Research On Translation Initiation Mechanism And Preparation Of Polyclonal Antibody Of Antheraea Pernyi Iflavirus

ApIV is one of the important pathogen tocause A.pernyi vomiting disease?Antheraea pernyi vomiting disease,AVD?.ApIV is a sense single-stranded RNA viruses.In the wild filed,larvae of the Antheraea pernyi are often affected by A.pernyi vomiting disease,which cause a serious economic loss.Its incidence can be 30-70%.Therefore,it is urgent to study the infection mechanism of ApIV virus and to find an effective detection and control method for the virus.In order to study the translation initiation mechanism of ApIV virus,this study firstly predicted the secondary structure of the 5'end of ApIV,which showed typical stem-loop structure and Y-type structure.According to their stem-loop structure,five fragments with different stem loops were amplified.The dual fluorescent protein reporter gene was used to verify its IRES activity,and the effects of different stem loops on its IRES activity were investigated.Fluorescence microscopy,microplate quantifier and flow cytometry were used to quantify the fluorescence,confirmed that the non-cap region of the non-cap-dependent endosome entry site?IRES?of the ApIV 5'non-coding region,and the stem ring I,IV pairs Its activity has a greater impact.Secondly,the bioinformatics method was used to analyze the interaction between each stem loop and several translation initiation factors by NPDock.The docking results showed that the stem loops I and IV had a great influence on the activity,which was consistent with the experimental results.In order to establish a rapid and concise ELISA method for ApIV virus,a specific polyclonal antibody against ApIV virus was prepared in this study.The ApIV leader protein gene and the capsid protein VP3 fragment were amplified,and the recombinant expression plasmid pET21-L^Pro-VP3 was constructed.The induction temperature,the inducer concentration and the induction time were optimized,and the recombinant protein was expressed in a large amount.The purified recombinant protein was used as an immunogen to immunize mice,and a polyclonal antibody against ApIV was prepared.Secondly,by measuring the titer,specificity,sensitivity and practical application of polyclonal antibodies,the results showed that the polyclonal antibody has a titer of 1:25600,which can specifically react with ApIV capsid protein and ApIV,and can detect Up to 0.4?g of the ApIV,and the tussah infected with the ApIV virus can be detected.