Construction Of Recombinant Canine Adenovirus Type 2 Containing Rabies SRV9 G Gene

Rabies is one of the natural epidemic zoonosis caused by rabies virus. Rabies extensively distribute all over the world, and because of the epidemic sources widely existing, there is no available ways and means to cure it. and it is very difficult to control and eliminate it too. To control and eliminate rabies, the most effective way is to carry out vaccination. The research of rabies vaccination focuses on the genetic engineering recombinant live vaccines at our time. The recombinant vaccine vectored by virus can express foreign immunogens well and cause high level of cell and body immunoreaction.Canine adenovirus type 2 is a kind of satisfied virus vector, CAV2 E3 region is not important for virus copy, to delete E3 or insert foreign DNA into E3 region, CAV-2 still can copy itself, so it is the suitable site to construct recombinant adenovirus in E3 region. Size of foreign DNA and deleted fragment of E3 is the key factor to construct recombinant adenovirus.For the first time, rabies glycoprotein gene has been successfully cloned into CAV-2 E3 region in this study. The ready CAV-2 plasmid vector pPoly â…¡-CAV-2 was modified by EcoRI, XbaI, AscI and XbaI,then a new pPoly â…¡-CAV-2 vector was obtained. The recombinant medium plasmid pVAX-E3 was digested by Draâ…¢ and Ssp â… , a piece of 1044bp fragment in length of E3 was deleted between 25097bp and 26141bp in CAV2 E3 region, then a piece of 2424bp Rgp expression cassette(CGS)containing CMV promoter, rabies virus strain SRV9 glycoprotein gene and SV40 polyA was cloned into the deleted site in pVAX-E3 at the same direction of duplication and pVAX-E3-CGS was obtained. Both pVAX-E3-CGS and pPolyâ…¡-CAV2 -new vector were digested by NruI and SalI, and then CGS was cloned into pPoly â…¡ -CAV2-new vector and the recombinant pPoly â…¡-CAV2-CGS wasobtained. Finally pPoly II-CAV2-CGS was digested by Asc I and Cla I to free a fragment of 32.7kb CAV2-CGS gene. And then, CAV2-CGS gene was transfected into MDCK cell with the participation of Lipofectamine?2000, 8 days later, the recombinant CAV-2-CGS virus was obtained, it can express rabies strain SRV9 glycoprotein improved by Western blotting assay.To evaluate its heredity stability, the recombinant virus genome was extracted and digested by Hindlll every 5 generations, and 40 generations later, its enzyme digestion sites almost keep stable. Preliminary immunization trial in dogs and kun'ming mouse showed that the recombinant virus could stimulate a specific immune response to both CAV-2 and rabies virus strain SRV9. In addition,different ways of vaccination produced different antibody titer, taking orally, dropping eyes and noses are satisfied. All of those have paved a way to prevent rabies by use of recombinant CAV2 contanining rabies SRV9 G gene.