[Construction of minireplicon of human respiratory syncytial virus]

The reverse genetics technology is an important way to develop genetically engineered attenuated living human respiratory syncytial virus (RSV) vaccine candidates. As the pilot experiment,it is necessary to prepare RSV minireplicon and investigate its biological activity.

After the gene start and gene end (GSGE) fragment containing the sequence of leader (Le), gene start (GS), multiple cloning sites (MCS), gene end (GE) and trailer (Tr) was synthesized and positioned under the control of T7 RNA promoter, we cloned this fragment further into px8deltaT vector. Then, the enhanced green fluorescent protein (EGFP) was cloned into the above recombinant px8deltaT vector, and RSV minireplicon plasmids px8deltaT/GSGE1/EGFP and px8deltaT/GSGE2/EGFP were finally obtained. Meanwhile, two ORFs of nucleocapsid proteins of large protein (L) and transcription elongation/antitermination factor (M2-1) were cloned to construct pcDNA3.1/L and pcDNA3.1/M2-1. At the end, we co-transfected a RSV minireplicon plasmid and four nucleocapsid protein plasmids into BSR T7/5 cell lines expressing T7 RNA polymerase by lipofectamine 2000, and analyed the expression of EGFP by inverted fluorescent microscopy and fluorescence activated cell sortor (FACS), respectively.

The px8deltaT/GSGE1/EGFP, px8deltaT/GSGE2/EGFP, pcDNA3.1/L and pcDNA3.1/M2-1 were successfully constructed. After co-transfecting, EGFP can be observed in the transfected BSR T7/5 cells under fluorescent microscopy and by FACS.

The constructed RSV minireplicon is able to replicate and transcript, which provides a solid foundation for further RSV vaccine development and drug selection by RSV reverse genetics.