表达增强型绿色荧光蛋白的重组脑心肌炎病毒的构建与鉴定OA

This study aimed to construct a recombinant encephalomyocarditis virus expressing enhanced green fluorescent protein(EGFP)using reverse genetics and to investigate its biological characteristics.An EGFP-expressing recombinant plasmid,p CI-EMCV-EGFP,was generated by overlap polymerase chain reaction(PCR)and transfected into baby hamster syrian kidney cells(BHK-21)to rescue the recombinant fluorescent virus rEMCV-EGFP.The rEMCV-EGFP was identified using a polyclonal antibody against the EMCV 2C protein by indirect immunofluorescence assay(IFA)and Western blot(WB).Its biological properties were further evaluated by growth curve analysis,plaque assays,and genetic stability tests.Sequencing confirmed the successful construction of the recombinant plasmid pCI-EMCV-EGFP.The rescued rEMCV-EGFP stably expressed EGFP.Typical cytopathic effects appeared at 36 h post-infection,which was approximately 6 h later than those observed for the EMCV wild-type strain and rEMCV.IFA and WB results demonstrated that rEMCV-EGFP could normally express the EMCV 2C protein while simultaneously expressing EGFP.Growth curve analysis and plaque assays showed that,compared with the EMCV wild-type strain and rEMCV,the viral titers at all time points after infection with rEMCV-EGFP were significantly reduced(P<0.000 1,P<0.001,P<0.01,or P<0.05),and both plaque size and plaque number were extremely significantly decreased(P<0.000 1).Genetic stability assays revealed that rEMCV-EGFP continuously expressed EGFP over six serial passages,and the EGFP gene could be stably detected by reverse transcription-PCR(RT-PCR).These results indicate that the recombinant fluorescent virus r EMCV-EGFP was successfully constructed using reverse genetics,providing a valuable platform for studies on EMCV pathogenic mechanisms,tissue tropism,antiviral drug screening,and the development of novel vaccines.