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J Microbiol Methods. 2015 Dec;119:228-32. doi: 10.1016/j.mimet.2015.11.009. Epub 2015 Nov 11.

Construction of bacterial ghosts for transfer and expression of a chimeric hepatitis C virus gene in macrophages.

Author information

1
Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
2
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.
3
Department of Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
4
Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
5
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

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.

KEYWORDS:

Bacterial ghost; Chimeric gene; HCV; SOEing PCR

PMID:
26578242
DOI:
10.1016/j.mimet.2015.11.009
[Indexed for MEDLINE]

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