哈维氏弧菌与溶藻弧菌共有核酸适配体的筛选及鉴定

引用本文: 刘慧敏, 郝聚敏, 许净, 鄢庆枇, 高晗, 郑江. 哈维氏弧菌与溶藻弧菌共有核酸适配体的筛选及鉴定[J]. 分析化学, 2020, 48(5): 623-631. doi: 10.19756/j.issn.0253-3820.191663 shu

Citation: LIU Hui-Min, HAO Ju-Min, XU Jing, YAN Qing-Pi, GAO Han, ZHENG Jiang. Selection and Identification of Common Aptamers Against Both Vibrio Harveyi and Vibrio Alginolyticus[J]. Chinese Journal of Analytical Chemistry, 2020, 48(5): 623-631. doi: 10.19756/j.issn.0253-3820.191663 shu

哈维氏弧菌与溶藻弧菌共有核酸适配体的筛选及鉴定

刘慧敏 ^1,2, ,
郝聚敏 ^1, ,
许净 ^1, ,
鄢庆枇 ^1, ,
高晗 ^1, ,
郑江 ^1,

1.
鳗鲡现代产业技术教育部工程研究中心, 福建省水产生物育种与健康养殖工程研究中心, 集美大学水产学院, 厦门 361021;
2.
福建省特种水产配合饲料重点实验室, 福建天马科技集团股份有限公司, 福清 350308
基金项目:
本文系福建省自然科学基金项目（No.2018J01455）、鳗鲡现代产业技术教育部工程研究中心开放基金项目（No.RE201808）、福建省水产生物育种与健康养殖工程研究中心开放基金课题项目（No.DF201901）和福建省特种水产配合饲料重点实验室开放课题项目（Mo.TMKJZ1909）资助

摘要: 共有位点是指不同微生物上结构相同或相似的位点，有些共有位点是同种或同属微生物所共有的，可以作为微生物种属鉴定的标记位点。因此，筛选这些共有位点的共有核酸适配体，不仅有助于对这些微生物进行总量分析，对于相关微生物的种属鉴定也具有重要意义。本研究以哈维氏弧菌（Vibrio harveyi）和溶藻弧菌（Vibrio alginolyticus）为靶目标，筛选这两种弧菌共有位点的共有核酸适配体。经过8轮筛选，适配体富集库的亲和力提高了24.1倍，同源性分析表明，适配体富集库呈明显的收敛性，表现了较好的筛选效率。对2个核酸适配体C14和C22的亲和性和特异性进行了研究，结果表明，这两个核酸适配体对哈维氏弧菌和溶藻弧菌都有较好的亲和性和特异性，但对哈维氏弧菌的亲和力要显著高于溶藻弧菌，而对非弧菌属的嗜水气单胞菌（Aeromonas hydrophila）和迟钝爱德华氏菌（Edwardsiella tarda）的亲和力较弱。C14对哈维氏弧菌和溶藻弧菌的亲和常数（K_d）分别为55.76和82.88 nmol/L，C22对哈维氏弧菌和溶藻弧菌的亲和常数分别为33.97和43.95 nmol/L。二级结构分析显示，两个核酸适配体都以茎环结构为主，其中C22的3'端结构比C14更复杂。荧光显微镜下，与核酸适配体孵育的哈维氏弧菌、溶藻弧菌、嗜水气单胞菌、迟钝爱德华氏菌和大肠杆菌（Escherichia coli）中，只有哈维氏弧菌和溶藻弧菌呈现出明显亮黄色的荧光，其它3种菌则无显著荧光，进一步证明了两个核酸适配体对这两种弧菌有较好的亲和性和特异性。本研究为这两种弧菌的识别检测及水产病原弧菌的种属鉴定奠定了基础。

关键词:

English

Selection and Identification of Common Aptamers Against Both Vibrio Harveyi and Vibrio Alginolyticus

1.
Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Engineering Research Center of Aquaculture Breeding and Healthy of Fujian, Fisheries College of Jimei University, Xiamen 361021, China;
2.
Fujian Province Key Laboratory of Special Aquatic Formula Feed, Fujian Tianma Science and Technology Group Co. Ltd., Fuqing 350308, China
Received Date: 11 November 2019
Revised Date: 16 December 2019
Fund Project:
This work was supported by the Natural Science Foundation of Fujian Province, China (No. 2018J01455), the Open Fund of Engineering Research Center of the Modern Technology for Eel Industry of Ministry of Education of China (No. RE201808), the Open Research Fund Program of Fujian Engineering Research Center of Aquatic Breeding and Healthy Aquaculture (No. DF201901), and the Open Research Fund from Fujian Province Key Laboratory of Special Aquatic Formula Feed (Fujian Tianma Science and Technology Group Co., Ltd.) (No. TMKJZ1909).

Abstract: Common sites are the sites of same or similar structure on different microorganisms. Some of the common sites can be shared by same genus of microorganisms, which can be used as the marker for the identification of the microorganism. Therefore, selection of common aptamers against these common sites is not only helpful to the analysis of the microorganisms, but also of great significance for the identification of the relative microorganisms. In the work, the common aptamers against both Vibiro harveyi and Vibiro alginolyticus were selected after 8 rounds of selection. The affinity of the enrichment pool of the aptamers increased by 24.1 times, and the analysis based on homologous tree showed that the sequences of the aptamer enriched pool showed obvious convergence, which showed a good selection efficiency. Two aptamers (C14 and C22) were proved for good affinity and specificity towards both V. harveyi and V. alginolyticus. The affinities between the two aptamers and V. harveyi were significantly stronger than those between the aptamers and V. alginolyticus. The affinity constants (K _d) of the aptamer C14 against V. harveyi and V. alginolyticus were 55.76 and 82.88 nmol/L respectively. And the affinity constants of the aptamer C22 against V. harveyi and V. alginolyticus were 33.97 and 43.95 nmol/L respectively. Secondary structures of the two aptamers were mainly composed of stems and loops. The 3'-end structure of aptamer C22 seemed to be more complex than that of aptamer C14. Significant fluorescence could be found only on V. harveyi and V. alginolyticus after the aptamers marked with fluorescence group were mixed with V. harveyi, V. alginolyticus, Aeromonas hydrophila, Edwardsiella tarda and Escherichia coli, which showed that the two aptamers had good affinity and specificity towards the two target bacteria. This studies laid a foundation for the identification and detection of the two microorganisms as well as the aquatic pathogenic vibrios.

Key words:

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哈维氏弧菌与溶藻弧菌共有核酸适配体的筛选及鉴定

English

Selection and Identification of Common Aptamers Against Both Vibrio Harveyi and Vibrio Alginolyticus

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