Construction Of PRV TK～- Transfer Vector Co-expressing P1-2A And 3C Genes Of FMDV

Swine Foot-and-Mouth disease is the great anthropozoonosis.We immunity or catch and kill the sick animal to resist it generally.PRV Ea strain is endemicity in our country,the homology is very high by comparing with the other endemical strains of PRV.So it possesses the utility by selecting PRV Ea strain to construct gene depletion vaccine.TK gene is the most important virulence gene,which can determine the virulence of PRV,It can keep the station of latent infection for long-term,increase in nervous tissue,and avitate the virion of latent infection.The depletion of TK gene can both loss or cut down remarkably the virulence of PRV and cut down the reproduction in nervous tissue,the dissemination from peripheral nerve to brain,and the baneful ability by causing encephalitis.It has nothing with the viral multiplication in cell.Meanwhile the PRV TK^- is the same as wild strain which can generate well in cell,but its virulence and the ability of latent infection depress largely.So the PRV TK^-strain is used for to study PRV gene deletion vaccine.At present,pseudorabies vaccine delete the TK gene mostly.Expressing the capsid protein precurson P1-2A gene and protease 3C gene of FMDV in viral live carrier vaccine or nucleonic acid vaccine,so as to make the expressed capsid protein precurson split to VP1 VP3 and VP0,and pkg virus-like particle,thereby it stimulus to produce humoral immunity and cell-mediated immunity with the similar to the real virus.According to the published gene order of PRV TK and PRV TK^- in GenBank,we design a pair of specificity primer to amplificate TK gene.The TK gene was connected with pTA2-T.After sequencing,we designed a pair of express primer containing EcoRI and HindIII site to amplificate TK gene,After cutting the gene and pcu18,we connected them.Than depleting the moiety of TK gene,inserting the express case with P_{CMV IE} promoter,MCS,IRES,3C,Fragment containing the bovine growth hormone poly A signal in depletion part.Than precurosor capsid protein P1-2A of FMDV inserted in multiple cloning site. Thereby we construct .the recombinate PRV TK^- transfer vector with 3C and P1-2A gene of FMDV.the vector was expressed in vero cell according to identified by indirect immunofluorescence. Success in vector construction establishes the foundation for obtaining in recombinant virus and prepareing gene engineering live carrier vector the next step.