以GRP78蛋白为靶向的胰蛋白酶抑制剂抗肠癌效应研究.docVIP

 以 GRP78 蛋白为靶向的胰蛋白酶抑制剂抗 肠癌效应研究# 赵超，付荣，赵亚蕊，史通麟，李卓玉，李宗伟** 5 10 15 20 25 30 35 40 （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：目的 构建以肿瘤细胞表面葡萄糖调节蛋白 78（GRP78）为靶向的重组绿豆胰蛋白酶 抑制剂活性片段 GBP-TI，并探讨其抗大肠癌的分子靶向效应。 方法 用带有 GRP78 结合肽 （GRP78 binding peptide，GBP）表达序列的引物 PCR 扩增胰蛋白酶抑制剂活性片段（TI）， 构建 GBP-TI 的原核表达载体；采用 GST 亲和层析柱纯化获得 GBP-TI 蛋白；激光共聚焦方法 检测 GBP-TI 与 GRP78 的相互作用；MTT 和 DAPI 细胞核染色方法分别检测 GBP-TI 对大肠癌 细胞存活和凋亡的影响。结果 GBP-TI 能够与大肠癌细胞表面的 GRP78 相互作用；与 GST-TI 相比，GBP-TI 能够剂量依赖性的诱导大肠癌细胞死亡，而对正常细胞生长无明显影响。结 论 GBP-TI 可以通过结合肿瘤细胞表面 GRP78 蛋白特异性杀伤肿瘤细胞，为肿瘤靶向治疗提 供一种新的策略。 关键词：胰蛋白酶抑制剂；葡萄糖调节蛋白 78；大肠癌；靶向药物 中图分类号：Q256 Anti-cancer effects of trypsin inhibitor targeting colorectal cancer cell surface GRP78 Zhao Chao, Fu Rong, Zhao Yarui, Shi Tonglin, Li Zhuoyu, Li Zongwei (The Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, TaiYuan 030006) Abstract: Objective The aim of this study was to construct the active fragment of recombinant trypsin inhibitor GBP-TI, which targets tumor cell surface glucose-regulated protein 78 (GRP78), and to investigate its molecular targeting effects on colorectal cancer cells. Methods To construct the GBP-TI expressing vector, the primer containing the expressed sequence of GRP78 binding peptide (GBP) was used to amplify the trypsin inhibitor active fragment (TI); GBP-TI protein was purified by GST-affinity chromatography; The interaction of GBP-TI with cell surface GRP78 was confirmed by confocal immunofluorescence analysis. The effects of GBP-TI on cell survival and apoptosis were detected by MTT and DAPI staining, respectively. Results GBP-TI was able to interact with colorectal cancer cell surface GRP78. In contrast to GST-TI, GBP-TI could induce apoptosis in colorectal cancer cells in a dose-dependent manner, but had a negligible effect on the growth of normal cells. Conclusion GBP-TI can specifically kill tumor cells through binding to cell surface GRP78. This study provides a new stra