| In order to study the biological function of Goatpox virus(GTPV)NIL gene(gN1L),this study constructed a vaccinia virus Tiantan(VTT)NIL gene(vN1L)fragment as the left and right homologous recombination arm.A transfer vector plasmid pLR-EGFP-gpt having a VACV early/late strong composite promoter(PE/L),a green fluorescent protein(EGFP)gene,and an Escherichia coli xanthine-guanine phosphoribosyltransferase(gpt)gene.The vN1L and gN1L gene expression cassettes were inserted into pLR-EGFP-gpt,respectively,and the transfer vector plasmids pLR-EGFP-gpt-N1Lrev and pLR-EGFP-gpt-gN1L were constructed.The constructed transfer vector and VTT were transfected together into BHK-21 cells,and the recombinant virus plaque expressing green fluorescent protein was screened and identified one by one,and finally the recombinant strain VTTAN1L lacking the vN1L gene was obtained.The recombinant strain VTT N1Lrev of the vN1L gene was rescued and the strain VTTgN1L of the vN1L gene was replaced with the gN1L gene.The stability,host range,replication,and virulence of the recombinant virus and the parent strain were evaluated by in vitro cell assays and animal in vivo assays.The results showed that the three recombinant viruses could be stably passaged at least 15 times in BHK-21 cells,and there was no significant difference in host range.VTT N1Lrev has the fastest replication rate in BHK-21 cells,followed by VTTgN1L,VTT and VTTAN1L.This study initially confirmed that the vN1L and gN1L genes have a function of promoting the speed of viral replication The virulence test in vitro and in vivo showed that VTT N1Lrev had the strongest virulence,followed by VTTgN1L,VTT and VTT?NIL.The virulence of VTTgN1L and VTT was not significantly different,and VTTAN1L had the lowest virulence.Conclusions,both vN1L and gN1L genes can significantly enhance viral virulence and replication,while the effect of gN1L gene on viral virulence and replication is weaker than that of vN1L gene. |
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