Selection And Preliminary Application Of The Aptamer Against The VP2 Protein Of Aleutian Mink Disease Virus

Aleutian disease of mink(AD)is a chronic,persistent,infectious disease caused by the infection of Aleutian mink disease virus(AMDV).AD caused autoimmune dysfunction and disorder in mink.After infection,young mink was dead,the quality and the reproductive ability of adult mink decreased significantly,which seriously affecting the development of mink breeding.There was no commercial vaccine,the main means of prevention and control is to eliminate the positive mink and establish the purification population through the regular screening,quarantine strategy.With the rapid development of mink breeding in China,it is imminent to develop the experimental animalization of mink.It has a wide application prospect to develop the diagnostic method and therapeutic drugs.Aptamer is a single chain nucleotide molecule(RNA or ssDNA)selected from the ligonucleotide library by SELEX(systematic evolution of ligands by exponential enrichment)strategy in vitro.It can fold into the advanced structure,effectively combining with a specific target and affecting the biological function of the target.Aptamer has more advantages of high affinity,high specificity,easy to modify,large synthesis with lower batch-to-batch variation,stable chemical property,it was widely used in the diagnosis of toxins,tumor cells,pathogenic bacteria and viruses.Therefore,aptamer can be used as a beneficial supplement to monoclonal antibodies,providing new ideas and tools for the study of veterinary pathogenic microorganisms.In this study,the aptamer against the AMDV VP2 protein were screened by magnetic beadsSELEX.After 10 rounds of screening,the enrichment library binding to the VP2 protein was obtained.After the high-throughput sequencing analysis,the homology comparison and the phylogenetic tree analysis,these sequences were divided into four different branches.Candidate sequences(AMDV-1#,AMDV-11#,AMDV-14# and AMDV-18#)were selected from four branches respectively to detect the ability.The result of enzyme-linked oligonucleotide sorbent assay(ELOSA)test showed that AMDV-1#,AMDV-14# had high affinity with the AMDV VP2 protein(Kd=565 nM and Kd=247 nM)and high affinity specificity(P<0.01).By comparing the advanced structure of AMDV-1# and AMDV-14#,there existed a common sequence "CCTATGCGTGCTACCGTG",and the sequence participated in the formation of a typical stem-ring structure,which may be directly related to the characteristics of aptamer.In order to develop the application value of aptamer in AMDV detection,a double sandwich ELOSA method combining aptamer and monoclonal antibody was established.By optimizing the reaction conditions,the optimal working concentration of monoclonal antibody was 10 ng/?L,the concentration of aptamer was 1 ??.The method had no cross reaction with canine parvovirus,canine adenovirus and BSA protein,and the detection limit was 50 ng/ ?L,By detecting 53 mink feces samples,the accordance rate was 89.66% between DAS-ELOSA and PCR detection.In order to select the aptamer with inhibition of viral replication,this study first established the infection system of AMDV-Utah strains on CRFK cells,and the MTT assay showed that different concentrations of aptamers had no significant effect on cell activity(P>0.05).According to the results of enrichment library sequencing and phylogenetic tree analysis,15 sequences were selected to carry out the antiviral activity research.The results showed that AMDV-12# could reduce the 22% viral load in the infection system,Other aptamers had no significant inhibitory effect on viral replication(P>0.05).Above all,the aptamer against the AMDV VP2 protein were successfully screened,the DAS-ELOSA method based on aptamer was established and the aptamer with inhibition of viral replication was screened,which could provide a new idea for the diagnosis and the antiviral research of AMDV.