Rescue Of The Minigenome Of Human Respiratory Syncytial Virus Based On T7Promoter Expression System

Object: In order to rescue the cDNA of human respiratory syncytial virus (RSV), weestablish a T7promoter based reverse genetics system and finish the rescue of theminigenome of RSV.Methods: We construct four helper plasmids of px8δT-PT1-N, px8δT-PT1-P,px8δT-PT1-M2-1and px8δT-PT1-L encoding RSV nucleocapsid proteins, respectively,and one mini-antigenome plasmid of pSC11-E containing open reading frame (ORF) ofthe enhanced green flurorecent protein (EGFP) and cis-acting elements including RSVleader region/promoter, gene start (GS), gene end (GE) and trailer region/antigenomicpromoter. All these plasmids are under the control of T7promoter, and identified byrestriction endonucleoase analysis and Western blot. The pSC11-E is rescued by RSVand the above helper plasmids in BSR T7/5cells. Then, the fluorescence expression isobserved over time with fluorescence microscopy.Result: We successfully constructed a reverse genetic system consisting of T7promoterbased5plasmids and finished the operation to rescue the minigenome of RSV.Conclusion: This system paves the way to further investigate the function of RSVgenome by deletion and mutation of its genes.