血管基膜衍生多功能肽的表达及其生物活性的鉴定

Expression of vascular basement membrane-derived multifunctional peptide in Pichia pastoris and identification of its bioactivity

目的：在毕赤酵母中表达血管基膜衍生多功能肽（vascular basement membrane—derived mulifunctional peptide，VB—MDMP），并鉴定其生物学活性。方法：利用PCR技术获得融合了谷胱甘肽转移酶（GST）的肿瘤抑素（tumstatin）活性成分的GST—VBMDMP基因，克隆到pPIC9K载体；然后原生质体转化法转化毕赤酵母细胞，行多克隆筛选和表型鉴定。获得His’Muts表型的转化子，在MGY培养液中30℃摇床培养，每24h从培养液中转移1ml培养液上清于-70℃保存，并保持培养瓶中培养液的量和培养液中甲醇0．5％的浓度不变，第9天行SDS—PAGE检测表达的蛋白并纯化，然后进行细胞和动物生物活性检测。结果：SDS—PAGE发现阳性重组子在MGY培养液中表达的蛋白量在1～7d逐渐增加，到第8天后开始减少，用Gluta—thioneSepharose4B亲和层析柱纯化获得GST—VBMDMP融合蛋白；GST—VBMDMP能抑制C57BL／6鼠动脉内皮细胞管状结构的形成；同时GST—VBMDMP（2、6、10mg／kg）对小鼠Lewis肺癌原发瘤（瘤重抑制率分别为96．6％、82．1％、61．2％）和自发性肺转移瘤（瘤结节抑制率分别为96．8％、87．9％、75、3％）均具有明显的抑制作用（P〈0.01）。结论：VBMDMP能够抑制鼠动脉内皮细胞管状结构的形成，并对小鼠Lewis肺癌原发瘤和肺转移具有明显的抑制作用。

Objective： To express vascular basement membrane-derived multiple peptide （VBMDMP） in Pichia pastoris and to identify its bioactivity. Methods： PCR technique was used to obtain the target fragment GST-VBMDMP, which was then cloned into pPICgK vector. The resultant vector pPICgK-GST-VBMDMP was transfected into Pichia pastoris cells with spheroplast and the positive recons was identified. The His ^＋ Muts transformant was shake-cultured in MGY at 30℃. The culture supernatant （ 1 ml） was collected for preservation at - 70℃ every 24 hours while maintaining the concentration of methanol at 0. 5% in the culture medium. SDS-PAGE analysis was used to detect the protein expression after 8 days and then animal and cell experiments were performed with GST-VBMDMP. Results： SDS-PAGE analysis found that protein express increased during 1-7 days and decreased after 8 days in MGY medium; GST-VBMDMP fused protein was obtained by Glutathione Sepharose 4B and it inhibited the artery endothelial cell tube structure formation in C57BL/6 mouse; GST-VBMDMP（ 2, 6, 10 mg/kg）also significantly inhibited the primary cancer Lewis mouse （tumor inhibitor rates being 96.6% , 82.1%, and 61.2%, respectively） and metastatic lung tumors（ tumor inhibitor rates being 96.8 %, 87.9 %, and 75.3%, respectively）（P 〈 0. 01 ）. Conclusion： VBMDMP can inhibit mouse artery endothelial cell tube structure formation and Lewis mouse primary, metastatic lung tumors.