significantly decreased, indicating the specificity (dependence) of the adhesion of TcfA protein. The competitive inhibition tests of short peptides containing RGD and short peptides containing RGE showed that TcfA proteins may function through RGD motifs at their amino acid sites. The extracellular killing test of gentamicin is one of the effective methods to detect whether the pathogen has the ability to invade the host cells, and the main method is to detect whether the adhesion molecule mediates the invasion of the pathogen. The results showed that TcfA protein did not mediate the invasion of HeLa cells by recombinant Escherichia coli. The adhesion of FHA,PRN gene deletion strain to polyclonal antibody against TcfA mice was significantly lower than that of FHA,PRN gene deletion strain and CS wild strain. As an adhesion factor of pertussis bacillus membrane, TcfA protein plays an important role in the process of bacterial adhesion. GST pull down assay showed that Myosin-9 protein may be the receptor of the interaction between TcfA protein and HeLa cells, but it needs further verification.
【学位授予单位】：中国食品药品检定研究院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

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