| Aeromonas hydrophila and Edwardsiella tarda are the two common pathogens inaquaculture activities, and may cause aquaculture animals to be sick or dead, bringing economiclosses to the aquaculture industry and health risk to humans. SELEX (Systematic Evolution ofLigands by Exponential Enrichment), a combination of separation, filtering and PCR(polymerase chain reaction), has found the satisfactory aptamers right for the target throughsimulating the Darwin’s Theory of Revolution. The aim of this study is to develop a technologyvia SELEX to detect Aeromonas hydrophila and Edwardsiella tardra, and provided a newmethod to detect the two germs.In our research, a synthesized78nt single stranded DNA random library which is containing35nt random sequences between the invariant primers was subjected to12rounds of selectionagainst Aeromonas hydrophila and14rounds of selection against Edwardsiella tardra by SELEX.According to the Digoxigenin Anti-Digoxigenin systerm, we finally got the aptamer againstAeromonas hydrophila and Edwardsiella tardra with high specificity and affinity. After clonedand sequenced, the primary structure and secondary structure were studied.1: The affinity of A. hydrophila has been increased2.25times from0.32in the first round to0.72in the ninth round. This study has analyzed the39clones of A.hydrophila by using DNAstarsoftware to find out that the Conserved Sequence GGTGG exists in all the39sequences whichwere divided into two groups based on the consistency of the lengths--33were in consistencywith the original length, while6were not. The analysis of the secondary structures has shownthat the stem-loop structure exists in all the secondary structures and this may be the structure ofconfirmations of aptamers and ligands. To analyze the secondary structures of39aptamers ofA.hydrophila, we divided the aptamers into seven groups based on the difference locations of thestem loops. The detection of idiosyncrasy on it of A. hydrophila has indicated that the bindingrate of the best aptamer and A.hydrophila is0.763, which has improved the detected aptamer hasa strong idiosyncrasy to A. hydrophila.2: After14rounds of selection, the affinity absorbance of E. tardra was increased from0.632in the first round to1.799in the thirteenth round. By using DNAstar and DNAMANsoftware, the analysis on the primary structure and secondary structure of the20aptamers ofEdwardsiella tardra has shown that the primary structure mainly consists of the threeconservative sequences--GGTGGG, TTTTX and GGAGGX. We divided the aptamers into5groups based on the difference locations of the steam loops. The analysis showed that in the secondary structure, the loop of the original secondary structure was the binding site, and thestructure of the stems was steady. Therefore, as the main structures in the secondary structure arestem loops, the stem loops may be the key point to the binding of the aptamers. Meanwhile,some pocket structures existing in the secondary structure may be the binding sites.3: The comparison of the purification steps during the selection showed that the homologyof the primary sequences of aptamers selected by selective purification was higher than thoseselected by purification in every round. And sequences with a100%homology could be moreeasily acquired in the selective purification. The analysis on secondary structures showed that wecan divide the secondary structures of aptamers selected by purification in every round into5groups, while those selected by selective purification into7groups based on the locations of thestem loops, which indicated that excessive purification would decrease the richness of selectionof the aptamers and reduce the efficiency of selection.4: The study has sorted the idiocratic aptamers for A.hydrophila and E.tarda and testifiedthe idiosyncrasy of the aptamers in A.hydrophila. |
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