Study on prokaryotic expression and partial biological activities for tissue factor pathway inhibitor-2 / 中华检验医学杂志

ABSTRACT

Objectives To express human TFPI-2 gene in E.coli and get the recombinant protein;to investigate the recombinant activities of antiplasmin and antitumor.Methods (1) Encoding region of mature protein of human TFPI-2 was amplified by PCR.The 645 bp PCR product was cloned into expression vector pET28a and transformed into BL21 strain to get expression.(2)Identifying the DNA segment of TFPI-2 with enzyme digestion and colony PCR and the TFPI-2 fusion protein with western blot respectively.(3)To establish the kinetics assay for detecting the TFPI-2 and test it′s inhibiting plasmin activities.(4)To assess ovarian tumor cell migratory and invasive behaviors with the Boyden chamber in vitro.Results (1)Expression plasmid of recombinant TFPI-2 was constructed and high-level expression of TFPI-2 was produced as fusion protein. (2) The TFPI-2 fusion protein was identified by western blot. (3) Antiplasmin activity of the TFPI-2 was confirmed in the fluid and on the cell surface. (4) In invasion assay, the number of the A2780 and A2780-TFPI-2 groups transferred the Matriged matrix-coated PVPF membrane was obvious decreased compared with that of A2780 group(P0.05).Conclusion (1) Prokaryotic expression of TFPI-2 gene was got, which can provide the rich experimental material for investigating the role of TFPI-2 in relative fields; (2)Recombinant TFPI-2 has antiplasmin activity, which provides the experiment basis for investigating the role of recombinant TFPI-2 in human ovarian tumor migration and invasion in vitro; (3) The recombinant TFPI-2 inhibits the invasive ability of human ovarian tumor cells in vitro, but has no effect on the migration, which may provide a target basis for treating human ovarian tumor with TFPI-2 protein therapy.