Construction And Expression Of Eukaryotic Expression Vector Of Serotype O Foot-and Mouth Disease Virus-like Particles Encoding Gene

At present,serotype O foot-and-mouth disease(FMD)is the most popular in China,and its control still depends mainly on the inoculation of inactivated vaccines.However,existing commercial FMD inactivated vaccines cannot induce effective cellular immune responses,at the same time,thermal instability,short protection period,and high cost in the preparation process also hinder the progress of prevention and treatment of foot-and-mouth disease.More importantly,several outbreaks of European FMD may be related to incomplete virus inactivation or live virus escape in vaccine production laboratories.These factors have promoted the development of new vaccines,recombinant vaccine as a new vaccine has a good market prospect.In order to construct an eukaryotic expression vector for the serotype O foot-and-mouth disease virus-like particle encoding gene,primers with EcoR V and BamH I restriction sites were first designed based on the pSMVP0/VP1/VP3 vector provided by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.A single FMDV structural protein VP0,VP1,VP3 gene fragment was amplified respectively,and the gene fragment and the eukaryotic expression vector pVAX1 were digested with EcoR V and BamH I,respectively,and ligated with T4 ligase to obtain a recombinant vector pVAX-VP0,pVAX-VP1 and pVAX-VP3.The amplified plasmids pVAX-VP0,pVAX-VP1,and pVAX-VP3 were digested by primers with BsmB I,EcoR I,and Sal I restriction sites,respectively,and were also digested with T4 ligase.The recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was obtained and identified by PCR and sequencing.The recombinant eukaryotic expression vector was transfected into BHK-21 cells by liposome transfection and its expression in cells was detected by indirect immunofluorescence assay(IFAT)and Western blot.The results showed that the recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was successfully constructed and successfully expressed in BHK-21 cells.In this experiment,the structural genes VP0,VP1,and VP3 of the serotype O foot-and-mouth disease virus were inserted into the eukaryotic expression vector pVAX1 simultaneously,and the recombinant expression vector pVAX-VP0VP1VP3 of the serotype O foot-and-mouth disease virus-like particles was successfully constructed to lay a foundation for the study of gene vaccine expressing FMDV virus-like particles.