Construction Of Recombinant PRV GG~- Gene Deleted Transfer Vector Expressing The Rabie Virus Glycoprotein

Rabies is an important zoonosis that causes severe infection to the central nervous system and is usually fatal.In recent years,the serious problem has ascended tendency year by year in China.China has reported the second highest rates of illness and death from human rabies worldwide.The domestic dog plays a pivotal role in rabies transmission;85-95%of human rabies cases are ascribed to dog bites,and 50-70%of human rabies cases are reported in rural areas.The reasons are all round.The main reasons are single type of vaccine and the low level of immunization.Lower vaccination coverage of dogs and stray dogs is widespread,largely because of poor awareness of precaution in rabies and the high cost of vaccination.So the control and eradication of human rabies depends on the development of safe,effective and economical vaccine that might be used in pre-exposure vaccination programs for animals.Dogs and wildlifes of canis familiaris are the main transmission intermediary of rabies,so the control and eradication of human rabies depends on the development of safe,effective and economical vaccine that might be used in expressing vaccination programs for animals.A recombinant expressing the rabies virus low virulent strain SRV₉ glycoprotein has been constructed by our lab.Pseudorabies virus(PRV) is the causative agent of Aujeszky's disease,which results in significant losses in pig husbandry.Protein kinase(PK) gene is one of the main virulent genes of PRV,and it is essential to the propagation of PRV in the nerve tissues,but dispensable for virus replication and infection in other tissues.In previous,the neutralizing antibody level is not as high as that induced by the conventional vaccines, i.e.,inactivated or attenuated live vaccines.It is broken though that viral vector contains only one expressing cassette,and it is constructed expressing cassette side by side in viral vector.In this way,not only have the theory materials been supply that researching capacity of PRV viral vector,but also has the level of glycoprotein antibody been study that recombinant virus induces organism.In this study,the genomic DNA of pseudorabies virus TK/gI gene-deleted strain was extracted and digested by Ascâ… ,the 8.7kb fragment,which containing the whole PK gene was reclaimed.Plasmid P8-AA was constructed by cloning the 8.7kb fragment into the Ascâ… site of pPolyâ…¡.The fragment containing gG gene was obtained by EcoRâ…¤and Stuâ… digestion with p8-AA,and was cloned into pIRESâ…¢.After gG gene of the pIRESâ…¢was deleted by BamHâ… /Ndeâ… digestion,the expressing cassette containing CMV promoter,EGFP gene,G gene and SV40 polyadenylation signal was inserted the deleted site.The recombinant plasmid, pIRâ…¤-SR,could express EGFP gene and G gene respectively.In the future,pIRâ…¤-SR and genome of pseudorabies virus should be transfected into PK-15 ceils to obstain recombinant pseudorabies virus by homologous recombination and develop genetically engineering vaccine against dog rabies.Then,we used the plasmid ZpIRV-SR and FpIRV-SR to transfect the PK-15 cells respectively,The fluorescence can be seen in plasmid ZpIRV-SR and FpIRV-SR.The results showed that the transfer vector ZpIRV-SR and FpIRV-SR can be used to homologous recombination to construct recombinants virus.