Design, construction and expression of recombinant vector containing the rabies virus nucleoprotein gene
Tehran University Medical Journal [TUMJ]. 2014; 72 (5): 294-300
em Persa
| IMEMR
| ID: emr-178174
ABSTRACT
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. 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]. 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. 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
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Índice:
IMEMR (Mediterrâneo Oriental)
Assunto principal:
Proteínas Virais
/
Vacina Antirrábica
/
Expressão Gênica
/
Imunofluorescência
/
Nucleoproteínas
Idioma:
Persa
Revista:
Tehran Univ. Med. J. [TUMJ]
Ano de publicação:
2014
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