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SHI Wei, SUN Li, LIN Sen-sen, YUAN Sheng-tao, ZHANG Lu-yong. Construction of eukaryotic expression plasmid for human TF full-length,TF truncated and characterization of their biological function[J]. Journal of China Pharmaceutical University, 2009, 40(5): 465-470.
Citation: SHI Wei, SUN Li, LIN Sen-sen, YUAN Sheng-tao, ZHANG Lu-yong. Construction of eukaryotic expression plasmid for human TF full-length,TF truncated and characterization of their biological function[J]. Journal of China Pharmaceutical University, 2009, 40(5): 465-470.

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

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  • 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.
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