Screening DNA Aptamers Of Anti-zearalenone Monoclonal Antibodies By Selex Technique

The zearalenone (ZEN)are secondary metabolites produced by Fusarium fungi.ZEN can not only directly contaminate a variety of grains, cereals, feed, but also can contaminate meat, milk and other animal derived food. ZEN has estrogenic activity, it can cause a variety of damage to humans and animals,including reproductive toxicity,DNA damage toxicity, immune suppression toxicity, it’s a serious threat to human and animal health.SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is also known as a new selection technique in vitro. And a specific nucleotide sequence can be screened from in vitro synthetic random oligonucleotide libraries by SELEX technique. Due to the interactions between the bases of oligonucleotides, spatial structures can be formed between the bases. These spatial structures were easily combined with various types of target molecules. And any aptamer which is DNA or RNA fragments that can bind to targets with high specificity and high affinitycan be screened from the random oligonucleotide library. Aptamer can precisely distinguish the targets and the short preparation cycle and easy synthesis in vitro make it become an alternative to the corresponding antigen or antibody. Zearalenone toxin monoclonal antibodies as target,After the DNA aptamers being screened, the toxins were no longer used as competition antigen when detected the zearalnenoe, thus developed a low-toxic, environmentally friendly detection kit.This can ensure the security of the detection method to humans and the environment.In the study, we in vitro synthesized a length of71bases, containing35random ssDNA sequence.After15rounds of selection against monoclonal antibody of zearalenone using SELEX technique, we applied biotin-streptavidin-horseradish peroxidase system to determine the binding affinity between the aptamers and monoclonal antibody against zearalenone,The final round of the enriched library were cloned, sequenced, and the use the DNAMAN software to analysis the structure and the secondary structure of the aptamer sequence.And then use aptamers to replace toxin to establish appropriate detection method.The test results show that the15rounds of SELEX screening that there is an increasing tendency of binding affinity between the the aptamers against monoclonal antibody of zearalenone. The absorbance value was from0.125of the first round to0.666of the fifteenth round which improved by5.328times.96aptamers were randomly selected to clone and sequence analysis,then use the correct sequence of aptamer to analyze the secondary structure and detect the affinity. Among the96aptamers,63aptamer were the same length with71bp single stranded DNA.The secondary structure predicted that stem-loop structure are the main structure, and the structure of the stem-loop are the base of aptamer and target molecule binding together.The number46aptamer have higher affinity and this aptamer was used to establish the indirect competition ELOSA(Enzyme-linked Oligonucleatide Assay, ELOSA) detection method of competitive inhibition curve.The titration to determine the best conditions was that concentration of coating monoclonal antibody was1μg/well,aptamer was diluted to1:10,and its concentration was80ng/ml.Streptavidin-HRP was diluted to1:300,and its concentration was3μg/ml.As a competitive antigen with a standard toxin solution concentration were50.00,10.00,5.00,1.00,0.10,0.01,0.00ng/mL,three parallel wells each concentration, repeated3times, the curve is stable.The intra-batch was7.6%,the inter-batch was9.3%,both the variance inter-batch and the variance intra-batch was less than10%.These show that the method has a better reliability and it establish the foundation for future applied research. Aptamers’ obtain provides a prerequisite for the future development of low-toxic, environmentally friendly immunological detectionkit.