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Production of human papilloma virus type 16 e6 oncoprotein as a recombinant protein in eukaryotic cells.

Mirshahabi H, Soleimanjahi H, Pourpak Z, Meshkat Z, Hassan ZM - Iran J Cancer Prev (2012)

Bottom Line: These oncogenic proteins represent ideal target antigens for developing vaccine and immunotherapeutic strategies against HPV-associated neoplasm.The transfected cells were harvested; mRNA detection and the interest protein production were confirmed by western blot analysis using specific anti E6 monoclonal antibody.Due to potential effect of this protein, its DNA construction can be used for DNA vaccine in future studies.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Virology and Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

ABSTRACT

Background: Cervical cancer is one of the most important and widespread cancer which affects women. There are several causes of cervical cancer; among them HPV types 16 and 18 are the most prominent ones which are recurrent and persistent infections. These genotypes are currently about 70% of cervical cancer causes in developing countries. Due to the importance of these viruses in cervical cancer, we pioneered the production of Human Papilloma Virus type16 E6 oncoprotein as a recombinant protein in order to develop a vaccine. Two HPV oncoproteins, E6 and E7, are consistently expressed in HPV-associated cancer cells and are responsible for malignant transformation. These oncogenic proteins represent ideal target antigens for developing vaccine and immunotherapeutic strategies against HPV-associated neoplasm.

Methods: In the present study, the cloned E6-oncoprotein of HPV16 in pTZ57R/T-E6 vector was used to produce professional expression vector. The target gene was subcloned in a eukaryotic expression vector. The pcDNA3-E6 vector was propagated in E.coli strain DH5α and transfected into CHO cells 72 hours post-transfection.

Results: The transfected cells were harvested; mRNA detection and the interest protein production were confirmed by western blot analysis using specific anti E6 monoclonal antibody.

Conclusion: HPV16-E6 target protein recognized by specific antibody could be an appropriate form of protein, which can be used for further studies. Due to potential effect of this protein, its DNA construction can be used for DNA vaccine in future studies.

No MeSH data available.


Related in: MedlinePlus

Shows agarose gel electrophoresis of colony PCR amplification products which stained with Ethidium Bromide and visualized under UV illumination.Lane 1: PCR negative control, lane 2: negative control plasmid (pcDNA3), lane M: DNA marker, lane 3: PCR positive control and lanes 4, 5, 6, 7: the colonies containing desired product of 600 bp belong to HPV16-E6 were confirmed.
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f1-IJCP-05-016: Shows agarose gel electrophoresis of colony PCR amplification products which stained with Ethidium Bromide and visualized under UV illumination.Lane 1: PCR negative control, lane 2: negative control plasmid (pcDNA3), lane M: DNA marker, lane 3: PCR positive control and lanes 4, 5, 6, 7: the colonies containing desired product of 600 bp belong to HPV16-E6 were confirmed.

Mentions: The expression vector containing HPV16-E6 gene was constructed as described in Material and Methods. The accuracy of resulted plasmid was confirmed by colony-PCR using designed specific E6 primers (Figure1).


Production of human papilloma virus type 16 e6 oncoprotein as a recombinant protein in eukaryotic cells.

Mirshahabi H, Soleimanjahi H, Pourpak Z, Meshkat Z, Hassan ZM - Iran J Cancer Prev (2012)

Shows agarose gel electrophoresis of colony PCR amplification products which stained with Ethidium Bromide and visualized under UV illumination.Lane 1: PCR negative control, lane 2: negative control plasmid (pcDNA3), lane M: DNA marker, lane 3: PCR positive control and lanes 4, 5, 6, 7: the colonies containing desired product of 600 bp belong to HPV16-E6 were confirmed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352521&req=5

f1-IJCP-05-016: Shows agarose gel electrophoresis of colony PCR amplification products which stained with Ethidium Bromide and visualized under UV illumination.Lane 1: PCR negative control, lane 2: negative control plasmid (pcDNA3), lane M: DNA marker, lane 3: PCR positive control and lanes 4, 5, 6, 7: the colonies containing desired product of 600 bp belong to HPV16-E6 were confirmed.
Mentions: The expression vector containing HPV16-E6 gene was constructed as described in Material and Methods. The accuracy of resulted plasmid was confirmed by colony-PCR using designed specific E6 primers (Figure1).

Bottom Line: These oncogenic proteins represent ideal target antigens for developing vaccine and immunotherapeutic strategies against HPV-associated neoplasm.The transfected cells were harvested; mRNA detection and the interest protein production were confirmed by western blot analysis using specific anti E6 monoclonal antibody.Due to potential effect of this protein, its DNA construction can be used for DNA vaccine in future studies.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Virology and Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

ABSTRACT

Background: Cervical cancer is one of the most important and widespread cancer which affects women. There are several causes of cervical cancer; among them HPV types 16 and 18 are the most prominent ones which are recurrent and persistent infections. These genotypes are currently about 70% of cervical cancer causes in developing countries. Due to the importance of these viruses in cervical cancer, we pioneered the production of Human Papilloma Virus type16 E6 oncoprotein as a recombinant protein in order to develop a vaccine. Two HPV oncoproteins, E6 and E7, are consistently expressed in HPV-associated cancer cells and are responsible for malignant transformation. These oncogenic proteins represent ideal target antigens for developing vaccine and immunotherapeutic strategies against HPV-associated neoplasm.

Methods: In the present study, the cloned E6-oncoprotein of HPV16 in pTZ57R/T-E6 vector was used to produce professional expression vector. The target gene was subcloned in a eukaryotic expression vector. The pcDNA3-E6 vector was propagated in E.coli strain DH5α and transfected into CHO cells 72 hours post-transfection.

Results: The transfected cells were harvested; mRNA detection and the interest protein production were confirmed by western blot analysis using specific anti E6 monoclonal antibody.

Conclusion: HPV16-E6 target protein recognized by specific antibody could be an appropriate form of protein, which can be used for further studies. Due to potential effect of this protein, its DNA construction can be used for DNA vaccine in future studies.

No MeSH data available.


Related in: MedlinePlus