Dnaaptamer Against EV-A71 VP1 Protein:Selecting And Application

Enterovirus 71(EV-A71)is one of the most important pathogens causing hand,foot and mouth disease(HFMD),and it has a high proportion of severe infections and a high mortality rate.Therefore,it is important for the control and treatment of the disease.Now RT-PCR is the mian method of EV-A71 detection,which comes with the disadvantages of high cost and complicated operation.It is necessary for better detection methods for EV-A71.At the same time,the treatments of HFMD,especially serious HFMD,are mainly base on symptomatic treatments,lacking of effective antiviral drugs.An aptamer is a nucleotide sequence that screened in vitro by the technology named systematic evolution of ligands by exponential enrichment technology(SELEX).Similar to antibodies,aptamers can bind to the targets with high specificity and affinity.However,aptamers have more advantages than antibodies,such as ease of synthesis and modification,having a wide variety of target materials.Therefore,aptamers are promising in virus detection and anti-virus therapy.In our study,we have expressed EV-A71 VP1 protein and used this protein to screen three DNA aptamers V7,V11,V21 with high specificity and affinity for EV-A71 VP1 protein.Based on the affinity of the aptamers,we selected V11 and V21 for subsequent researches,including the utilization of aptamers to design a rapid chemiluminescence aptamer biosensor(aptasensor)for EV-A71 and the preliminary exploration of the feasibility of aptamers for anti-EV-A71 infection.In the study of constructing the aptasensor,we coated the amino-modified aptamer V21 on the carboxyl-modified magnetic beads(MBs)to obtain immunomagnetic MBs for capturing EV-A71 virus particles or VP1 protein in the sample,and then adding biotin-modified V11 binding to the VP1 protein on the MBs,the streptavidin-modified horseradish peroxidase(SA-HRP)sequentially joined the system and combined with biotin-V11 to form a MBs-V21-virus-V11-HRP sandwich structure.Finally,chemiluminescent substrates A and B was added.The HRP could act on the substrates causing an enzymatic reaction and producing a strong 425 mm fluorescence.A detector was used to detect the fluorescence intensity,Different fluorescence intensity is derived from the amount of EV-A71 virus or vp1 protein in the sample.The entire inspection process takes less than 40 minutes.In the exploratory study on the feasibility of aptamer in anti-EV-A71 infection,we pretreated the virus with aptamers V11 and V21 respectively,and infected the humanrhabdomyosarcoma(RD)cells with the pretreated virus.We found that,comparing to the CPE in non-treatment group,the CPE of RD cells in aptamer treatment group is milder.The expression of EV-A71 RNA and protein in RD cells of treatment groups was also significantly less than that in the non-treatment group.It shows that V11 and V21 can depress the infection of EV-A71 in RD cells.