[Cloning of expression vector of human tissue factor gene and its expression in human ovarian cancer cell line].
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.
Fang
Jun
J
Institute of Hematology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. fangjunfj@163.net
Wei
Wen-Ning
WN
Liu
Zhong-Ping
ZP
Song
Shan-Jun
SJ
D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005260:Female; D006801:Humans; D051379:Mice; D010051:Ovarian Neoplasms / Q000378:metabolism* / Q000473:pathology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013925:Thromboplastin / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D011994:Recombinant Proteins; D013925:Thromboplastin