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

• Autorzy: Ye Z , Peng H. , Fang Y. , Feng J. , Yang D.S.
• Języki publikacji: EN

Abstrakty

EN

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.

Słowa kluczowe

EN

green fluorescent protein; osteosarcoma; inhibitor apoptosis protein; reverse transcription; human cytomegalovirus; plasmid; flow cytometry; azurin; polymerase chain reaction; apoptosis; transfection

• Wydawca: -
• Czasopismo: Cellular and Molecular Biology Letters
• Rocznik: 2007
• Tom: 12
• Numer: 3
• Opis fizyczny: p.407-421,fig.,ref.

Twórcy

autor

Ye Z

• Zhejiang University, 88 Jie Fang Road, Hangzhou, 310009, Zhejiang, P.R.China

autor

Peng H.

autor

Fang Y.

autor

Feng J.

autor

Yang D.S.

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