Construction And Identification Of Recombinant Adenovirus Containing The G And N Gene Of Rabies Virus

Rabies is a kind of deadly zoonosis caused by rabies virus. Vaccine inoculation is a useful way to control this disease. Although traditional vaccines have shown their efficiency in controlling the incidence of rabies, they are too expensive and fragile to be used for wildlives and cattles. To control and eradicate rabies need novel effective and economical rabies vaccines. Recombinant adenovirus vaccine based on rabies is safer and more effective than other recombinant vaccines. It could be used as a good candidate for such purposes.Previous study demonstrated that rabies virion is composed of a single molecule of genomic RNA and 5 structure proteins. The glycoprotein(GP),which is the only rabies antigen that consistently induces virus-neutralization antibody,is the hotspot of researching recombinant vaccine against rabies. Furthermore,the nucleoprotein(NP)could enhance the T-helper cell immune response to rabies vaccination. In this paper,mice were attacked by rabies virus CVS, total viral RNA was isolated from the brain tissues of rabid mouse, and the nucleoprotein gene and glycoprotein gene were obtained by RT-PCR and cloned into pMD18-T plasmid,respectively. After sequencing and analyzing with DNAstar,it was indicated that the glycoprotein gene and nucleoprotein gene were derived from rabies CVS strain with high neuropathogenicity.In this study,the recombinant adenovirus containing the GP and NP genes simultaneously was successfully obtained for the first time. To construct recombinant adenovirus,first, the genes of GP and NP were respectively cleaved by double restriction endonuclease digestion from their pMD18-T plasmids,and then cloned into shuttle vector pAdTrack-CMV coinstantaneously, being selected with kanamycin and screened by restriction endonuclease digestion,a recombinant shuttle plasmid of pAdTrack-CMV/G+N was obtained; Secondly,using the pAdEasy system,the clonal human type 5 recombinant adenovirus DNA bearing exogenous rabies genes in early 1 region by employing homologous recombination was efficiently constructed,the resultant construction was linearized and transformed together with a supercoiled adenoviral vector pAdEasy-1 into E. coli strain BJ5183,which is short of growth period and easily manipulated; Third,the recombinant adenoviral construct was cleaved with PacI to expose its inverted terminal repeats,and transfected into 293 packaging cell line. The strain of recombinant adenovirus containing glycoprotein gene and nucleoprotein gene from CVS strain was successfully obtained.