| 摘要: |
| 变形假单胞菌（Pseudomonas plecoglossicida）是大黄鱼内脏白点病的主要致病菌。核酸适配体，因其亲和力高、特异性好等多个优点，在微生物检测等多个领域都有较好的应用前景。以变形假单胞菌为靶目标，采用SELEX筛选方法，从高频序列中筛选获得了变形假单胞菌的5个核酸适配体，测定了这些核酸适配体对变形假单胞菌的亲和特异性及其亲和常数（Kd）和最大亲和力（Am），并对其二级结构进行了分析研究。结果表明，这5个核酸适配体对变形假单胞菌的亲和力是其他非目标菌（嗜水气单胞菌、溶藻弧菌、哈维氏弧菌、大肠杆菌、迟钝爱德华氏菌、铜绿假单胞菌）的10倍以上，体现出了较好的亲和特异性。测定了5个核酸适配体（#1、#2、#3、#4、#5）的Kd和Am，相应的Kd值分别为（25.77±6.11）、（7.14±1.86）、（19.59±3.01）、（83.22±22.45）、（34.31±8.76） nmol/L，Am值分别为（789.23±38.46）、（1392.27±58.03）、（3012.50±106.19）、（1457.99±171.32）、（1070.91±70.16） nmol/L。二级结构模拟发现核酸适配体对靶标的结合能力可能主要与其二级结构中的大中型环有关。相关研究对于变形假单胞菌的快速检测及其病害防控都具有重要意义。 |
| 关键词: 变形假单胞菌 核酸适配体 亲和常数 最大亲和力 二级结构 |
| DOI：10.11693/hyhz20220700194 |
| 分类号:Q939;S917.1 |
| 基金项目:福建省自然科学基金项目，2021J01823号；鳗鲡现代产业技术教育部工程研究中心开放基金项目，RE202205号，RE202308号；福建省特种水产配合饲料重点实验室开放课题项目，TMKJZ1909号。 |
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| SELECTION OF APTAMERS OF PSEUDOMONAS PLECOGLOSSICIDA BY SELEX |
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LIN Xiao-Jun1,2, XU Jing1, ZHENG Jiang1,2, YAN Qing-Pi1, GAO De-Ji1, FAN Yun-Ting1, HUANG Jiang-Yuan1
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1.Fisheries College of Jimei University, Xiamen 361021, China;2.Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Xiamen 361021, China
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| Abstract: |
| Pseudomonas plecoglossicida is the main pathogen of visceral white spot disease in large yellow croaker. Aptamer shows good application prospects in microbial detection due to many advantages such as high affinity and good specificity. Five aptamers of P. plecoglossicida were selected from the high-frequency sequences by SELEX. The affinities of the five aptamers towards P. plecoglossicida were more than 10 times those of the non-target bacteria (Aeromonas hydrophila, Vibrio alginolyticus, Vibrio harveyi, Escherichia coli, Edwardsiella tarda and Pseudomonas aeruginosa), which proved that the aptamers had good affinities and specificities. The affinity constants (Kd) and maximum affinities (Am) of the five aptamers (#1, #2, #3, #4, #5) were determined, and their Kd were (25.77±6.11), (7.14±1.86), (19.59±3.01), (83.22±22.45), (34.31±8.76) nmol/L, respectively, and their Am were (789.23±38.46), (1 392.27±58.03), (3 012.50±106.19), (1 457.99±171.32), and (1 070.91±70.16) nmol/L, respectively. Finally, the secondary structures of the aptamers were analyzed. It was found that the binding ability of aptamer with its target was related mainly to the large and medium rings in the secondary structure. The relative researches shall have important significance for the rapid detection of P. plecoglossicida and the control of disease caused by the pathogen. |
| Key words: Pseudomonas plecoglossicida aptamer affinity constant maximum affinity secondary structure |
