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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

SDS-PAGE and b) western blotting of expression in E. coli and baculovirusProtein MWM, Lane 1 and 2: Expressions in E. coli with 0.5 and 1 mM IPTG, respectively, used for induction, Lane 3: BL21 strain transformed by mock plasmid as a negative control, Lane 4-7: Expressions in baculovirus with 1/20, 1/100, 1/500, 1/2500, respectively as a folding dilution of the initial transfection mixture. Lane 8: Sf9 cells transfected by mock plasmid as a negative control. Expressed subunit 32kDa is shown by arrows in SDS-PAGE and western blot.
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Figure 3: SDS-PAGE and b) western blotting of expression in E. coli and baculovirusProtein MWM, Lane 1 and 2: Expressions in E. coli with 0.5 and 1 mM IPTG, respectively, used for induction, Lane 3: BL21 strain transformed by mock plasmid as a negative control, Lane 4-7: Expressions in baculovirus with 1/20, 1/100, 1/500, 1/2500, respectively as a folding dilution of the initial transfection mixture. Lane 8: Sf9 cells transfected by mock plasmid as a negative control. Expressed subunit 32kDa is shown by arrows in SDS-PAGE and western blot.

Mentions: The recombinant clones with A subunit was selected and induced by addition of IPTG. The expressed protein was analyzed on SDS-PAGE electrophoresis and detected by western blotting. Western blot analysis was done by antibody against A subunit of shiga toxin that was raised previously (10). The induced clones in E. coli and baculovirus were assessed and the A subunit peptide as a 32KD protein was observed (Fig. 3). Expressions in baculovirus was detected with 1/20, 1/100, 1/500, 1/2500 dilutions as a folding dilution of the initial transfection mixture, respectively, in the Sf9 cells. In Baculovirus expression two bands was detected in comparison with E. coli STXA expression.


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)

SDS-PAGE and b) western blotting of expression in E. coli and baculovirusProtein MWM, Lane 1 and 2: Expressions in E. coli with 0.5 and 1 mM IPTG, respectively, used for induction, Lane 3: BL21 strain transformed by mock plasmid as a negative control, Lane 4-7: Expressions in baculovirus with 1/20, 1/100, 1/500, 1/2500, respectively as a folding dilution of the initial transfection mixture. Lane 8: Sf9 cells transfected by mock plasmid as a negative control. Expressed subunit 32kDa is shown by arrows in SDS-PAGE and western blot.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: SDS-PAGE and b) western blotting of expression in E. coli and baculovirusProtein MWM, Lane 1 and 2: Expressions in E. coli with 0.5 and 1 mM IPTG, respectively, used for induction, Lane 3: BL21 strain transformed by mock plasmid as a negative control, Lane 4-7: Expressions in baculovirus with 1/20, 1/100, 1/500, 1/2500, respectively as a folding dilution of the initial transfection mixture. Lane 8: Sf9 cells transfected by mock plasmid as a negative control. Expressed subunit 32kDa is shown by arrows in SDS-PAGE and western blot.
Mentions: The recombinant clones with A subunit was selected and induced by addition of IPTG. The expressed protein was analyzed on SDS-PAGE electrophoresis and detected by western blotting. Western blot analysis was done by antibody against A subunit of shiga toxin that was raised previously (10). The induced clones in E. coli and baculovirus were assessed and the A subunit peptide as a 32KD protein was observed (Fig. 3). Expressions in baculovirus was detected with 1/20, 1/100, 1/500, 1/2500 dilutions as a folding dilution of the initial transfection mixture, respectively, in the Sf9 cells. In Baculovirus expression two bands was detected in comparison with E. coli STXA expression.

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