1. Construction and identification of helper plasmids of newcastle disease virus Italien strain
- Author
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Zhen REN, Ding WEI, Gang NAN, Fu-quan HU, Zhi-nan CHEN, and Hui-jie BIAN
- Subjects
lcsh:R5-920 ,animal structures ,oncolytic viruses, recombinant ,viruses ,lcsh:R ,genetic techniques ,lcsh:Medicine ,helper plasmid ,newcastle disease virus ,lcsh:Medicine (General) - Abstract
Objective Newcastle disease virus (NDV) is a naturally oncolytic virus that has been shown to be safe and effective for cancer therapy. NDV virions possess a non-segmented negative-sense single-stranded RNA genome which contains six genes encoding the nucleocapsid protein (NP), phosphoprotein (P), large polymerase protein (L), matrix protein, fusion protein, and hemagglutinin-neuraminidase. The ribonucleoprotein (RNP) complex consisting of the genomic RNA and the three proteins NP, P, and L are the active template for transcription and replication of the viral genome. The purpose of this study was to construct the expression plasmids of NP, P and L genes of NDV Italien strain in which phage T7 promoter was a transcription promoter for the aim of generation of recombinant NDV. Methods NP, P and L genes were cloned from the genome RNA of NDV Italien followed by introduction into the downstream of T7 promoter and internal ribosome entry sites to construct the expression plasmids of NP, P and L, respectively. Expression of exogenous gene in BSR-T7/5 cells which constitutively express phage T7 RNA polymerase and transfected with plasmids of NP and P was detected by indirect immunofluorescence assay. The function of NP, P and L proteins expressed by constructed plasmids to facilitate the genomic RNA to form RNP complex was tested using minigenome of NDV Italien carrying firefly luciferase as a reporter gene. Results The expression plasmids of NP, P and L genes were confirmed by DNA sequencing. Using the indirect immunofluorescence assay, we detected the expression of viral NP and P proteins in BSR-T7/5 cells. When the helper plasmids were co-transfected with NDV minigenome plasmid, the expression of firefly luciferase was more significant compared with the control group (P < 0.001). Conclusion The helper plasmids of NDV Italien strain using T7 promoter as a transcription promoter has been constructed successfully, and it provides a basis for the construction of recombinant NDV.
- Published
- 2012