Design, construction and expression of recombinant vector containing the rabies virus nucleoprotein gene

Fanayi, Khadijeh; Ajorloo , Mehdi; Mozhgani , Sayed Hamid Reza; Irani , Shiva; Gholami , Alireza

Volume 72, Issue 5 (August 2014) Tehran Univ Med J 2014, 72(5): 294-300 | Back to browse issues page

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Fanayi K, Ajorloo M, Mozhgani S H R, Irani S, Gholami A. Design, construction and expression of recombinant vector containing the rabies virus nucleoprotein gene. Tehran Univ Med J 2014; 72 (5) :294-300
URL: http://tumj.tums.ac.ir/article-1-6143-en.html

Design, construction and expression of recombinant vector containing the rabies virus nucleoprotein gene

Khadijeh Fanayi¹

, Mehdi Ajorloo²

, Sayed Hamid Reza Mozhgani³

, Shiva Irani¹

, Alireza Gholami ^*

⁴

1- Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
2- Human Rabies Vaccine Labora-tory, Pasteurs Production and Research Complex, Institute of Iran, Tehran, Iran. Department of Virology, School of Medical Sciences, Tarbiat Mo-dares University, Tehran, Iran.
3- Department of Virology, Faculty of Public Health, Tehran Univer-sity of Medical Sciences, Tehran, Iran.
4- Human Rabies Vaccine Labora-tory, Pasteurs Production and Research Complex, Institute of Iran, Tehran, Iran.WHO CC For Reference and Research on Rabies, Pasteur Institute of Iran, Tehran, Iran. , agholami@pasteur.ac.ir

Abstract: (8353 Views)

Background: Rabies is an acute encephalitis that causes more than 60,000 deaths worldwide. The only way to save individuals bitten by a rabies-infected animal is the timely use of effective vaccines. Treatment with new generation vaccines is expensive. Therefore, there is a global movement towards the production of less expensive vaccines which retain and improve upon the quality and effectiveness of the vaccine. Production and evaluation of non-classical vaccines is one of the approaches taken in this regard. In this study, we describe a new eukaryotic expression system to express the nucleoprotein N gene of rabies virus which, if suitable, may be evaluated for anti-rabies vaccine production. Methods: The complete sequence of the N gene of rabies virus PV subtype was amplified by real-time polymerase chain reaction and cloned into the pCDNA3.1(+) vector. The cloned gene was excised from the vector by restriction enzyme digestion and sequenced. Due to mutations detected in the N gene, the gene coding sequence was purchased as a recombinant pGH/N vector. Vector pGH/N was amplified and following enzymatic digestion, the excised N gene was once again cloned into vector pCDNA3.1(+). Successful cloning was confirmed using restriction digests and quick check. The recombinant vector pCDNA3.1(+)/N was transformed into cultured BSR cells and protein N expression was analyzed using fluorescent antibody test (FAT). Results: Electrophoresis confirmed amplification of the nucleoprotein N gene and subsequent restriction enzyme digestion showed that the N gene had been successfully cloned into the recombinant pCDNA3.1(+)/N vector. However, DNA sequencing revealed the presence of mutations within the N gene. Restriction digest of the commercial pGH/N vector showed that the N gene had been excised from the vector. Successful cloning of the N gene into the pCDNA3.1(+) expression vector was confirmed using restriction digests and quick check. Protein expression in BSR cells was assayed by immunostaining with anti-ribonucleocapsid FITC-conjugated antibody and visually confirmed by fluorescence microscopy. Conclusion: This study showed that the protein N of rabies virus subtype PV can be expressed in a eukaryotic expression system using the pCDNA3.1(+) expression vector.

Keywords: FAT, rabies virus, ribonucleoproteins, sequence, vaccines

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Type of Study: Original Article |