Screening Of Nucleic Acid Aptamers Against Porcine Epidemic Diarrhea Virus Infected-cells And Antiviral Study Of Manganese Dioxide Nanosheets

Porcine epidemic diarrhea virus（PEDV）and pseudorabies virus（PRV）are two typical viruses that seriously endanger the pig industry and cause significant economic losses to the pig industry in my country and the world.The establishment of new virus detection methods and the development of new anti-virus means are of great significance for controlling the prevalence of the virus.In this paper,cell-systematic evolution of ligands by exponential enrichment（Cell-SELEX）technology was used to successfully select nucleic acid aptamer candidates for PEDV-infected cells;the inhibitory effect of manganese dioxide（Mn O₂）nanosheets on PEDV and PRV were studied.The specific contents are as follows:Using Cell-SELEX technology,we successfully selected aptamer candidates against African green monkey kidney cells（VERO）infected by porcine epidemic diarrhea virus.Based on the synthesis of oligonucleotide library,upstream primers and downstream primers,the initial ss DNA sequence was identified by agarose gel electrophoresis characterization method,and the appropriate time and dosage of PEDV-infected VERO were explored by the indirect immunofluorescence method.Using PEDV-infected VERO as positive screening cells and selecting normal VERO for anti-screening cells.After 13rounds of screening,a nucleic acid aptamer enrichment library was obtained.The preferred sequences in the high-throughput sequencing results of the 11th pool were analyzed and structurally predicted with the help of NUPACK software and finally three nucleic acid aptamer candidates were obtained.The research results have certain reference significance for the discovery of biomakers on the surface of PEDV-infected VERO and the early diagnosis and treatment of PEDV.The inhibitory effect of manganese dioxide nanosheets on the proliferation of Porcine epidemic diarrhea virus and pseudorabies virus were studied.The Mn O₂nanosheets were successfully synthesized by using the synthesis technique reported in the reference,and their morphology and structure were characterized accordingly.The results of transmission electron microscope（TEM）and scanning electron microscopy（SEM）showed that the Mn O₂nanosheets exhibited a two-dimensional micro-scale sheet-like structure.It could be seen from ultraviolet-visible absorption spectra（UV-vis absorption spectra）of Mn O₂nanosheets that a strong broad absorption peak appeared 376 nm,which was caused by the d-d transition.The absorbance at 376 nm was plotted against the various concentrations of Mn O₂nanosheets.The characteristic peaks of Fourier transform-infrared spectroscopy（FT-IR）at 700,523 and 474 cm^-1were related to the bending vibration and stretching vibration of Mn-O bond.Element mapping analyzed the distribution characteristics and abundance of Mn and O elements.All these confirmed the formation of two-dimensional nanomaterial Mn O₂nanosheets.The MTT method was used to study the toxicity of Mn O₂nanosheets on VERO and porcine kidney cells（PK-15）,and the results showed that Mn O₂nanosheets with a concentration of less than200μM treated VERO within 48 h or Mn O₂nanosheets with a concentration of less than80μM treated PK-15 within 48 h,the cell survival rates all reached about 90%.Using PEDV and PRV as RNA and DNA virus models,the indirect immunofluorescence test was performed to explore the antiviral effects of Mn O₂nanosheets on these two viruses.The results confirmed that Mn O₂nanosheets had a significant inhibitory effect on both PEDV（RNA virus）and PRV（DNA virus）.This not only provides new ideas for the development of PEDV and PRV antiviral drugs,but also shows the application prospects of Mn O₂nanosheets as a broad-spectrum antiviral nanomaterial.