【作者】 汤学敏； 郑江； 鄢庆枇； 黎中宝；

【Author】 TANG Xue-min, ZHENG Jiang *, YAN Qing-pi, Li Zhong-bao (Fisheries College of Jimei University, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Xiamen 361021, China.)

【机构】 集美大学水产学院福建省高校水产科学技术与食品安全重点实验室；

【摘要】 利用SELEX技术筛选到的适配子已经成功应用于人类药物筛选和疾病治疗,但在水产中的应用还处于起步阶段。本研究以灭活的溶藻弧菌(Vibrioal ginolyticus)为靶目标,利用SELEX技术,经过15轮的筛选,其中每隔3轮分别用迟钝爱德华氏菌(Edwardsiella tarda)、嗜水气单胞菌(Aeromonas hydrophila)、哈维氏弧菌(Vibrio harveyi)进行反筛。随后进行了ssDNA富集文库与灭活溶藻弧菌的亲和力、亲和常数的测定。将最后一轮的PCR产物送上海美吉生物医药科技有限公司进行克隆测序,测序结果用DNAMAN和RNAstructure5.3分别进行一级和二级结构分析。研究结果表明:经过15轮筛选,随机ssDNA文库与灭活溶藻弧菌的亲和力从0.02507上升至0.4516,上升了18倍;15轮随机ssDNA富集文库的亲和常数为:Kd=27.5±9.2nM。根据软件分析,我们将克隆测序的53条序列去重后整理成32条,并按一级结构将其分成9个家族,其中的8个有共同的保守序列。这32条序列中有6条序列具有较高的重复率,我们初步认为其具有进一步研究的价值。更多还原

【Abstract】 Aptamers against inactive Vibrio alginolyticus were selected from a 82nt ssDNA random library by systematic evolution of ligands by exponential enrichment (SELEX). In order to obtain the anti-inactive Vibrio alginolyticus aptamers with high affinity, we add the negative selection with Edwardsiella tarda, Aeromonas hydrophila, Vibrio harveyi every three selection rounds. After 15 rounds of selection and amplification, the affinity of aptamers in the last enriched pool increased by about 18 times. The last enriched pool was also proved to have good specificity to its target, inactive V. alginolyticus, and its Kd was equal to 27.5 ± 9.2 nM. The PCR products of the last round was given to Shanghai Majorbio Bio-pharm Technology Co.,Ltd for cloning sequencing. After cloning and sequencing, the primary and secondary structures of sequences were analyzed by DANMAN and RNA structure 5.3 software. The whole fifty-three clone sequences were reorganized into thirty-two by eluting the identical sequences, and which were further divided into nine families. Eight of them have the common conserved sequence. Among the thirty-two sequences, there were six sequences (with high repetition rate) were thought to be the specific to V. Alginolyticus.更多还原