Construction and application of dicistronic minigenome of human respiratory syncytial virus

Abstract:

[Objective] To clarify the role of nucleocapsid/polymerase proteins or helper proteins of human respiratory syncytial virus (RSV) in rescuing recombinant RSV, plasmids of RSV dicistronic minigenome encoding enhanced green fluorescent protein (EGFP) gene was constructed and rescued.[Methods] Based on the methods of gene synthesis and molecular cloning, RSV monocistronic minigenome plasmids, pUC57-RSV-EGFP encoding EGFP and pUC57-RSV-ORF1 encoding pseudo-virus protein, were constructed. Then, RSV dicistronic minigenome was further cloned through these two monocistronic minigenome plasmids and pBR322B vector, and identified by the analyses of restriction endonuclease and nucleotide acid sequence. Following co-transfecting helper plasmids to BHK-T7 cells together with RSV dicistronic minigenome plasmid, the function of helper proteins was evaluated based on the transcribed EGFP mRNA by real-time quantitative polymerase chain reaction (RT-qPCR) and the expressed EGFP by fluorescent microscope.[Results] The recombinant plasmid of RSV dicistronic minigenome encoding EGFP gene and driven by T7 promoter, pBR322B-RSVⅡ-EGFP, was constructed successfully. The differential expressions of EGFP both transcriptionally and translationally occurred following the dicistronic minigenome rescued by different combinations of the helper plasmids, which showed the helper proteins functioned differently in the replication of RSV.[Conclusion] The constructed RSV dicistronic minigenome containing EGFP gene is a useful tool for function analysis of the helper proteins, and the M2-1 protein, capable of elongating the transcription, is confirmed.

Key words:human respiratory syncytial virus;dicistronic;minigenome;helper proteins