【作者】 张纯；

【导师】 邓乐；

【作者基本信息】 湖南师范大学 ， 生物学·微生物学， 2020， 硕士

【摘要】 杀鲑气单胞菌（Aeromonas salmonicida）与嗜水气单胞菌（Aeromonas hydrophila）是海洋与湖泊生态系统中鱼类的主要致病菌,可引发鱼类患疖疮病和出血性败血症甚至死亡,同时也能通过水源、食物使人类患病。因此,建立快速检测水体中这两种致病菌的技术与方法对水产养殖及公共健康安全具有非常重要的意义。适配体是利用指数富集的配体系统进化技术（Systematic evolution of ligands by exponential enrichment,SELEX）获得的一段单链DNA（Single-stranded DNA,ssDNA）或RNA序列。适配体具有高特异性、高亲和力、稳定性强等优点,在疾病诊断、生物传感以及靶向治疗等领域得到了广泛应用。类石墨相氮化碳（Graphene-like carbon nitride,g-C₃N₄）是一种新型的高分子纳米材料,因其持续发光、无毒、优良的生物相容和吸附ssDNA等优势,在生物传感和生物成像等领域显示出巨大的应用潜力。本论文以杀鲑气单胞菌作为靶标,利用Cell-SELEX得到其特异性识别的适配体,结合g-C₃N₄的独特性质,构建了一种比值荧光纳米生物传感器,并用于水体中杀鲑气单胞菌的快速检测。同时,以嗜水气单胞菌鞭毛蛋白FlgE为靶标,通过SELEX筛选获得了高特异适配体。具体研究内容如下:（1）杀鲑气单胞菌适配体的筛选。利用Cell-SELEX筛选获得一个特异结合杀鲑气单胞菌的适配体群。经高通量测序及同源性分析,挑选了3条适配体进行结合性能的鉴定。其中,适配体A.s-2与杀鲑气单胞菌结合效果较好（Kd值为41.78±3.6 nM）,且具有较强的特异性。（2）基于类石墨相氮化碳的杀鲑气单胞菌比值荧光传感器的构建。该检测体系以捕获功能的适配体A.s-2连接DNA酶序列的核酸片段为探针,与g-C₃N₄纳米片非共价结合,构建了一种比值荧光纳米生物传感器。实验结果表明,该方法对杀鲑气单胞菌在10³～10⁷CFU/mL范围内的检测有良好的线性关系（R²=0.9655）,且检测下限为10³ CFU/mL;并成功应用于患病斑马鱼中杀鲑气单胞菌的检测,为鱼类疾病诊断提供了一个高效、快速的方法。（3）嗜水气单胞菌鞭毛蛋白FlgE适配体的筛选。以嗜水气单胞菌鞭毛蛋白Flg E为靶标,利用筛选试剂盒进行SELEX筛选,得到具有高特异性和高亲和力的适配体。首先,利用构建的pET-28a-flgE重组质粒导入受体细胞,对诱导表达的重组蛋白FlgE进行纯化、浓缩。然后,利用筛选试剂盒以重组蛋白FlgE为靶标进行SELEX筛选,获得一个适配体群。通过高通量测序、亲和力和特异性的分析,得到一条对嗜水气单胞菌具有高特异和高亲和力（Kd值为40.3±2.8 nM）的适配体A.h-1,为后续发展基于适配体的嗜水气单胞菌的检测传感器奠定了基础。更多还原

【Abstract】 Aeromonas salmonicida and A.hydrophila are the main pathogens of fish in marine and lake ecosystems,which can cause furunculosis,hemorrhagic septicemia and even death in fish,as well as human disease through water and food.Therefore,it is significant for aquaculture,public health and safety to establish techniques and methods for fast detection of these pathogens in water.Aptamer is a single-stranded DNA（ssDNA）or RNA sequence which was obtained by systematic evolution of ligands by exponential enrichment（SELEX）technique.Because of its advantages in high specificity,high affinity and strong stability,aptamer has been widely used in disease diagnosis,biosensor,targeted therapy and other fields.As a new type of polymer nano-materials,graphite-like carbon nitride（g-C₃N₄）has showed a great application potential in the fields of biosensor and biological imaging due to its advantages in continuous luminescence,non-toxicity,excellent biological compatibility and adsorption of ssDNA.In this thesis,a specific aptamer against A.salmonicida was successfully obtained by Cell-SELEX.Combined with the unique properties of g-C₃N₄,a ratio fluorescence nano-biosensor was established and applied to the rapid detection of A.salmonicida in water.At the same time,the aptamer with specific binding to A.hydrophila flagellin FlgE was also screened by SELEX.The main works are summarized as follows:（1）Screening the aptamer for A.salmonicida.A group of aptamers specifically binding to A.salmonicida were obtained by Cell-SELEX.After high-throughput sequencing and homology analysis,three aptamers sequences were selected to identify their binding properties.Among them,the aptamer A.s-2 had the highest binding effect（Kd=41.78±3.6 nM）and specificity combining A.salmonicida.（2）Construction of a ratio fluorescence sensor based on graphite-like carbon nitride for detection of A.salmonicida.A ratio fluorescence nano-biosensor was constructed of the nucleic acid fragments containing the capture function of aptamer A.s-2 linked to the DNAzyme sequences as probes,followed by non-covalent binding to g-C₃N₄ nanosheets.The results indicated that the sensing strategy had high sensitivity toward A.salmonicida in a range of 10³～10⁷ CFU/mL（R²=0.9655）with a detection limit of 10³ CFU/mL.Besides,the proposed method has been successfully applied to the detection of A.salmonicida in diseased zebrafish.Thus,the system may provide an efficient and rapid method for disease diagnosis in fish.（3）Selecting the aptamer for A.hydrophila flagellin FlgE.A group of aptamers specifically binding to A.hydrophila flagellin FlgE were obtained by the screening kit of SELEX.After recombinant plasmid pET-28a-flgE was transformed into competent cells,the induced recombinant protein FlgE was purified and concentrated.In addition,a group of aptamers with high specifically binding to the protein FlgE were selected by the screening kit of SELEX.Among them,the selected aptamer A.h-1 had the highest affinity（Kd=40.3±2.8 nM）and specificity toward A.hydrophila through high-throughput sequencing,affinity and specificity analysis.Therefore,the results laid the foundation for the subsequent development of aptamer-based sensor for the detection of A.hydrophila.更多还原