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

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.

Gene disruption in Salmonella typhimurim by modified λ Red disruption system.

There are many techniques to knock out directed genes in bacteria, some of which have been described in Salmonella species. In this study, a combination of SOEing PCR method and the λ Red disruption system were used to disrupt phoP gene in wild type and standard strains of Salmonella typhimurium. Three standards PCR and one fusion PCR reactions were performed to construct a linear DNA including upstream and downstream of phoP gene and Kanamycin cassette. As a template plasmid, we used pKD4 which carries kanamycin gene flanked by FRT (FLP recognition target) sites. The resulting construct was electroporated into prepared competent cells of S. typhimurium. The transformants colonies related to the standard strain appeared on the LB-Km-agar plates after incubation, but there was no colony on LB-Km-agar plates corresponding to the wild type strain. The failure in transformation of the wild type strain may be because of inflexibility of the λ Red disruption system in this strain or its unique restriction-modification system. However, by this construct we are able to generate phoP mutant in many of the Salmonella species due to high homology of the phoP gene which exists in different species.

Construction and eukaryotic expression of recombinant large hepatitis delta antigen.

BACKGROUND: Hepatitis delta virus (HDV) is a subviral human pathogen that exploits host RNA editing activity to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the large form (L-HDAg), which is required for RNA packaging. METHODS: In this study, PCR-based site-directed mutagenesis by the overlap extension method was used to create the point mutation converting the small-HDAg (S-HDAg) stop codon to a tryptophan codon through three stages. RESULTS: Sequencing confirmed the desirable mutation and integrity of the L-HDAg open reading frame. The amplicon was ligated into pcDNA3.1 and transfected to Huh7 and HEK 293 cell lines. Western blot analysis using enhanced chemiluminescence confirmed L-HDAg expression. The recombinant L-HDAg localized within the nuclei of cells as determined by immunofluorescence and confocal microscopy. CONCLUSION: Because L-HDAg requires extensive post-translational modifications, the recombinant protein expressed in a mammalian system might be fully functional and applicable as a tool in HDV molecular studies, as well as in future vaccine research.

Replacing CTL epitope of recombinant HBsAg by SOEing PCR / 第三军医大学学报

Objective To replace the endogenous CTL epitope LLD in HBsAg with EYILSLEEL of glypican(GPC3)in order to prepare a GPC3-HBsAg multiple peptides vaccine for HBV infection.Methods The HBsAg/GPC3 DNA was amplified by gene splicing by overlap extension(SOEing)PCR from pcHBsAg plasmid and then inserted into pBSSK+ vector to construct a pBSSK/GPC3 vector.The vector was then identified by PCR,double digesting and sequencing.The fragment encoding GPC3 CTL epitope EYILSLEEL was obtained by double digesting the plasmid pBSSK/GPC3 and then inserted into pcDNA3.1+ vector.Results Sequencing and restrict endonulease digestion indicated that eukaryotic expression vector pcDNA-HBsAg/GPC3 was constructed successfully.Conclusion The endogenous CTL epitope LLD of HBsAg is replaced by the EYILSLEEL,and an eukaryotic expression vector pcDNA-HBsAg/GPC3 is constructed.