title: Construction of bacterial ghosts for transfer and expression of a chimeric hepatitis C virus gene in macrophages.
abstract: 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.
authors.affiliation: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: M R
authors.initials: MR
authors.lastname: Miri
authors.affiliation: 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.
authors.forename: A
authors.initials: A
authors.lastname: Behzad-Behbahani
authors.affiliation: Department of Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Fardaei
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: A
authors.initials: A
authors.lastname: Farhadi
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Y
authors.initials: Y
authors.lastname: Talebkhan
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Mohammadi
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Tayebinia
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: F
authors.initials: F
authors.lastname: Farokhinejad
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: P
authors.initials: P
authors.lastname: Alavi
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Fanian
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: F
authors.initials: F
authors.lastname: Zare
authors.affiliation: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: J
authors.initials: J
authors.lastname: Saberzade
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: N
authors.initials: N
authors.lastname: Nikouyan
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: M A
authors.initials: MA
authors.lastname: Okhovat
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: R
authors.initials: R
authors.lastname: Ranjbaran
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: G
authors.initials: G
authors.lastname: Rafiei Dehbidi
authors.affiliation: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: S
authors.initials: S
authors.lastname: Naderi
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques / Q000295:instrumentation; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000821:virology*; D051379:Mice; D000067996:RAW 264.7 Cells; D014162:Transfection
keywords: Bacterial ghost; Chimeric gene; HCV; SOEing PCR