体外筛选铜绿假单胞菌适体的研究及初步应用

发布时间：2018-01-17 22:06

本文关键词：体外筛选铜绿假单胞菌适体的研究及初步应用　出处：《福建医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 【目的】体外筛选出高亲和力高特异性针对铜绿假单胞菌的适体群;测定适体与铜绿假单胞菌的结合活性,确定其结合率及特异性。【方法】 1.在体外构建两端为固定序列,中间为35个碱基随机区,全长78个碱基的寡核苷酸库,以纯培养的铜绿假单胞菌为靶标,将二者混合,经过九轮的吸附-漂洗-洗脱-扩增的SELEX过程,筛选针对铜绿假单胞菌高亲和力高特异性的适体,并从第五轮起每隔一轮分别用荧光假单胞、斯氏假单胞和产碱假单胞菌进行反筛,以提高筛选的特异性。每隔一轮利用地高辛-抗地高辛抗体-碱性磷酸酶系统测定ssDNA富集库与铜绿假单胞菌的结合率。 2.将第九轮的筛选产物——寡核苷酸片断进行克隆、测序并用相关软件分析其一级和二级结构。 3.用微量荧光分光光度计检测荧光素标记适体与铜绿假单胞菌的结合率,在5个测序克隆中选出结合率最高的一条适体,分别与铜绿假单胞、门多萨假单胞、斯氏假单胞和产碱假单胞菌做结合反应,以初步检测其特异性。【结果】 1.经过九轮的筛选和扩增,使得针对铜绿假单胞菌的适体逐步得到富集,每轮的ssDNA文库与铜绿假单胞菌的结合率呈上升趋势。将每轮的筛选产物用标有地高辛的引物扩增后,与铜绿假单胞菌进行结合实验,地高辛-抗地高辛抗体-碱性磷酸酶显色系统提示,第九轮筛选获得的富集库与铜绿假单胞菌结合后显色的OD值较第一轮的OD值提高将近3.2倍。 2.在200个克隆子中随机选取27个克隆进行了测序,将测序结果按碱基多少分成A、B两群,A群19条与预期片段大小一致;B群为中间随机区少于35个碱基的片段。将测序适体以计算机软件分析其保守序列及二级结构。二级结构预测以茎-环结构为主,这可能是适体与靶标作用的结构基础。其中适体序列完全相同的共有6条,此序列随机区富含20个G,可能为G四聚体的形成基础。此外,有些序列是单个序列,与任何家族无同源。某些一级结构无同源性的适体克隆二级结构模拟却极为相似,如2-26、2-61、2-119号克隆,2-24、2-46号克隆,2-12、2-135克隆序列等。 3.结合实验显示,2-61号克隆适体与铜绿假单胞菌的结合率最高,为15.3%,2-106号克隆适体与铜绿假单胞菌的结合率最低,为5.6%;2-61号克隆适体与铜绿假单胞、斯氏假单胞菌、产碱假单胞菌和门多萨假单胞,进行结合反应后,测得F值分别为16.6%、3.8%、2.6%和4.5%。【结论】本研究在国内首次利用反向-指数富集配体系统进化(SELEX)技术筛选出针对铜绿假单胞菌的ssDNA适体,并对筛选出的适体进行了结合率及特异性的检测,初步确定了结合率较高特异性较好的适体分子,为荧光基团标记的适体快速鉴定铜绿假单胞菌检测方法的建立奠定了基础。
[Abstract]:[purpose] In vitro, the aptamer groups with high affinity and specificity against Pseudomonas aeruginosa were screened, and the binding activity between aptamer and Pseudomonas aeruginosa was determined, and the binding rate and specificity of the aptamer and Pseudomonas aeruginosa were determined. [methods] 1. A 78 base oligonucleotide library with a fixed sequence at both ends and 35 base random regions in the middle was constructed in vitro. The two oligonucleotides were mixed with pure cultured Pseudomonas aeruginosa as the target. After nine rounds of SELEX process of adsorption, rinsing, elution and amplification, the aptamers with high affinity and high specificity for Pseudomonas aeruginosa were screened, and fluorescent pseudomonas were used every other round from 5th rounds. Pseudomonas sp. And Pseudomonas sp. In order to improve the specificity of screening, the binding rate of ssDNA enrichment library with Pseudomonas aeruginosa was determined by using digoxigenin-anti-digoxin antibody alkaline phosphatase system every other round. 2. The oligonucleotide fragment of 9th rounds of screening product was cloned and sequenced and its primary and secondary structures were analyzed by software. 3. The binding rate of fluorescein labeled aptamer to Pseudomonas aeruginosa was detected by microfluorimetric spectrophotometer. One aptamer with the highest binding rate was selected among the five sequencing clones, and the aptamer was associated with the Pseudomonas aeruginosa (Pseudomonas aeruginosa). Mendoza pseudomonas, Stemont's pseudomonas and Pseudomonas alcaligenes were combined to detect the specificity of Mendoza pseudomonas. [results] 1. After nine rounds of screening and amplification, the aptamer for Pseudomonas aeruginosa was gradually enriched. The binding rate of each ssDNA library to Pseudomonas aeruginosa was on the rise. The screening products were amplified with digoxigenin primers and then combined with Pseudomonas aeruginosa. The results of digoxigenin-anti-digoxin antibody alkaline phosphatase system suggested that the OD value of the enrichment library combined with Pseudomonas aeruginosa was 3.2 times higher than that of the first round. 2. Among the 200 clones, 27 clones were randomly selected and sequenced. According to the number of bases, the sequencing results were divided into two groups of Agna B and A groups, 19 of which were consistent with the expected size of the fragments. Group B was a fragment with less than 35 bases in the middle random region. The sequenced aptamer was analyzed by computer software for its conserved sequence and secondary structure. The secondary structure was predicted mainly by stem-loop structure. This may be the structural basis of the interaction between aptamer and target. There are 6 aptamers with identical aptamer sequences, and the random region of the aptamer sequence is rich in 20 Gs, which may be the basis for the formation of G tetramer. Some sequences are single sequences and have no homology with any family. The secondary structure simulation of some aptamer clones with no homology in some primary structures is very similar, such as clone 2-262-61C2-119. Cloning sequence of 2-24N 2-46, 2-12, 2-135, et al. 3. The binding rate between clone aptamer No. 2-61 and Pseudomonas aeruginosa was the highest, and the binding rate between clone aptamer No. 2-106 and Pseudomonas aeruginosa was the lowest (5.6B). After the binding reaction of aptamer No. 2-61 with Pseudomonas aeruginosa, Pseudomonas sp., Pseudomonas sp. And Pseudomonas monocytogenes Mendoza, the F values were 16. 6% and 3. 8%, respectively. 2.6% and 4.5. [conclusion] In this study, ssDNA aptamers against Pseudomonas aeruginosa were screened by reverse exponential enrichment ligand phylogeny (SELEX) technique for the first time in China. The binding rate and specificity of the selected aptamers were determined, and the aptamer molecules with higher binding rate and better specificity were preliminarily determined. The results laid a foundation for rapid identification of Pseudomonas aeruginosa by fluorescent group labeling.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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