哈维氏弧菌适配子的SELEX筛选及其亲和特异性研究

哈维氏弧菌是水产养殖中的重要条件致病菌,对其进行快速、准确地检测和鉴定是相关病害防治的基础和关键.适配子具有亲和力高、特异性强、稳定性好等优点,在微生物的检测和鉴定方面呈现出广泛的应用前景.本研究以哈维氏弧菌为靶目标,采用SELEX技术,即指数级富集配体的系统进化技术,筛选其特异性适配子.经15轮筛选后,随机ssDNA文库的亲和力从3.51上升到58.95,提高了15.8倍.筛选出的适配子富集库经克隆、测序后得到52条不同序列,根据同源性将这些序列分成8个家族,其中第1和第2家族的适配子数量最多,超过总数的50%.通过深入分析,筛选出6个对哈维氏弧菌有显著亲和特异性(P0.01)的高频适配子,其中5个高频适配子(S1、S25、S26、S27、S35)对哈维氏弧菌有较高的亲和力,相应的亲和常数Kd值分别为(32.6±7.1)、(45.3±10.1)、(24.7±5.8)、(34.8±5.6)、(12.9±4.0)nmol/L.本文还对高频适配子的产生机制及其应用价值进行了探讨.本文首次筛选出了对哈维氏弧菌具有较高亲和特异性的适配子,为后续利用适配子进行哈维氏弧菌的检测和鉴定奠定了基础.

Selection and Characterization of Aptamers Against Vibrio harveyi by SELEX

The disease caused by the opportunistic pathogen Vibrio harveyi is a serious infection which brings a heavy damage to aquaculture. Accurate and rapid detection of the microorganism is the first and necessary step to control the disease. Aptamers have a good potential application in the detection and identification of microorganisms because of their strong affinity, high specificity and good stability. In the present paper, aptamers targeting to V. harveyi were selected by the technique of systematic evolution of ligands by exponential enrichment (SELEX), and their affinities and specificities to V. harveyi were also evaluated. After 15 rounds of selection, the affinity of the aptamers in the enriched pool increased to 58.95, which was 16.8 times of that (3.51) in the original pool. After cloning and sequencing, 52 supposed aptamers were obtained from the enriched pool. These aptamers were divided into 8 families based on the homology analysis. Over 50% of these aptamers in the first and the second families shared higher homology, suggesting the selection was convergent and efficient. Six high-frequency aptamers were also found to have significant affinities and specificities to V. harveyi (P < 0.01). Five of them (S1,S25, S26, S27, S35) were proved to have higher affinities, and their affinity constants (K_d) were as followed: (32.6±7.1), (45.3±10.1), (24.7±5.8), (34.8±5.6) and (12.9±4.0) nmol/L, respectively. The production mechanism and the application values of high-frequency aptamers were also discussed. The present paper was the first report of aptamers with high affinity and specificity to the target V. harveyi, which would lay the foundation for the detection of the pathogenic microorganism based on aptamers.