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Construction of a Baculovirus vector containing A subunit of Shiga toxin for protein delivery.

Oloomi M, Bouzari S, Imani M, Akhtarian N - Iran J Microbiol (2013)

Bottom Line: The expression of STXA peptide (32kDa) was confirmed by SDS-PAGE and western blotting in both expression systems.The A subunit challenge to human cell Lines was applied as a delivery system by baculoviruses.Furthermore, it was shown that A subunit of Shiga toxin delivered by baculovirus can inhibit cell proliferation in HeLa cells and leading to cell death.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Department, Pasteur Institute of Iran, Pasteur Ave. 13164.

ABSTRACT

Background and objectives: Baculovirus can be used as a vector in gene delivery system. Viral envelope of baculovirus would display expressed protein/peptide and it could render as a potential vaccine delivery system. In this regard, the gene coding for A subunit of shiga toxin (StxA) from Escherichia coli (E. coli) strain was cloned in a baculovirus expression system. StxA subunit has the ability to inhibit protein synthesis and this ability applied in cancer therapy. In this study, expression of StxA in baculovirus as a protein delivery system was assessed in vitro.

Material and methods: StxA gene was cloned in pTriEx™ multisystem expression vector. This vector enables the protein expression in multisystem, E. coli and baculovirus. This construct was used to express the gene in E. coli and baculovirus. The construct containing StxA gene was made in baculovirus and expression was confirmed, then baculovirus expressing STXA transfect HeLa cells.

Results: The expression of STXA peptide (32kDa) was confirmed by SDS-PAGE and western blotting in both expression systems. The A subunit challenge to human cell Lines was applied as a delivery system by baculoviruses. On the other hand, the inhibition of cell proliferation was also demonstrated by baculovirus containing STXA subunit.

Conclusion: STXA peptide expression in baculovirus was shown in E. coli and baculovirus expression system. Furthermore, it was shown that A subunit of Shiga toxin delivered by baculovirus can inhibit cell proliferation in HeLa cells and leading to cell death. Therefore, this prototype system could be a promising model for in vivo cancer therapy and targeted protein delivery system.

No MeSH data available.


Related in: MedlinePlus

Plaque assay;a) Sf9 cells negative control (Mock)b) one day after virus dilution added to Sf9 cellsc) two days after virus dilution added to Sf9 cellsd) four days after virus dilution added to Sf9 cellsPlaques are shown by the arrows in the figure
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Figure 4: Plaque assay;a) Sf9 cells negative control (Mock)b) one day after virus dilution added to Sf9 cellsc) two days after virus dilution added to Sf9 cellsd) four days after virus dilution added to Sf9 cellsPlaques are shown by the arrows in the figure

Mentions: Recombinant plaques were visualized 3-4 days after adding transfection mixture directly to a Sf9 monolayer insect cell (Fig. 4). In Fig. 4 formation of plaques was shown gradually on monolayer of Sf9 cells, one day after adding virus to cells. Plaque formation was started, two days after adding virus dilution to Sf9 cells and it continues four days after infection. The formed plaques were calculated to determine the infectious titer.


Construction of a Baculovirus vector containing A subunit of Shiga toxin for protein delivery.

Oloomi M, Bouzari S, Imani M, Akhtarian N - Iran J Microbiol (2013)

Plaque assay;a) Sf9 cells negative control (Mock)b) one day after virus dilution added to Sf9 cellsc) two days after virus dilution added to Sf9 cellsd) four days after virus dilution added to Sf9 cellsPlaques are shown by the arrows in the figure
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4385160&req=5

Figure 4: Plaque assay;a) Sf9 cells negative control (Mock)b) one day after virus dilution added to Sf9 cellsc) two days after virus dilution added to Sf9 cellsd) four days after virus dilution added to Sf9 cellsPlaques are shown by the arrows in the figure
Mentions: Recombinant plaques were visualized 3-4 days after adding transfection mixture directly to a Sf9 monolayer insect cell (Fig. 4). In Fig. 4 formation of plaques was shown gradually on monolayer of Sf9 cells, one day after adding virus to cells. Plaque formation was started, two days after adding virus dilution to Sf9 cells and it continues four days after infection. The formed plaques were calculated to determine the infectious titer.

Bottom Line: The expression of STXA peptide (32kDa) was confirmed by SDS-PAGE and western blotting in both expression systems.The A subunit challenge to human cell Lines was applied as a delivery system by baculoviruses.Furthermore, it was shown that A subunit of Shiga toxin delivered by baculovirus can inhibit cell proliferation in HeLa cells and leading to cell death.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Department, Pasteur Institute of Iran, Pasteur Ave. 13164.

ABSTRACT

Background and objectives: Baculovirus can be used as a vector in gene delivery system. Viral envelope of baculovirus would display expressed protein/peptide and it could render as a potential vaccine delivery system. In this regard, the gene coding for A subunit of shiga toxin (StxA) from Escherichia coli (E. coli) strain was cloned in a baculovirus expression system. StxA subunit has the ability to inhibit protein synthesis and this ability applied in cancer therapy. In this study, expression of StxA in baculovirus as a protein delivery system was assessed in vitro.

Material and methods: StxA gene was cloned in pTriEx™ multisystem expression vector. This vector enables the protein expression in multisystem, E. coli and baculovirus. This construct was used to express the gene in E. coli and baculovirus. The construct containing StxA gene was made in baculovirus and expression was confirmed, then baculovirus expressing STXA transfect HeLa cells.

Results: The expression of STXA peptide (32kDa) was confirmed by SDS-PAGE and western blotting in both expression systems. The A subunit challenge to human cell Lines was applied as a delivery system by baculoviruses. On the other hand, the inhibition of cell proliferation was also demonstrated by baculovirus containing STXA subunit.

Conclusion: STXA peptide expression in baculovirus was shown in E. coli and baculovirus expression system. Furthermore, it was shown that A subunit of Shiga toxin delivered by baculovirus can inhibit cell proliferation in HeLa cells and leading to cell death. Therefore, this prototype system could be a promising model for in vivo cancer therapy and targeted protein delivery system.

No MeSH data available.


Related in: MedlinePlus