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  

Cloning,Expression and Biological Activity of Mung Bean Trypsin Inhibitor Derivatived Argc Fragment

作　　者：付荣[1] 李宗伟[1] 赵超[1] 单树花[1] 武海丽[1] 袁琳洁[1] 李卓玉[1] 

机构地区：[1]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,山西太原030006

出　　处：《山西大学学报（自然科学版）》2013年第1期102-107,共6页Journal of Shanxi University(Natural Science Edition)

基　　金：国家自然科学基金(31040018;31271516;8115026);国家大学生创新性实验项目基金(2012B25);高等学校博士学科点专项科研基金(20111401110011)

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.通过化学合成MBTI-Argc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.表达的可溶性融合蛋白GST-Argc经GST层析柱纯化后体外测定活性表明,该片段3.3分子对1分子胰蛋白酶活力的抑制率不足5%.DTNB法定量测定二硫键表明,与天然产物相比,Argc的二硫键个数有所减少.GST-Argc对细胞增殖和迁移生物学效应的检测实验发现,GST-Argc能显著促进细胞的增殖和迁移效应.上述结果表明,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.Mung bean trypsin inhibitor contains lysine and arginine active centers. To study the biological ac- tivity of arginine active fragment （MBTI-Arg）, the coding sequence of MBTI-Argc by chemical synthesis was cloned into the prokaryotic expression vector pGEX-4T-1 and transformed into E. coli DH5a. After IPTG induction,the fusion protein GST-Argc was purified by GST affinity column. The assay for trypsin inhibitory activity of GST-Argc showed that it had a weak trypsin inhibitor activity. DTNB assay was used to detect the number of free thiols. The result showed that compared with natural products,the number of disulfides were changed into two from three in Argc. Moreover,the biological effects of GST-Argc on cell proliferation and migration were detected by MTT test and scratching wound healing assay, respectively. The results indicated that GST-Argc could significantly promote cell proliferation and migration. Further- more,the proliferation effects of GST-Argc on normal cells were more significant than that of cancer cells. The findings suggest MBTI-Argc exhibits a growth factor-like activity with a relatively low trypsin inhibi- tory activity and the MBTI-Argc may play a role in cellular and tissue injury recovery.

关 键 词：胰蛋白酶抑制剂 Argc活性片段 二硫键 细胞增殖 细胞迁移 

分 类 号：Q814[生物学—生物工程]