绿豆胰蛋白酶抑制剂精氨酸活性片段衍生物的克隆表达及生物学活性研究.docVIP

 绿豆胰蛋白酶抑制剂精氨酸活性片段衍生 物的克隆表达及生物学活性研究# 付荣，赵超，单树花，赵亚蕊，林凯丽，武海丽，李宗伟，李卓玉** 5 10 15 20 25 30 35 （山西大学生物技术研究所，化学生物学与分子工程教育部重点实验室，太原 030006） 摘要：绿豆胰蛋白酶抑制剂（MBTI）具有赖氨酸和精氨酸两个活性中心，目前针对精氨酸活 性中心的生物活性研究较少。本研究通过化学合成 MBTI-Argc 的编码基因，克隆到原核表达 载体 pGEX-4T-1 并转化到大肠杆菌 DH5α，IPTG 诱导表达的可溶性融合蛋白 GST-Argc，GST 层析柱分离、纯化目的蛋白。体外活性测定表明，该蛋白几乎不具有胰蛋白酶抑制剂活性。 DTNB 法定量测定二硫键表明，获得的 Argc 与天然产物相比，总巯基数不变，但二硫键个数 由原来的三对变为两对。采用 MTT 法和细胞划痕实验分别检测 GST-Argc 对细胞增殖和迁移 生物学效应的影响，发现 GST-Argc 能够明显的促进细胞的增殖和迁移效应，与肿瘤细胞相 比，该片段对正常细胞增殖的促进效应更为明显。研究结果表明，MBTI-Argc 与天然产物相 比在结构和功能上都发生了显著变化，表现出类生长因子活性，可能在组织细胞损伤修复及 创伤愈合中具有广泛应用前景。 关键词：Argc 活性片段；二硫键；增殖；迁移 中图分类号：Q256 Cloning expression and biological activity of Mung bean trypsin inhibitor derivative Argc fragment Fu Rong, Zhao Chao, Shan Shuhua, Zhao Yarui, Lin Kaili, Wu Haili, Li Zongwei, Li Zhuoyu (Key Laboratory for Chemical Biology and Molecular Engineering of Ministry of Education,Institute of Biotechnology, Shanxi University, TaiYuan 030006) Abstract: Mung bean trypsin inhibitor contains lysine and arginine active centers. To study the biological activity of arginine active fragment (MBTI-Arg), the coding sequence of MBTI-Argc by chemical synthesis is cloned into the prokaryotic expression vector pGEX-4T-1 and transformed into E.coli DH5α. After 0.5mM IPTG induction, the soluble fusion protein GST-Argc was purified by column separation. The assay for trypsin inhibitory activity of GST-Argc showed that GST-Argc had a weak trypsin inhibitor activity. DTNB assay was used to detect the number of free thiol. The result showed that, compared with natural products, the number of disulfides were changed into two from three in Argc. Next, the biological effects of GST-Argc on cell proliferation and migration were detected by MTT test and scratching wound assay, respectively. The results showed that GST-Argc could significantly promote cell proliferation and migration. Furthermore, the proliferation effect of GST-Argc on normal cells are more significant than that of cancer cells. Our fi