Selection And Analysis Of Aptamers Against Vibrio Harveyi By SELEX

Vibrio harveyi is an important conditional pathogenic bacteria in aquaculture and the control of the diseases caused by the bacteria is based on the rapid identification and detection of the microorganism. Aptamer has a great application prospect in detection because of many advantages such as high affinity and strong specificity.The present thesis explores Systematic Evolution of Ligands by Exponential Enrichment(SELEX) to select specific aptamers against Vibrio harveyi and inactivate Vibrio harveyi respectively, and the common aptamers against both Vibrio harveyi and Vibrio alginolyticus. The primary and secondary structure of these aptamers was analyzed by softwares DNAMAN and RNAstructure respectively. Affinity and specificity of part aptamers were verified and their dissociation constants were determined. In the end, a series of aptamers with high affinity and specificity were obtained.The research showed that, after 15 rounds selection targeting Vibrio harveyi, the affinity of the random ssDNA enriched library increased from 3.51 to 58.95, 16.8 times the affinity of the original library. After cloning and sequencing the last enriched library, 52 different sequences were obtained. Six sequences which had more than one copy in the library were tested and four sequences or aptamers(S1, S26, S27, S35) were proved to have high affinity and specificity with Vibrio harveyi, whose dissociation constant(Kd) are(32.57±7.07),(24.69±5.78),(34.75±5.61),(12.85±4.00) nmol/L respectively.In a similar manner, after 15 rounds selection targeting inactivate Vibrio harveyi, the affinity of the enriched library increased from 3.36 to 47.62, 14.2 times the affinity of the original library. After cloning and sequencing, 35 different sequences were obtained. Eight sequences with more than one copy were tested and five sequences or aptamers(I6, I9, I22, I27, I28) were proved to have high affinity and specificity with the inactivate Vibrio harveyi, whose dissociation constant(Kd) are(25.07±7.51),(29.17±7.24),(28.65±8.71),(26.05±7.42),(30.99±7.34)nmol/L respectively.In the same way, after 8 rounds selection targeting both Vibrio harveyi and Vibrio alginolyticus, the common aptamers against both vibrio could be obtained. The affinity of the ssDNA enriched library rose from 2.10 to 49.88, 23.8 times the affinity of the original library. After cloning and sequencing, 48 different sequences were obtained and seven sequences with more than one copy were found. Four sequences with more copies among the seven ones were chosen to verify and two sequences or aptamers(C7, C14) were proved to have high affinity with both Vibrio harveyi and Vibrio alginolyticus, whose Kd with Vibrio harveyi are(40.36±7.55),(55.76±12.45) nmol/L respectively, and with Vibrio alginolyticus are(53.02±13.52),(82.88±21.02) nmol/L respectively.Comparing the aptamers′ sequences obtained from the previous three SELEXs, two aptamers(S16, S31) were all found in the three independent selection, and proved to have certain affinities with Vibrio harveyi, Vibrio alginolyticus and their inactivate bacteria. In addition, four aptamers(S12, S25, S36, S47) were found in the selection of the aptamers against both Vibrio harveyi and inactivate Vibrio harveyi, which were proved to have higher affinities and specificities with both Vibrio harveyi and inactivate Vibrio harveyi. The discovery of these aptamers would provide a good basis for the rapid detection of Vibrio harveyi.a...