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Cellular and Molecular Biology Letters

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Volume 12, Issue 3

Issues

The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it

Zhaoming Ye
  • Department of Orthopedics, Second Affiliated Hospital, Medical College, Zhejiang University, 88 Jie Fang Road, Hangzhou, 310009, Zhejiang, P.R. China
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/ Huiqin Peng / Yongming Fang / Jie Feng
  • Department of Orthopedics, Second Affiliated Hospital, Medical College, Zhejiang University, 88 Jie Fang Road, Hangzhou, 310009, Zhejiang, P.R. China
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/ Di-Sheng Yang
  • Department of Orthopedics, Second Affiliated Hospital, Medical College, Zhejiang University, 88 Jie Fang Road, Hangzhou, 310009, Zhejiang, P.R. China
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Published Online: 2007-04-06 | DOI: https://doi.org/10.2478/s11658-007-0012-3

Abstract

In our previous study, we demonstrated that azurin could selectively trigger apoptosis in human osteosarcoma cell line U2OS cells. However, the rate of apoptosis (35.8 ± 3.2%) is not very high, and azurin is too expensive to obtain readily. To solve these problems, we constructed a eukaryotic expression plasmid containing the azurin gene with an influenza virus haemagglutinin 9 peptide HA epitope tag, and transfected the recombinant plasmid pcDNA3.1(+)/azurin into U2OS cells. RT-PCR and Western blot analysis validated the successful transfection and the expression of the azurin-HA protein. Conspicuous apoptosis of the transfected cells was detected by flow cytometry (FCM) and the DNA ladder test. The apoptosis rate reached 64.3 ± 13.1%. The transcriptional levels of the Bax and p53 genes increased significantly in U2OS cells transfected with pcDNA3.1(+)/azurin, but the Bcl-2 mRNA level decreased. There was no difference in the levels of Bcl-xl mRNA and Survivin mRNA. We propose that the transfection of the recombinant plasmid pcDNA3.1(+)/azurin can significantly induce apoptosis in U2OS cells. This is closely associated with the up-regulation of the transcriptional level of the Bax and p53 genes, and the down-regulation of that of the Bcl-2 gene.

Keywords: Azurin; Transfection; Osteosarcoma; Apoptosis

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About the article

Published Online: 2007-04-06

Published in Print: 2007-09-01


Citation Information: Cellular and Molecular Biology Letters, Volume 12, Issue 3, Pages 407–421, ISSN (Online) 1689-1392, DOI: https://doi.org/10.2478/s11658-007-0012-3.

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© 2007 University of Wrocław, Poland. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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