Construction and identification of recombinant canine adenovirus type 2 expressing exogenous rabies glycoprotein (Rgp)

Abstract:

Safe and effective vaccination is important for rabies prevention. Here, genetically engineered rabies vaccine CAV2-ΔE3-Rgp was developed and characterized. The recombinant genome pPoly2-CAV2-ΔE3-Rgp carrying the rabies glycoprotein (Rgp) cDNA was generated by a series of strictly gene cloning steps and infectious recombinant virus CAV2-ΔE3-Rgp was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. To efficiently construct cloned recombinant canine adenovirus type 2 genome pPoly2-CAV2-ΔE3-Rgp bearing exogenous Rgp gene, The Rgp gene was first subcloned from the clone vector pMD18-T into the eukaryon expression vector pVAX1. The Rgp expression cassette was then subcloned into the shuttle vector pVAXΔE3 and subsequently into the canine adenovirus type 2 backbone vector pPoly2-CAV2. To indirectly confirm pPoly2-CAV2-ΔE3-Rgp, conventional restriction endonuclease digestion was performed. CAV2-ΔE3-Rgp can generate typical CPE of CAV-2. CAV2-ΔE3-Rgp was tested by restriction endonuclease digestion, PCR, DNA sequencing. As a result, The Rgp expression cassette was successfully integrated into the target region of the CAV2 genome. It is confirmed by RT-PCR, Western blot that CAV2-ΔE3-Rgp can express Rgp antigen in MDCK cell. This recombinant virus, CAV2-ΔE3-Rgp, was intramuscularly injected into dogs. All vaccinated dogs produced effective antibodies against CAV and RV after three inoculations. This recombinant virus would be prospective in immunizing dogs against CAV and RV.