Construction and rescue of the minigenome of bovine parainfluenza virus type 3 based on T7 promoter expression system

Abstract:

[Objective] To establish a T7 promoter based reverse genetics system competent for the rescue of bovine parainfluenza virus type 3 (BPIV3).[Methods] We constructed three helper plasmids of px8δT-PT1-bPIV3-NP, px8δT-PT1-bPIV3-P and px8δT-PT1-bPIV3-L and one minigenome plasmid of pSC11-bPIV3-EGFP containing open reading frame (ORF) of the enhanced green fluorescent protein (EGFP) and cis-acting elements including BPIV3 leader region, gene start (GS), gene end (GE) and trailer region. All these plasmids are under the control of T7 promoter and identified by restriction endonuclease analysis. We rescued the pSC11-bPIV3-EGFP by two different methods. Then, we observed the fluorescence expression over time with fluorescence microscopy.[Results] We successfully constructed a reverse genetic system based 4 plasmids under the control of T7 promoter and finished the rescue operation to the minigenome of BPIV3.[Conclusion] This system can be further applied to investigate the function of BPIV3 genome by deletion and mutation of its genes.

Key words:bovine parainfluenza virus type 3;minigenome;reverse genetics;rescue