Screening And Activity Research Of The RNA Aptamers Of Newcastle Disease Virus

Newcastle disease virus (NDV) belongs to Paramyxoviridae, is a highly contagiousvirus which causes respiratory, nervous, intestinal and other diseases in poultrys. NDVis a single stranded, nonsegmented, negative strand RNA virus envelope. NDV standardvirulent strain F48E9was first isolated in1948in China, which was endemic to China.The technology aptamers was made use of the systematic evolution of ligands byexponential enrichment (SELEX) to synthesize oligonucleotide fragment in vitro. In thisstudy, we make a deep analysis on the RNA aptamers we screened from the optimalligand specific three-dimensional structure, specificity and affinity and the biologicalactivity so as to provide basis for inhibiting the interaction between the Newcastledisease virusand host.Firstly,we identified and purified the target viral and protein, then constructed atotal length of random oligonucleotide library with132bp by in vitro synthesis method,and there were58fixed sequences in the two end and74random sequences in middle.Using SELEX cellulose nitrate membrane filtration to make a12repeated rounds ofscreening and enrichment, finally we got an enriched library special to the F48E9virusand HN protein, then connected, transformed, cloned and sequenced the last round ofscreening RNA library, and made a homology analysis on the primary structure of thesequencing results through the DNAMAN software, at last made a two stage structureprediction analysis on the successfully sequenced aptamer. AS a result, we got3advantage aptamers respectively against the F48E9strain and HN protein.ELISA andDot-Blot test were used to analyze the binding affinity and specificity of the advantagesaptamers, and the results proved that the aptamer had a higher affinity and specificity.The result of the biological activity test on the advantages of aptamers showed thataptamer FE12-4can reduce hemagglutination of F48E9virus on the titer of4units,FHN12-7aptamers can reduce that of F48E9virus on the titer of2units, preliminarysuggested that screening aptamers have a certain inhibition for F48E9. In the virusplaque experiment, FE12-4aptamers can reduce the virus plaque of F48E9strain about84.62%on the DF1cells culture, at the same time aptamer FHN-7can reduce virusplaque of F48E9about55.36%,futher suggested that screening two types of aptamershave different inhibitory for the same virus. In the fluorescence quantitative RT-PCR experiment, we firstly obtained the linear regression equation: Y=-3.089x+28.259,detected that aptamer FE12-4and FHN12-7can reduce77.73%and49.78%virussystem load in the culture respectively, which suggested that screened RNA aptamer caneffectively inhibit the replication of F48E9strain in cell level,aptamer FE12-4andFNH12-7of advantage aptamers in the selected have obvious characteristics. differentaptamers of F48E9strain virus showed different specificity, affinity and biologicalactivity.