Screening The Aptamers Against Listeriolysin O

Listeria monocytogenes is a foodborne pathogen.It widely exists in nature environment and may infect human and animals.Listeria monocytogenes can produce several virulence factors.Among these virulence factors,listeriolysin O(LLO)is found to be essential for the virulence of Listeria monocytogenes as it is responsible for the lysis of phagocyte vacuole.LLO coded by hly gene is a relevant marker for the identification of pathogenic strains.Therefore,this research aimed to get single strand DNA(ssDNA)aptamers against LLO using the systematic evolution of ligands by exponential enrichment(SELEX),which would provide the improtant materials for detecting the pathogenic Listeria monocytogenes.The main researches were as followed.The recombinate LLO was expressed in prokaryotic cells.After the hly gene was cloned and sequenced,the result showed it was made of 1590 base pairs and was 99%homology compared with the other hly genes by BLAST.Then,hly gene was inserted into the vector pET-30a(+)to construct the prokaryotic expression plasmide p-hly.Escherichia coli BL21(DE3)with plasmide p-hly were induced by IPTG to express the recombinant LLO protein.The expressed protein was produced in the form of inclusion body.The recipient bacterium was induced by IPTG(0.5mmol/L)for 10 h at 28?to get much more LLO.The LLO was extracted from inclusion body after ultrasonic disruptor.Finally,we obtained 6.774mg from 1L bacteria culture solution.The hemolytic activity was4.72×10~4HU/mg by hemolysis test.The aptamers were screened against LLO by SELEX.After 16 rounds selection,the binding rate of ssDNA sub-library reached saturation(35.51%).The plasmids p MD-19T linked with aptamers was transformed into E.coli DH5?.Thirty-nine positive clones were randomly picked to be sequenced.The results showed the sequence of 7 clones(7/39)was identical,named aptamer 1,and other four groups had four clones(named aptamer 2),three clones(named aptamer 3)and two clones(named aptamer 4),which showed the ssDNA library was enriched at a certain extent.The secondary structure predicted by DNAMAN software had a closely relationship with the primary structure.The aptamer 1and aptamer 2 were chosen to identify its specificity by ELISA.The results showed that the aptamer 1 recognized specifically Listeria monocytogenes.Therefore the aptamers binded specificly with LLO was obtained successfully.