基于外膜蛋白OmpC识别的沙门氏菌噬菌体受体结合蛋白结合机制研究

Bacteriophage can infect host bacteria with a high specificity, which mainly depends on the specific recognition and binding of the receptors on the surface of host bacteria through the receptor binding protein (RBP). Therefore, RBP can be used as a novel molecular recognition probe for the detection of pathogenic bacteria. The binding domain of RBP is generally located at the C-terminus of RBP, and can also be used alone as a capture and detection probe for bacteria with advantages. This project intends to take the bacteriophage PS1706, which uses Salmonella typhimurium ATCC14028 as the host bacteria and has a broad host and strong lytic ability, as the research object. The binding domain sequence of RBP (RBPc gene) is obtained by whole-genome sequencing of the bacteriophage PS1706, and a phage surface display library of RBPc is constructed by random mutagenesis; RBPc with different binding characteristics are screened by using conserved outer membrane protein OmpC of Salmonella typhimurium ATCC14028 as the representative surface receptor of host. The mechanism of RBP binding to OmpC was revealed by computer-aided molecular design and site-directed mutagenesis technology. The present research will lay the foundation for studying the molecular mechanism of the interaction between RBP and host bacteria, and provide insights into the preparation of novel molecular recognition probes for pathogens.

噬菌体能高度特异性的侵染宿主细菌，这主要依赖于噬菌体受体结合蛋白（receptor binding protein，RBP）特异性识别并结合宿主菌表面的受体，因此，RBP可作为新型分子识别探针应用于病原菌检测。RBP的宿主结合功能域一般位于RBP的C端，也可单独作为检测细菌的识别探针，且更具优势。本项目拟将前期以鼠伤寒沙门氏菌ATCC14028为宿主菌筛选的具有宽宿主谱和较强裂解能力的噬菌体PS1706作为研究对象，经全基因组测序获得其RBP结合功能域序列（RBPc基因），通过随机突变构建RBPc噬菌体表面展示文库；以ATCC14028保守性外膜蛋白OmpC为宿主菌代表性表面受体，筛选不同结合能力的RBPc；结合计算机辅助分子设计和特定位点突变技术定向改造RBPc，揭示RBP结合OmpC的机制，为研究RBP与宿主菌相互作用的分子机制奠定基础，也为制备新型病原菌分子识别探针提供技术支撑。

沙门氏菌作为一种常见的人畜共患病原菌，严重影响食品安全，开展沙门氏菌的防控和检测具有十分重要的意义。噬菌体的受体结合蛋白（receptor binding protein，RBP）对其识别宿主菌发挥着重要作用，因此RBP可作为沙门氏菌识别探针。沙门氏菌外膜蛋白OmpC是一种重要的表面抗原，本项目以沙门氏菌噬菌体为研究对象，挖掘与OmpC具有不同结合活性的RBP，分析最高结合活性RBP与OmpC亲和力，并基于同源模建和分子对接技术探讨RBP结合OmpC的机制。项目研究获得了结合力较高的RBP62，其与OmpC按1：1模式结合，且RBP62（200 μg/mL）与OmpC（150 μg/mL）相互作用亲和力为70.1 nM。RBP62可识别OmpC的多个位点，其关键作用氨基酸可能为Asn49、Phe59。本项目的研究结果为噬菌体-沙门氏菌识别机制研究提供了参考。