In Vitro Selection Of Aptamers To N.gonorrhoeae By SELEX

N . gonorrhoeae is the non-spore-forming Gram-negative bacterial pathogen that causes the Gonorrhea that is the STD of highest incidence in China. the passion aggrandize the dangerous of infection to HIV. N. gonorrhoeae has serious inperil the chinese public health.SELEX (Systematic Evolution of Ligands by Exponential Enrichment), is also known as a new selection technique in vitro. Aptamer, which is screened from the large random nucleic acids libraries by SELEX technique, is oligonucleotide that bind to targets with high affinity and specificity.Aptamers are powerful candidates for molecular detection of targets due to their unique recognition properties. These affinity probes can be used to recognize and bind their targets in the various types of assays that are currently used to detect and capture moles of china. As aptamers resembled with antibody but superior to antibody for having a large number of target molecules, high affinity and specificity, stability and ease of production, regeneration. Aptamer has promising applications prospect in clinical therapy and laboratory diagnosis. This study aim to screen the aptamer with high affinity and specificity for N. gonorrhoeae and set a base for establishing relative easy and quick laboratory detection methods.In present study, ssDNA pools are used in the selection process, ssDNA is heat-denatured at 95℃for 5 min ,and then cooled immediately to 4℃in binding buffer. Selection is performed by incubating ssDNA pools with bacterial at room temperature for 25-30 minute in binding buffer by gentle rotation. After 30 min the aptamers-bacterial antigen complex is centrifuging and washed four times with washing buffer, ssDNA retained on the bacterial is elute after heat by 99℃ddH₂O. Selected ssDNAs are amplified by PCR and used for the next round selection. After round 10th, we use M. catarrhalis, N. sicca. N. cinerea in counter selection. PCR products of the last round of selection are cloned and sequenced relative software is employed to analyze the primary structure and prediction secondary structure of the aptamers.After 13 rounds of selection and amplification, we obtaine the aptamers against N. gonorrhoeae. Meanwhile, we can see the sequences of the 12 aptamer clones are 78 nt and the sequences are divided into some groups on the basis of primary sequence homology. These will be the focus of our study in future. Hairpin loop is the main motif in prediction secondary structure which may be the binding structure between the aptamers and target peptides. Based on the selection of the aptamers against cyclosporin A, we can set up relative analysis systens for detection.