The Study On The Rescue Of Foot-and-Mouth Disease Virus By Amber Orthogonal System

Foot-and-mouth disease(FMD)is a highly contagious animal disease,which is caused by foot-and-mouth disease virus.At present,vaccine immunization is the main measure for prevention and control of foot-and-mouth disease.However,conventional vaccines cause outbreaks of foot-and-mouth disease due to various reasons.Therefore,in-depth study of the pathogenesis of FMDV and the preparation of a vaccine with better protection rate and more stable immune effect is essential for the prevention and control of FMD.In this study,the pCMV-EGFP-Tyr40-TAG mutant plasmid and the orthogonal aminoacyl tRNA synthetase/tRNA plasmid were constructed and co-transfected into BHK-21 to get a stable expression cell line by antibiotic screen and Western-blot identification.The wild-type plasmid of FMDV was constructed.The wild-type plasmids expressing VP1,VP2,VP3 and 3D proteins were constructed respectively,and the TAG stop codon was introduced at different sites to obtain the mutant plasmids of VP1,VP2,VP3 and 3D.The wild type plasmid and the mutant plasmids were transfected into a stable expression of the Orthogonal Acyl-tRNA synthetase/tRNA cell line respectively,and identified by Western-blot.The mutant plasmid capable of expressing the target protein in the stable expression of the orthogonal aminoacyl tRNA synthetase/tRNA cell line was screened.And the introduction site for the TAG stop codon on the foot-and-mouth disease virus genome was determined.Based on the results of the mutation site,the amber stop codon(TAG)was introduced into the foot-and-mouth disease virus genome by point mutation technique to obtain FMDV-TAG infectious clones.UAG-containing mRNA was transcribed in vitro and transfected into cells.In normal cell lines,the translation of FMDV will be terminated prematurely due to the introduction of TAG,so that the complete virions cannot be packaged;However,in a cell line that introduces a specific orthogonal system,the orthogonal aminoacyl tRNA synthetase/tRNA can recognize the non-natural amino acids,which in turn continues the translation of FMDV and packages the virus particles carrying the TAG mutation with complete function and activity.In this study,The FMDV-TAG mutant virus could be controlled.The FMDV-TAG mutant virus with immunogenicity will be obtained which could not proliferate in normal mammalian cells.All of these will provide a new idea and platform for the study of FMDV pathogenesis and new vaccine development.This platform is suitable for all viruses that can be reverse genetically manipulated.