Construction and Growth Characteristics of Recombinant Lumpy Skin Disease Virus Carrying eGFP Gene
[Objective]The aim of this experiment was to screen different promoter/cell combinations that could efficiently express recombinant Lumpy skin disease virus(LSDV),and thus construct recombinant LSDV carrying enhanced green fluorescent protein(eGFP)gene.The growth characteristics of the virus were studied,which laid the foundation for the development of antiviral drugs and the study of viral replication and mechanism of action.[Method]The Orf virus(ORFV)promoter and LSDV promoter were linked to eGFP gene by fusion PCR to replace LSDV 005 gene and construct homologous recombinant fragment,and transfect it into African green monkey kidney cells(Vero),African green monkey kidney cell derived line(Vero-E6),Madin-Darby bovine kidney cell(MDBK),hamster ovary cells(CHO-K1),lamp testicular cells(LT),and primary lamp testicular cells(PLT)infected with LSDV,respectively.The transfection effect was observed by fluorescence microscopy,and the optimal promoter/cell combination was selected by using ImageJ software to calculate the percentage of fluorescence area.The recombinant virus was transfected with the optimal combination,and the purification effect of the virus was tested.The growth characteristics and stability of recombinant virus were further studied.[Result]The transfection effect of LSDV promoter was superior to that of ORFV promoter in all 6 kinds of cells,and the fluorescence area percentage of LSDV promoter in PLT cells was the highest(8.38％),which was significantly different from that of other groups(P＜0.05).The recombinant virus LSDV-A005/eGFP was constructed using the combination of LSDV promoter/PLT cells.After purification,green fluorescence was observed in all the lesion sites under inverted fluorescence microscope,and the fluorescence area percentage was about 50.8％.The growth characteristics and stability analysis of the virus showed that the pathological effect of the recombinant virus LSDV-A005/eGFP in 6 kinds of cells and the trend of viral titer change over time was similar to that of the wild virus LSDV/Heilongjiang/2022,and the eGFP gene was statically expressed in PLT cells after 10 consecutive generations.[Conclusion]The recombinant LSDV was constructed using the combination of LSDV promoter/PLT cells,and the growth characteristics of the recombinant LSDV-A005/eGFP were consistent with those of wild LSDV,and the genetic stability was good after 10 passages.
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