Cloning of expression vector of human tissue factor gene and its expression in human ovarian cancer cell line.
- Author:
Jun FANG
1
;
Wen-Ning WEI
;
Zhong-Ping LIU
;
Shan-Jun SONG
Author Information
1. Institute of Hematology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. fangjunfj@163.net
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Line, Tumor;
Cloning, Molecular;
Female;
Humans;
Mice;
Ovarian Neoplasms;
metabolism;
pathology;
Recombinant Proteins;
biosynthesis;
Thromboplastin;
analysis;
biosynthesis;
genetics;
Transfection
- From:
Journal of Experimental Hematology
2003;11(6):579-582
- CountryChina
- Language:Chinese
-
Abstract:
The aim was to construct the expressive vector of human tissue factor (TF), and determine its expressive level in stable-transfected human ovarian cancer cell line. The human TFcDNA was obtained from human placenta by RT-PCR and then inserted into eukaryotic expressive vector pcDNA3 to obtain the TF-pcDNA3 recombinant. This recombinant gene was introduced into human ovarian cell line A2780 through transfection mediated by lipofectamine. Stable-transfected cells were screened by G418. The TF expressive levels were detected by RT-PCR and flow cytometry. The results showed that: (1) the constructed product was identified as TF-pcDNA3 recombinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780 cell (transfected cell 3.99 +/- 0.15, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. It was concluded that TF gene was successfully cloned, and was introduced into human ovarian cancer cell, and the subline A2780/TF which stably expresses TF at high level was obtained. It will provide good experimental basis for investigating new mechanisms of tumor growth, invasion, metastasis, hypercoagulability, and for exploring a new strategy of gene therapy.
