[Sequencing the complete genome of rabies virus CVS-11 strain and constructing its full-length infectious cDNA clone].
摘要
OBJECTIVE: To sequence the complete genome of CVS-11 strain and establish a reverse genetic system of CVS-11 to further study its pathogenic mechanism, virulence genes and antigenic sites. METHODS: We amplified12 fragments covering the complete genome of the CVS-11 strain by RT-PCR, and then cloned to pEASY-Blunt vector for sequencing the complete genome of CVS-11. We analyzed single restriction enzyme sites of the full length cDNA of the CVS-11 strain by DNAMAN and designed 4 pairs of specific primers. We amplified the full-length cDNA of CVS-11 by RT-PCR. We obtained four fragments and cloned into pcDNA3. 1. We named the full-length cDNA plasmid pcDNA3. 1-CVS-11. We also cloned helper plasmids pcDNA3.1-N, P, L and G expressing N, P, L and G protein of CVS-11 strain, respectively. We co-transfected NA cells with the full-length plasmid and four helper plasmids. We identified the supernatant of the transfected and then passaged NA cells by immunofluorescence staining and RT-PCR and found the recombinant virus rCVS-11 rescued successfully. RESULTS: Sequencing results showed that the complete genome of CVS-11 was composed of 11 927 nucleotides. The complete genome of CVS-11 encoded 5 structure proteins and gene array was the same as other reported rabies viruses. We successfully constructed a reverse genetic system of CVS-11, namely the full length plasmid pcDNA3. 1-CVS-11 and 4 help plasmids pcDNA3. 1-N, P, L, G and rescued the rCVS-11 from a full-length infectious cDNA clone. CONCLUSION: The reverse genetic system of the CVS-11 strain laid the foundation for future studies on rabies virus.