Construction of eukaryotic expression plasmid for human TF full-length，TF truncated and characterization of their biological function

Aim ：To investigate the function of tissue factor(TF) cytoplasmic domain，and study its biological functions in breast cancer migration. Methods ：Total RNA was isolated from human breast cells(MDA-MB-231) with Trizol，and the full-length TF and truncated TF genes were amplified by RT-PCR，and then inserted into pGEM-T vectors.DNA sequencing was performed before the amplified products were cloned into the eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid was transiently transfected into MCF-7 cells by means of liposome and the expression levels were examined by RT-PCR and Western blotting.And the TF procoagulant activities were detected by chromogenic substrate assay after being transfected with TF full-length，TF truncated cDNA and empty vector.A transwell assay was employed to examine the over-expression of TF full-length and TF truncated on breast cancer cells migration. Results ：The amplified products were confirmed as the cDNA of TF full-length and TF truncated by DNA sequencing.Positive clones pcDNA3.1(+)-TF full-length and pcDNA3.1(+)-TF truncated were verified by endonuclease digestion.With the increased TF expression，procoagulant activity was also augmented.TF truncated had no effect on MCF-7 cells migration，but over-expression of TF full-length significantly increased MCF-7 cell migration. Conclusion ：The eukaryotic expression plasmids for TF full-length and TF truncated were constructed successfully，which provides a basis for further investigation on the role of TF and its cytoplasmic phosphorylation domain in cancers.