The aim of the present study was to develop a new therapeutic
drug to improve the prognosis of
ovarian cancer patients. Human
urokinase-type plasminogen activator (uPA)17-34-kunitz-type
protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino
acids were created according to the native
amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of
methanol. The bioactivities of a
recombinant fusion protein were detected with
trypsin inhibition analysis, and the inhibitory effects on the growth of
ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and
Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino
acids, 285-288 amino
acids of
amyloid precursor
protein (APP) and 1-57 amino
acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion
protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The
recombinant fusion protein was able to inhibit the activity of
trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and
metastasis of
ovarian cancer cells. By considering uPA17-34 amino
acid specific binding uPAR as the targeted part of fusion
protein and utilizing the
serine protease inhibitor activity of KPI, it was found that the
recombinant fusion protein uPA17-34-KPI inhibited the invasion and
metastasis of ovarian
tumors, and may therefore be regarded as effective in targeted treatment.