Selection Of SsDNA Aptamers Recognizing Newcastle Disease Virus

Newcastle Disease(ND) caused by Newcastle disease virus(NDV) is a highly contagious and cute infectious disease of poultry. It causes respiratory, gastrointestinal and central nervous system damage. It distributes worldwide and causes great economic losses to the poultry industry. Aptamer is an oligonucleotide ligand binding to the target molecules specifically, which is selected from a random synthetically oligonucleotide library by SELEX. Aptamers have many biochemical merits, such as low immunogenicity, wide range of target molecules, small molecular weight, easily reconstructed and marked, etc.Consequently, aptamers possess an important application value in the field of clinical diagnosis and therapy of disease.In the study, we constructed a total length of random oligonucleotide library with 81 nt by synthesis method in vitro. This library contains up to 40 oligonucleotides in random region,which are flanked by short constant regions, usually used to anneal primers during PCR. The optimum annealing temperature Tm=65?, reaction cycle number is 12 cycles.ssDNA is obtained by asymmetric PCR and PCR primers in the ratio of F: R = 100:1. We got an enriched library special to the purified target HN protein andDNA after 10 repeated rounds of screening and enrichment ssDNA aptamers by cellulose nitrate membrane filtration and microwell plate. ssDNA was obtained from dsDNA by asymmetry PCR. Then the last round of screeningDNA library, connected, transformed, cloned and sequenced,and made a homology analysis on the primary structure of the sequencing results through theDNAMAN software, and made a two stage structure prediction analysis by Mfold.The affinity and specificity of aptamers is analyzed by ELISA and Dot blot and the results show that B53 aptamer appeared higher affinity and be able to specific identification of HN protein and GM virus, but without specific recognition of AIV,IBV and SPF allantoic fluid.The results of HI show that the adapters against GM have different degrees of inhibitory activity of coagulation. Hemagglutination titer of control group was 11log2,hemagglutination titer of A1 treatment group was 9log2, A3 treatment group was 7log2,A20 treatment group was 10log2, B7, B53 and B60 treatment group was 8log2. The result of the plaque test show that aptamer B53 can reduce 44.2% plaque of GM strains.In this study, the aptamer Screened by SELEX can specifically bind to NDV and having a hemagglutination inhibition and plaque forming inhibition activity. The result shows that the aptamer having the potential for NDV diagnosis and antiviral research.