Su Manman, Chang Weiqin, Wang Dingding, Cui Manhua, Lin Yang, Wu Shuying, Xu Tianmin
Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin, P.R. China.
Department of Biotechnology, College of Life Sciences and Biopharmacology, Guangdong Pharmaceutical University, Guangzhou, Guangdong, P.R. China.
Oncol Rep. 2015 Sep;34(3):1337-44. doi: 10.3892/or.2015.4118. Epub 2015 Jul 9.
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.
本研究的目的是开发一种新的治疗药物，以改善卵巢癌患者的预后。构建了人尿激酶型纤溶酶原激活剂（uPA）17 - 34库尼茨型蛋白酶抑制剂（KPI）真核表达载体，并在巴斯德毕赤酵母中表达了重组人uPA17 - 34 - KPI（rhuPA17 - 34 - KPI）。在本研究中，根据天然氨基酸序列创建了编码uPA 17 - 34个氨基酸的DNA序列，并将其插入构建好的KPI - pPICZαC载体中。然后，将uPA17 - 34 - KPI - pPICZαC转化到巴斯德毕赤酵母X - 33中，通过甲醇诱导表达rhuPA17 - 34 - KPI。用胰蛋白酶抑制分析检测重组融合蛋白的生物活性，并用TUNEL检测、体外伤口愈合检测和基质胶模型分析鉴定其对卵巢癌细胞生长的抑制作用。重组载体uPA17 - 34 - KPI - pPICZα的DNA序列分析结果表明，编码人uPA 17 - 34个氨基酸、淀粉样前体蛋白（APP）285 - 288个氨基酸和KPI 1 - 57个氨基酸的DNA已正确插入pPICZαC载体。经甲醇诱导后，用SDS - PAGE和蛋白质免疫印迹分析观察到分子量为8.8 kDa的融合蛋白。rhuPA17 - 34 - KPI在50 ml试管中于巴斯德毕赤酵母中的表达产量为50 mg/l。重组融合蛋白能够抑制胰蛋白酶的活性，抑制SKOV3细胞的生长并诱导其凋亡，还能抑制卵巢癌细胞的侵袭和转移。通过将uPA17 - 34氨基酸特异性结合uPAR作为融合蛋白的靶向部分，并利用KPI的丝氨酸蛋白酶抑制剂活性，发现重组融合蛋白uPA17 - 34 - KPI可抑制卵巢肿瘤的侵袭和转移，因此可被视为在靶向治疗中有效。
