Construction On Rearrangement Plasmid Of N-G Gene And Helper Expression Plasmids In SRV₉Rabies Virus

The open reading frame sequences of N, P, M, G and L genes in rabies virus SRV9were amplified byRT-PCR. The amplified fragments was1352bp(N)、893bp (P)、608bp (M)、1572bp (G), and6384bp(L), respectively, then amplified fragments were ligated into pMD19-T vectors in turn, andidentified b y PCR,enzyme digestion, and DNA sequencing. The results were shown N gene of SRV9washighly conservative, and no mutation was found, whereas two sites of mutations in M gene were seen,belonging to the nonsense mutation.Five auxiliary Expression Plasmids containing nucleoprotein (N), phosphoprotein (P), matrix protein (M),glycoprotein (G) and large protein (L) of SRV9were constructed with pcDNA3.1(+) vectors, and namedpcDNA-N, pcDNA-P, pcDNA-M, pcDNA-G, pcDNA-L, respectively. The results of enzyme digestionand sequence identification were shown there was one site of gene mutation in pcDNA-P and pcDNA-Meach，whereas there were two sites of gene mutation in pcDNA-G.The two fusion genes SRV9N-G and SRV9P-M were constructed by recombinant PCR, and cloned intopMD19-T vector, respectively, and identified by enzyme digestion and DNA sequencing. Fusion genesSRV9N-G and SRV9P-M were completely overlapped, besides of a few of nonsense mutation. The effectof fidelity in fused genes indicated that recombinant PCR was a convenient, effective and shortcut tool toconstruct the full-length cDNA of SRV9.