Khadijeh Fanayi, Mehdi Ajorloo , Sayed Hamid Reza Mozhgani , Shiva Irani , Alireza Gholami ,
Volume 72, Issue 5 (August 2014)
Abstract
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.
Bita Soltanian , Shiva Irani , Sarvenaz Hashemi , Seyed Hamid Reza Mozhgani , Mehdi Ajorloo, Yoosef Cheraghi , Alireza Gholami ,
Volume 72, Issue 11 (February 2015)
Abstract
Background: Mycoplasma contamination in cell cultures is considered as a major economic, research and production problem. In this study, mycoplasma-infected Vero cell lines were treated by various dilutions of ciprofloxacin and enrofloxacin in a timely manner. Removal of mycoplasma contamination from infected cell cultures was evaluated and demonstrated by polymerase chain reaction (PCR) method.
Methods: This study was done from October 2013 to May 2014, in Human Rabies Vaccine Laboratory, Pasteur Institute Production and Research Complex, Tehran, Iran. Different dilutions of ciprofloxacin and enrofloxacin were used in sequential passages for treatment of infected Vero cell line. Based on lowest passages of the cell line, antibiotic treatment with ciprofloxacin and enrofloxacin was done. Amelioration of the infection and removal of mycoplasma contamination was confirmed in each step by PCR method. The technique for order of preference by similarity to ideal solution, TOPSIS method, was used to suggest the most efficient concentration of ciprofloxacin and enrofloxacin.
Results: Proposed concentration of ciprofloxacin is 20 μg/ml, and in the second order is 200 μg/ml. For enrofloxacin the best proposed concentrations are 30, 300 and 3 μg/ml respectively. Ciprofloxacin and enrofloxacin and ability of them for removal of mycoplasma and also the time of treatment were verified by evaluation of the recurrence of infection through consecutive subcultures of the treated cell line.
Conclusion: Our results showed that 20 μg/ml of ciprofloxacin was the dilution of choice for mycoplasma elimination followed by 200 μg/ml of ciprofloxacin. Concentrations of 3, 30 and 300 of enrofloxacin, respectively, are appropriate for mycoplasma removal. More detailed works would be needed to verify the authenticity of the proposed simple and affordable way of mycoplasma elimination.
