鼠伤寒沙门氏菌适配体的筛选及应用研究

发布时间：2018-01-03 14:41

本文关键词：鼠伤寒沙门氏菌适配体的筛选及应用研究　出处：《湖南师范大学》2014年硕士论文　论文类型：学位论文

【摘要】：沙门氏菌病是公共卫生学上最有重要意义的的人畜共患病之一,其病原沙门氏菌属于肠杆菌科,是一大群寄生于人类和动物肠道内,抗原构造和生化反应相类似的革兰氏阴性杆菌的统称。尤其以鼠伤寒沙门氏菌为代表的病原菌在食品安全、环境监测、疾病预防等方面造成了巨大的威胁,对人体健康造成了很大危害。如何实现沙门氏菌的快速检测成为当前研究的热点问题。适配体是通过指数富集的配体系统进化技术(SELEX)筛选得到的一段短的单链DNA或RNA序列,它能够与相应的靶标分子如金属离子、有机小分子药物、蛋白质乃至整个细胞进行高亲和力和强特异性的结合。由于适配体几乎可与每一个目标分子进行化学修饰,在生物分子的分析检测和治疗诊断等方面开创了一个新的局面。石墨烯是一种由纯碳原子紧密堆积成单层蜂窝网状结构的新型二维纳米材料。由于具有非常大的比表面积、优良的导热性和导电性、使得石墨烯在诸多领域都得到了广泛应用。氧化石墨烯是石墨烯的一种重要衍生物,表面大量存在的含氧基团能够增加其亲和性及水溶性。近年来,通过利用氧化石墨烯良好的生物相容性、高的荧光猝灭效率以及大的比表面积等优点,相继开发了各种荧光生物传感器。本论文以鼠伤寒沙门氏菌特异性识别、灵敏检测这一热点问题为切入点,主要开展了以下两个方面的研究工作：一、SELEX筛选鼠伤寒沙门氏菌适配体方法的建立及适配体克隆、测序、鉴定及分析以鼠伤寒沙门氏菌(S. typhimurium)为靶标,利用体外细胞SELEX筛选技术,构建了全长为78个碱基,中间含35个碱基随机序列的单链核苷酸文库,通过11轮反复常规筛选以及以肠炎、猪霍乱、甲型副伤寒和亚利桑那沙门氏菌为靶标的4轮消减筛选,初步筛选到一组能特异性结合S. typhimurium的核酸适配体群。对最后一轮筛选得到的核酸适配体的PCR产物进行克隆测序,获得了39条适配体序列。随后对它们进行一级结构的同源性分析及二级结构的模拟分析,最终获得了6条对S. typhimurium亲和力高、特异性好的序列。利用荧光分析法对其亲和力进行测定,Kd值均为0～100nM,其中Apt6的亲和力最高,Kd值仅为30±4nM。随后通过选择特异性实验分析表明,适配体与S. typhimurium的结合具有很强的特异性且对同种属的沙门氏菌不会有交叉结合。二、构建基于纳米材料氧化石墨烯的荧光适配体传感器利用氧化石墨烯荧光猝灭剂的性质和高特异性适配体,构建了快速、简单、高灵敏的荧光适配体传感器。在没有靶标S. typhimurium时,荧光标记的适配体会自动吸附到氧化石墨烯表面,适配体与氧化石墨烯之间发生荧光能量共振转移(FRET),适配体的荧光由于能量转移被迅速猝灭；当加入靶标菌后,适配体会与靶标形成特殊的空间结构,从而使适配体从氧化石墨烯表面释放出来,荧光恢复。结果表明这种传感器的检测下限为102cfu/mL,并在103—108cfu/mL浓度之间呈线性关系,同时有很高的选择性。因此,这种基于氧化石墨烯的荧光适配体传感器可以有效的用于病原菌的快速检测。
[Abstract]:Salmonellosis is one of the most important public health is the zoonotic disease of the pathogenic Salmonella bacteria belonging to the Enterobacteriaceae, is a large group of parasites in the intestinal tract of human and animal, referred to as the antigenic structure and biochemical reactions similar to gram negative bacteria. Especially the pathogens in Salmonella typhimurium as a representative in food safety, environmental monitoring, disease prevention and other aspects caused a huge threat, causing great harm to human health. How to achieve the rapid detection of Salmonella has become a hot issue in current research.
Aptamers are through the evolution of ligands by exponential enrichment system (SELEX) screened a short single stranded DNA or RNA sequence, it can with the corresponding target molecules such as metal ions, small organic molecules, and the whole cell binding protein with high affinity and high specificity. The aptamer almost by chemical modification and each target molecule in biomolecular detection and analysis of diagnosis and treatment so as to create a new situation.
Graphene is a pure carbon atoms tightly packed into a two-dimensional model of nano materials. Due to single-layer cellular network structure has a very large surface area, thermal conductivity and excellent conductivity of graphene, which has been widely applied in many fields. The graphene oxide is an important derivative of Shi Moxi, oxygen there are a large number of surface groups can increase their affinity and solubility in water. In recent years, through the use of graphene oxide with good biocompatibility, fluorescence quenching of high efficiency and large surface area and so on, have developed a variety of fluorescence biosensor.
In this paper, the specific identification of Salmonella typhimurium, sensitive detection of this hot issue as the breakthrough point, mainly carried out the following two aspects of the research work:
Establishment of a SELEX screening method for Salmonella typhimurium and its aptamer cloning, sequencing, identification and analysis
Salmonella typhimurium (S. typhimurium) as the target, using in vitro SELEX screening technology, constructed a full-length 78 BP single nucleotide intermediate library containing 35 BP random sequence, through 11 rounds of repeated routine screening as well as enteritis, hog cholera, Salmonella paratyphi A and Alexander Sangnashamenshi bacteria as the targets of the 4 round subtractive screening, preliminary screening to a set of specific binding S. typhimurium nucleic acid aptamer group. PCR products of the last round of nucleic acid aptamers were cloned and sequenced, obtained 39 aptamer sequences. Then simulate the homology analysis of primary structure and secondary structure of two of them. Won the 6 S. typhimurium of high affinity and specificity. The sequence of their affinity was determined by fluorescence analysis method, Kd value was 0 ~ 100nM, which Apt6 the highest affinity, Kd value is only 30 4N. M. followed by specific experimental analysis showed that the combination of aptamers and S. typhimurium was highly specific and had no cross linking to Salmonella belonging to the same genus.
Two, a fluorescent aptamer sensor based on nanomaterial graphene oxide
By using properties of graphene oxide fluorescence quenching and high specificity of aptamer constructs, rapid, simple, high sensitive fluorescent aptamer sensor. In the absence of target S. typhimurium, fluorescence labeled adaptation of automatic adsorption onto graphene oxide and graphene oxide between the aptamer and fluorescence resonance energy transfer (FRET), aptamer fluorescence energy transfer due to rapid quenching; when added to the target bacteria after adaptation of formation of special structures and targets, so that the aptamer released from the surface of graphene oxide, fluorescence recovery. The results showed that the detection limit of the sensor is 102cfu/mL, and a linear relationship between the 103 108cfu/mL concentration, and has very high selectivity. Therefore, the fluorescence of graphene oxide based on aptamer sensor can be used for rapid detection of pathogenic bacteria effectively.

【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R378

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