Jeju Special Self-Governing Province Agricultural Research & Extention Services, Jeju 690-909, Republic of Korea.
J Biosci Bioeng. 2012 Jan;113(1):106-11. doi: 10.1016/j.jbiosc.2011.09.008. Epub 2011 Oct 21.
Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots by fibrinolysis and in activating plasminogen to plasmin in blood vessels. t-PA and synthetic t-PA (st-PA) genes were expressed as enzymatically active form in hairy roots of Oriental melon (Cucumis melo L. cv. Geumssaragi-euncheon) infected by Agrobacterium rhizogenes. The insertion of the t-PA genes in genomic DNA of transgenic hairy roots was verified by PCR. The presence and expression of t-PA-specific transcripts in the total RNAs of transgenic hairy roots were confirmed by RT-PCR. Western blot analysis of the transgenic hairy roots showed a single major band of 59-kDa recombinant t-PAs. ELISA demonstrated that the highest level of recombinant t-PA (798 ng mg⁻¹) was detected in hairy roots expressing t-PA. Similarly, the maximum fibrinolysis of recombinant t-PAs was observed in hairy roots transformed with t-PA. WPM medium was found to be more suitable for rapid growth of hairy roots among all the seven media types tested. The hairy root production was 5.8 times higher than that of White medium. The total yield of hairy roots grown on WPM medium was 621.8±8.7 g L⁻¹ at pH 7.0. These studies demonstrate that the hairy roots could be employed for the mass production of enzymatically active t-PA.
人组织型纤溶酶原激活物（t-PA）是一种溶栓蛋白，通过纤维蛋白溶解在血管中发挥溶解纤维蛋白栓子的积极作用，并将纤溶酶原激活为纤溶酶。发根农杆菌感染的东方香瓜（Cucumis melo L. cv. Geumssaragi-euncheon）的毛状根中表达具有酶活性的 t-PA 和合成 t-PA（st-PA）基因。通过 PCR 验证了 t-PA 基因在转基因毛状根基因组 DNA 中的插入。通过 RT-PCR 证实了 t-PA 特异性转录物在转基因毛状根总 RNA 中的存在和表达。转基因毛状根的 Western blot 分析显示 59 kDa 的重组 t-PA 存在单一大带。ELISA 表明，在表达 t-PA 的毛状根中检测到重组 t-PA 的最高水平（798ng mg⁻¹）。同样，在转化有 t-PA 的毛状根中观察到重组 t-PA 的最大纤维蛋白溶解活性。在测试的七种培养基类型中，WPM 培养基最适合毛状根的快速生长。毛状根的产量比 White 培养基高 5.8 倍。在 pH 值为 7.0 时，在 WPM 培养基上生长的毛状根的总产量为 621.8±8.7gL⁻¹。这些研究表明，毛状根可用于大规模生产具有酶活性的 t-PA。
