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Construction and preparation of three recombinant adenoviruses expressing truncated NS3 and core genes of hepatitis C virus for vaccine purposes.

Hosseini SY, Sabahi F, Moazzeni SM, Modarressi MH, Saberi Firoozi M, Ravanshad M - Hepat Mon (2012)

Bottom Line: The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared.The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells.These adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Tarbiat Modares University, Tehran, IR Iran ; Gastroentero -Hepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran.

ABSTRACT

Background: In spite of dozens of clinical trials to establish effective therapeutic and/or preventive vaccine to resolve HCV infection, no real vaccine has been proved to date. Genetic vaccines based on replication-defective adenoviruses have proved to elicit strong and long lasting T-cell responses against a number of viral antigens and are even currently being used for vaccine trials in humans. According to the controversy in the immune modulatory effects of both core and NS3 full length genes, it seemed more practical to employ some parts of these HCV proteins for vaccine design.

Objectives: To generate recombinant Adenoviral vectors containing new overlapping-truncated region of NS3 gene or both the N- and C-terminal deleted parts of core gene, as well as a fusion fragment derived from both of them.

Materials and methods: The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared. The integrity and sequence of the transfer vectors were confirmed, and followed by experiments involving homologous recombination between them and the adenovirus backbone plasmid in the bacterial host. Recombinant Ad-pNS3, Ad-pCore and Ad-pNS3pCore viruses were prepared by transfection of these new recombined constructs into 293 packaging cell lines. The virus titer was then calculated by an immunohistochemistry based method. The RT-PCR, Real-Time PCR and western blotting were used to evaluate gene expression by all recombinant constructs. The production of complete virion particles was evaluated by detailed electron microscopy in addition to the appearance of typical cytopathic effects (CPE) and GFP expression patterns in 293 cells. The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells.

Results: RT-PCR, Real-Time PCR or western blotting confirmed expression of truncated fragment of NS3, core or a fusion fragment of theirs by newly constructed Ad-pNS3, Ad-pCore, Ad- pNS3pCore particles. Electron microscopy, which revealed many adenovirus-like particles and characteristics of CPE in infected cells in addition to GFP detection, confirmed the infectivity, potency and integrity of recombinant adenoviral particles.

Conclusions: These adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.

No MeSH data available.


Related in: MedlinePlus

Digestion Analysis of Recombinant Adenovirus Constructs by BstXI and PacI(A) Restriction Pattern for recombinant Ad-pCore construct, 1: negative control including pAdenovator Ad5 genome digested with BstXI and appearance of special 6 band pattern. 2 and 3: Two true colonies of Ad-pCore construct digested with BstXI. Appearance of a nearly 700 bp band as evidence of Core gene insertion, presence of a 2.1 kb fragment as recombination clue as well as disappearance of original 8 kb band(bracket) in recombinant construct approved vector, 4: DNA ladder 1kb Fermentas cop., Cat. Number: SM1163, 5 and 6: true colonies of Ad-pCore constructs digested with PacI. Appearance of a 4.5 kb and big fragment (upper than 10kb) was a sign of gene insertion and vector integrity, 7: DNA ladder, (B) Restriction Pattern for recombinant Ad-pNS3 construct 8: digestion of Ad-pNS3 construct with PacI showed the appearance of a 4.5 kb and a big fragment (upper than 10kb) which implied the vector integrity, 9: ladder, 10: a false/ wrong colony digested with BstXI, 11: digestion of right Ad-pNS3 construct by BstXI, detection of 2.1 kb fragment and disappearance of original 8 kb (bracket) band was evidence of gene insertion as well as construct integrity.
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fig37: Digestion Analysis of Recombinant Adenovirus Constructs by BstXI and PacI(A) Restriction Pattern for recombinant Ad-pCore construct, 1: negative control including pAdenovator Ad5 genome digested with BstXI and appearance of special 6 band pattern. 2 and 3: Two true colonies of Ad-pCore construct digested with BstXI. Appearance of a nearly 700 bp band as evidence of Core gene insertion, presence of a 2.1 kb fragment as recombination clue as well as disappearance of original 8 kb band(bracket) in recombinant construct approved vector, 4: DNA ladder 1kb Fermentas cop., Cat. Number: SM1163, 5 and 6: true colonies of Ad-pCore constructs digested with PacI. Appearance of a 4.5 kb and big fragment (upper than 10kb) was a sign of gene insertion and vector integrity, 7: DNA ladder, (B) Restriction Pattern for recombinant Ad-pNS3 construct 8: digestion of Ad-pNS3 construct with PacI showed the appearance of a 4.5 kb and a big fragment (upper than 10kb) which implied the vector integrity, 9: ladder, 10: a false/ wrong colony digested with BstXI, 11: digestion of right Ad-pNS3 construct by BstXI, detection of 2.1 kb fragment and disappearance of original 8 kb (bracket) band was evidence of gene insertion as well as construct integrity.

Mentions: The IR-pCore, IR-pNS3 and IR-pNS3pCore transfer vectors were separately transformed with supercoiled pAdenovator Δ E1/E3 into BJ bacterial host and, consequently, by homologous recombination, new Adenovirus recombinant plasmids were created as shown in Figure 1. As a confirmatory step to evaluate recombinant Ad-pCore, Ad-pNS3 and Ad-pNS3pCore integrity, these constructs were treated and analyzed with BstXI and PacI restriction enzymes according to companies’ guidelines. Based on the guidelines, digestion of an Adenovirus containing gene of insert with BstXI enzyme should appear in a six band pattern, especially a 2.1 kb fragment as evidence of gene insertion after a long run on 1% agarose gel. In the case of Ad-pNS3, the result was the same as expected but for Ad-pCore and Ad-pNS3pCore, because of the presence of the newly emerged BstXI restriction site at original core gene, Other bands around 700 and 900 base pairs were detected, which confirmed gene insertions as shown in Figure 2. Digestion analysis by PacI also showed the presence of a 4.5 kb fragment below a big plasmid backbone fragment as further evidence for integrity and also right insertion of genes into the construct (Figure 2).


Construction and preparation of three recombinant adenoviruses expressing truncated NS3 and core genes of hepatitis C virus for vaccine purposes.

Hosseini SY, Sabahi F, Moazzeni SM, Modarressi MH, Saberi Firoozi M, Ravanshad M - Hepat Mon (2012)

Digestion Analysis of Recombinant Adenovirus Constructs by BstXI and PacI(A) Restriction Pattern for recombinant Ad-pCore construct, 1: negative control including pAdenovator Ad5 genome digested with BstXI and appearance of special 6 band pattern. 2 and 3: Two true colonies of Ad-pCore construct digested with BstXI. Appearance of a nearly 700 bp band as evidence of Core gene insertion, presence of a 2.1 kb fragment as recombination clue as well as disappearance of original 8 kb band(bracket) in recombinant construct approved vector, 4: DNA ladder 1kb Fermentas cop., Cat. Number: SM1163, 5 and 6: true colonies of Ad-pCore constructs digested with PacI. Appearance of a 4.5 kb and big fragment (upper than 10kb) was a sign of gene insertion and vector integrity, 7: DNA ladder, (B) Restriction Pattern for recombinant Ad-pNS3 construct 8: digestion of Ad-pNS3 construct with PacI showed the appearance of a 4.5 kb and a big fragment (upper than 10kb) which implied the vector integrity, 9: ladder, 10: a false/ wrong colony digested with BstXI, 11: digestion of right Ad-pNS3 construct by BstXI, detection of 2.1 kb fragment and disappearance of original 8 kb (bracket) band was evidence of gene insertion as well as construct integrity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475015&req=5

fig37: Digestion Analysis of Recombinant Adenovirus Constructs by BstXI and PacI(A) Restriction Pattern for recombinant Ad-pCore construct, 1: negative control including pAdenovator Ad5 genome digested with BstXI and appearance of special 6 band pattern. 2 and 3: Two true colonies of Ad-pCore construct digested with BstXI. Appearance of a nearly 700 bp band as evidence of Core gene insertion, presence of a 2.1 kb fragment as recombination clue as well as disappearance of original 8 kb band(bracket) in recombinant construct approved vector, 4: DNA ladder 1kb Fermentas cop., Cat. Number: SM1163, 5 and 6: true colonies of Ad-pCore constructs digested with PacI. Appearance of a 4.5 kb and big fragment (upper than 10kb) was a sign of gene insertion and vector integrity, 7: DNA ladder, (B) Restriction Pattern for recombinant Ad-pNS3 construct 8: digestion of Ad-pNS3 construct with PacI showed the appearance of a 4.5 kb and a big fragment (upper than 10kb) which implied the vector integrity, 9: ladder, 10: a false/ wrong colony digested with BstXI, 11: digestion of right Ad-pNS3 construct by BstXI, detection of 2.1 kb fragment and disappearance of original 8 kb (bracket) band was evidence of gene insertion as well as construct integrity.
Mentions: The IR-pCore, IR-pNS3 and IR-pNS3pCore transfer vectors were separately transformed with supercoiled pAdenovator Δ E1/E3 into BJ bacterial host and, consequently, by homologous recombination, new Adenovirus recombinant plasmids were created as shown in Figure 1. As a confirmatory step to evaluate recombinant Ad-pCore, Ad-pNS3 and Ad-pNS3pCore integrity, these constructs were treated and analyzed with BstXI and PacI restriction enzymes according to companies’ guidelines. Based on the guidelines, digestion of an Adenovirus containing gene of insert with BstXI enzyme should appear in a six band pattern, especially a 2.1 kb fragment as evidence of gene insertion after a long run on 1% agarose gel. In the case of Ad-pNS3, the result was the same as expected but for Ad-pCore and Ad-pNS3pCore, because of the presence of the newly emerged BstXI restriction site at original core gene, Other bands around 700 and 900 base pairs were detected, which confirmed gene insertions as shown in Figure 2. Digestion analysis by PacI also showed the presence of a 4.5 kb fragment below a big plasmid backbone fragment as further evidence for integrity and also right insertion of genes into the construct (Figure 2).

Bottom Line: The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared.The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells.These adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Tarbiat Modares University, Tehran, IR Iran ; Gastroentero -Hepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran.

ABSTRACT

Background: In spite of dozens of clinical trials to establish effective therapeutic and/or preventive vaccine to resolve HCV infection, no real vaccine has been proved to date. Genetic vaccines based on replication-defective adenoviruses have proved to elicit strong and long lasting T-cell responses against a number of viral antigens and are even currently being used for vaccine trials in humans. According to the controversy in the immune modulatory effects of both core and NS3 full length genes, it seemed more practical to employ some parts of these HCV proteins for vaccine design.

Objectives: To generate recombinant Adenoviral vectors containing new overlapping-truncated region of NS3 gene or both the N- and C-terminal deleted parts of core gene, as well as a fusion fragment derived from both of them.

Materials and methods: The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared. The integrity and sequence of the transfer vectors were confirmed, and followed by experiments involving homologous recombination between them and the adenovirus backbone plasmid in the bacterial host. Recombinant Ad-pNS3, Ad-pCore and Ad-pNS3pCore viruses were prepared by transfection of these new recombined constructs into 293 packaging cell lines. The virus titer was then calculated by an immunohistochemistry based method. The RT-PCR, Real-Time PCR and western blotting were used to evaluate gene expression by all recombinant constructs. The production of complete virion particles was evaluated by detailed electron microscopy in addition to the appearance of typical cytopathic effects (CPE) and GFP expression patterns in 293 cells. The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells.

Results: RT-PCR, Real-Time PCR or western blotting confirmed expression of truncated fragment of NS3, core or a fusion fragment of theirs by newly constructed Ad-pNS3, Ad-pCore, Ad- pNS3pCore particles. Electron microscopy, which revealed many adenovirus-like particles and characteristics of CPE in infected cells in addition to GFP detection, confirmed the infectivity, potency and integrity of recombinant adenoviral particles.

Conclusions: These adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.

No MeSH data available.


Related in: MedlinePlus