Construction Of N1L Gene Deletion Recombinant Goatpox Virus And Research On Its Biological Properties

In the research, we constructed the transfer vector plasmid pLR-EGFP-gpt expressing reporter gene EGFP and screening gene gpt with GTPV antigen as a template and N1L gene flanking sequences as the homologous recombinant arms.Vero cells pre-infected with GTPV were subsequently transfected with resulting plasmid.Finally,through recombination,selection culture,purification and homologous identification, recombinant GTPV without N1L gene were obtained. While we constructed recombinant transfer vector pLR-EGFP-gpt-N1Lrev after the N1 gene deletion by the reverse insertion of N1L gene in pLR-EGFP-gpt and screened the revertant virus GTPV N1Lrev after co-transfection with GTPV. Genetic stability and biological characteristics show that GTPV △N1L could be genetic stably at least 10 generations with the growth characteristics of the parental strains in cell cultures,but the copies of GTPV △ N1L were 30 times of parental strains in the same titer. N1L may be one of the virulence factor of GTPV.GTPV N1L gene fragment was amplified by PCR and cloned to eiakaryotic expression vector pcDNA3.0(-).After the the acquired plasmid transfected into Hela cells,G418 selection medium was used to screen, and the cell strains expressing N1 protein was obtained. Identification and analysis results of the cell line by RT-PCR and Western-blotting, The results show that the Hela cell lines stably expressing GTPV Nl protein were established successfully.The research will lay a foundation for exploring the function of N1 protein in the process of GTPV infection host cells.