Cloning Structural Genes Of Rabies Virus And Construction On Recombinant Plasmid With Gene N In Canine Distemper Virus And EGFP Gene

The total RNA was extracted from cultured rabies virus SRV9by BHK-21cell. The N gene, P gene, MGene, G gene, L1and L2gene from the total RNA were amplified by reverse PCR (RT-PCR) and ligatedinto pMD19-T vectors. The amplified fragments were1352bp (nucleoprotein gene, N gene),893bp(phosphoprotein gene, P gene),608bp (matrix protein gene, M gene),1574bp (glycoprotein gene, G gene),3372bp (large protein gene1, L1gene)and3012bp (large protein gene2, L2gene), respectively. The resultsof sequencing and compared with SAD B19sequence were shown the nucleotide homology of N gene, Pgene, M gene, G gene, L1gene and L2gene was100%,99.66%,99.67%,99.75%,100%and99.90%,respectively, and amino acid homology was100%,98.99%,100%,98.65%,100%,99.91%separately.The recombinant plasmids of enhanced green fluorescent protein eGFP gene and N gene in caninedistemper virus (pT-eGFP-CDVN), rabies virus fusion gene P-M (pT-PM) and L (pT-L) were constructedby recombinant PCR. The results were shown the fusion gene fragments of eGFP-CDV N, P-M and L were2292bp,1501bp and6384bp, respectively. The three fusion genes were completely overlapped, no genemutation and mispairing was found.The three fusion genes were successfully constructed by recombinant PCR in this study. It is a good toolto construct full-length cDNA of rabies virus SRV9, and even to develop an attenuated recombinant vaccineagainst rabies.