Abstract: The purpose of this study was to construct a recombinant feline panleukopenia virus (FPV) carrying green fluorescent protein (GFP) in order to detect neutralizing antibody more quickly and effectively.GFP specific primers were designed by referring to the GFP gene sequence in GenBank,and the GFP gene carrying AgeⅠ and NheⅠ digestion sites was amplified by PCR,and plasmid pFPV preserved in laboratory was subjected to site-directed mutagenesis to obtain the Age Ⅰ and Nhe Ⅰ restriction sites.pFPV and GFP genes were double-digested with Age Ⅰ and Nhe Ⅰ,and ligated at 4 ℃ overnight and transformed to obtain the recombinant plasmid pFPV-GFP.The recombinant plasmid pFPV-GFP was successfully transfected into feline kidney cells (F81) by Lipofection 3000.Using the unique luminescence mechanism of GFP,the labeled recombinant virus rFPV-GFP could be observed in vitro under fluorescence microscope,and Western blotting was used to identify the expression of GFP in the recombinant virus rFPV-GFP.The results showed that green fluorescent protein could be observed when the recombinant plasmid pFPV-GFP was transfected into F81 cells for 24 hours,and with the extension of transfection time,the amount of green fluorescent protein was increased,indicating that the recombinant plasmid pFPV-GFP was successfully transfected into F81 cells and GFP was stably expressed in the recombinant virus rFPV-GFP.In this study,GFP was successfully inserted into pFPV and rescued recombinant virus,indicating that foreign genes could be inserted into pFPV,laying a foundation for the insertion of other foreign genes into pFPV.At the same time,GFP insertion provided a more favorable research tool for the research of FPV,which laid the foundation for the basic research of FPV and the rapid detection of neutralization antibodies.

Key words: feline panleukopenia virus (FPV); green fluorescent protein (GFP); recombinant virus; rescue