Construction of bacterial ghosts for transfer and expression of a chimeric hepatitis C virus gene in macrophages.
M R Miri, A Behzad-Behbahani, M Fardaei, A Farhadi, Y Talebkhan, M Mohammadi, M Tayebinia, F Farokhinejad, P Alavi, M Fanian, F Zare, J Saberzade, N Nikouyan, M A Okhovat, R Ranjbaran, G Rafiei Dehbidi, S Naderi
Author Information
M R Miri: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
A Behzad-Behbahani: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran; Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran. Electronic address: behzadba@sums.ac.ir.
M Fardaei: Department of Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
A Farhadi: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Y Talebkhan: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
M Mohammadi: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
M Tayebinia: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
F Farokhinejad: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
P Alavi: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
M Fanian: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
F Zare: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
J Saberzade: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
N Nikouyan: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
M A Okhovat: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
R Ranjbaran: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
G Rafiei Dehbidi: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
S Naderi: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
The bacterial ghost (BG) production is a field of biotechnology for applications in vaccine and drug delivery. We assessed the capacity of BG for delivery of a recombinant gene encoded for both cell mediated and antibody dependent epitopes of hepatitis C virus (HCV) into murine macrophages. Escherichia coli (E. coli) cells were transformed with the lysis plasmid (pHH43). To produce chimeric gene, NS3 (non-structural protein 3) and core regions of HCV genome were fused together by splicing by overlap extension (SOEing) PCR and were cloned into plasmid pEGFP-C1. Bacterial ghosts were loaded with recombinant pEGFP-C1 and then were transferred to murine macrophages (RAW 264.7). To investigate plasmid transfection and chimeric mRNA transcription, fluorescent microscopy and RT-PCR were used. In vitro studies indicated that bacterial ghosts loaded with pEGFP-C1 plasmid were efficiently taken up by murine macrophages and indicated a high transfection rate (62%), as shown by fluorescent microscopy. RT-PCR from extracted intracellular mRNAs for chimeric Core-NS3 gene showed a specific 607 bp fragment of the gene. The sequence analysis of purified PCR products demonstrated the expected unique mRNA sequence. We constructed a chimeric HCV gene containing both cell mediated and antibody dependent epitopes with a significant expression in murine macrophages delivered by bacterial ghost.
