甘蓝型油菜Kunitz蛋白酶抑制剂的异源表达与理化特征分析  

Heterologous expression and physicochemical characterization of Brassica napus Kunitz protease inhibitor

作　　者：冯玮[1] 李朋朋 宋鹏 FENG Wei;LI Pengpeng;SONG Peng(School of Life Sciences, Liaocheng University, Liaocheng 252000, Shandong, China;Bureau of Agricultural and Rural Affairs in Dongchangfu, Liaocheng, Liaocheng 252000, Shandong, China)

机构地区：[1]聊城大学生命科学学院,山东聊城252000 [2]聊城市东昌府区农业农村局,山东聊城252000

出　　处：《浙江农业学报》2022年第1期112-119,共8页Acta Agriculturae Zhejiangensis

摘　　要：利用毕赤酵母表达系统首次克隆表达了一个油菜籽来源的Kunitz蛋白酶抑制剂基因(RTI;rti),分离纯化后对该抑制剂进行了理化特征分析。结果显示:在摇瓶发酵水平,重组RTI诱导表达水平达到628 mg·L^(-1),抑制剂比活性达到69.6 TIU·mg^(-1)蛋白;重组RTI在30~90℃可保持70%以上抑制活性,在pH 2.0~11.0保持80%以上抑制活性;金属离子Cu^(2+)和Co^(2+),有机试剂甲醇、乙醇、丙酮和氯仿对其有抑制作用;重组RTI以非竞争性抑制方式与胰蛋白酶作用,对胰蛋白酶具有很强的、专一性抑制作用,属于典型的植物Kunitz型胰蛋白酶抑制剂。结合其良好的理化特性,重组RTI具有开发为抗虫蛋白的潜力。A Kunitz inhibitor gene(RTI;rti)from rapeseed was cloned and expressed in Pichia pastoris expression system for the first time.Its physicochemical characteristics were investigated after separation and purification.The results showed that the expression level of recombinant RTI reached 628 mg·L^(-1) and its inhibitory activity reached 69.6 TIU·mg^(-1) in shaking flask fermentation.The recombinant RTI maintained over 70%inhibitory activity between 30-90℃and retained over 80%inhibitory activity between pH 2.0-11.0.The metal ions Cu^(2+) and Co^(2+),the organic reagents methanol,ethanol,acetone or chloroform inhibited the activity of RTI.The recombinant RTI reacted with trypsin in a non-competitive manner,and demonstrated a strong and specific inhibitory effect on trypsin.These results indicated that RTI was a typical plant Kunitz trypsin inhibitor.Combined with good physicochemical properties,RTI has the potential to be developed as insect resistant protein.

关 键 词：油菜籽Kunitz蛋白酶抑制剂 毕赤酵母 克隆表达 理化特征 

分 类 号：S436.421[农业科学—农业昆虫与害虫防治]