Cloning of nucleoprotein gene of rinderpest virus and expression in eucaryotic cells

2. ABSTRACT In the present study, cloning and expression nucleoprotein (NP) gene of rinderpest virus (RPV) RBOK vaccine strain in eucaryotic cells are reported. For this purpose, total RNA extracted from RPV-RBOK infected Vero cells was subjected to reverse transcription-polymerase chain reaction (RT-PCR) with the NP gene spesific primers. After RT-PCR, a 1575 bp amplicon was yielded. The NP gene of RPV-RBOK vaccine strain was cloned into pGEM-3Zf (-) cloning vector. The presence of the NP gene was confirmed by PCR-screening and enzyme digestion assays. The recombinant plasmid was cut with Nhel and Sail enzymes to obtain the NP gene. Then, the NP gene was placed into pCI-neo mammalian expression vector. To confirm the presence of the NP gene in pCI-neo vector; PCR-screening and enzyme digestion assays were performed. So, the resulting recombinant plasmid was named as pSV-NP. Afterwards, pSV-NP was transiently transfected into Vero cells. The presence of the NP proteins was showed by Western Immunoblotting assay demonstrating that Vero cells express a 60 kDa protein reacting with anti-NP antibody. Key words: Rinderpest virus, nucleoprotein gene, cloning, protein expression.