Screening Of Specific Aptamers For Rabbit Hemorrhagic Disease Virus

Rabbit hemorrhagic disease(RHD)is an acute,lethal and contact-induced disease of adult rabbits caused by rabbit hemorrhagic disease virus(RHDV).It brings rabbit breeding industry to a great economic loss.At present,the laboratory commonly used "O" type erythrocyte of hunman agglutination(HA)to confirm whether rabbits are infected with RHDV.However,sometimes it brings false positives or false negatives.RHDV also can be diagnosed by electron microscopy which has high demands on devices.Reverse transcription polymerase chain reaction(RT-PCR)is highly sensitive but it wastes time.The immunological methods include agar diffusion,enzyme-linked immunosorbent assay(ELISA),Western blot,in-situ hybridization and other methods.These methods require antibodies and cost is higher.Since the 1990 s,the aptamer has been proposed for more than 20 years,whose advantage was gradually emerging in treatment and diagnosis.Aptamers have many advantages comparing to antibodies.It includes easy synthesizing,less costs,repeatable using,and easy storing.Therefore,aptamers have been widely used in many fields,such as the bioscience research,target identification,targeted therapy of tumor,drug screening,environmental monitoring and others.The method for screening the specific aptamers of the target substance from the library is called SELEX technology.The SELEX technology has been applied to screen a wide variety of virus aptamers including human immunodeficiency virus(HIV),hepatitis virus(HBV),avain influenza(AI),etc.But until now no RHDV aptamers have been reported.To screen highly specific and sensitive aptamers of RHDV,we synthesized a random single-stranded oligonucleotide library(ssDNA)in vitro,which was 80 nt in length containing a random sequence of 40 nt internally and 2 constant regions at flank..RHDV capsid protein VP60 acted as the target molecule,which was transferred to PVDF membrane after SDS-PAGE.After 20 rounds of screen,we got the secondary library with the biotion markers.We used the biotin-labeled secondary library as primary antibody to recognize VP60 on PVDF.The results showed that the secondary library contain the aptamers that can recognize the target molecule.The library was ligated into pMD18-T vector,then it was transformed and identified,in which 50 clones were sequenced.Biotinylated aptamers were generated based on positive clones,and the target molecules were identified by these aptamers.The results showed that three aptamers were able to recognize the target molecules,meanwhile they did not recognize other proteins of RHDV virion on the PVDF membrane,healthy rabbit liver tissue proteins or BSA proteins,which indicate the three aptamers have specificity and affinity to the target molecule.By analysis of the aptamer squences,we found that guanine(G)and thymine(T)accounted for more than 70% of the random sequences We predicted that these bases are important components of the aptamers conserved sequence,which are specific to the target molecule.Through the mfold website(http://unafold.rna.albany.edu/?q=mfold),We found that the three aptamers contain stem and loop structure,and we speculated that the structure is a basic structure for recognizing VP60 protein of the target molecule by aptamers.Collectively,we find the condition of screening specific aptamers and obtain the specific and affnic library to target protein VP60 by combined the Western blot method with the traditional SELEX technique.In addition,we successfully screened three aptamers for target protein VP60 with specificity and affinity.These aptamers have proved to be clinically useful as therapeutic molecules with specific antiviral potential against RHDV infections..