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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (10): 15-20    DOI: 10.13523/j.cb.20161003
研究报告     
决明胰蛋白酶抑制剂1活性相关残基的定点突变与抑制活性分析
向缅, 朱建全, 俞继华, 李洋洋, 李娟娟, 刘祖碧, 王万军, 廖海, 周嘉裕
西南交通大学生命科学与工程学院 成都 610031
The Site-directed Mutation of Key Residues and the Analysis about Inhibitory Activity of Cassia obtusifolia Trypsin Inhibitor
XIANG Mian, ZHU Jian-quan, YU Ji-hua, LI Yang-yang, LI Juan-juan, LIU Zu-bi, WANG Wan-jun, LIAO Hai, ZHOU Jia-yu
Southwest Jiaotong University, Chengdu 610031, China
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摘要：

决明胰蛋白酶抑制剂1（CoTI1）属于Kunitz胰蛋白酶抑制剂家族成员，通过序列比对预测Arg86、Leu84和Thr88等3个氨基酸残基可能是CoTI1发挥抑制作用的关键残基。通过定点突变的方法将Arg86、Leu84与Thr88残基分别突变为Asp残基，并考察各突变体对胰蛋白酶及棉铃虫等鳞翅目害虫消化酶的抑制作用。与CoTI1相比，CoTI1R86D、CoTI1T88D与CoTI1L84D突变体对胰蛋白酶的抑制活性分别下降了93%、64%与59%；对棉铃虫、甜菜夜蛾、斜纹夜蛾等3种鳞翅目害虫消化酶的平均抑制活性分别下降了88.7%、57%与60.7%。以上结果表明Arg86、Leu84与Thr88是CoTI1发挥抑制作用的关键残基，这为CoTI1的抑制分子机制及抗虫研究提供了重要的理论依据。
关键词： 胰蛋白酶抑制剂;  定点突变;  决明    
Abstract:

A trypsin inhibitor (CoTI1) from Cassia obtusifolia was attributed to the Kunitz-type trypsin inhibitor family. According to the sequence alignment, Arg86, Leu84 and Thr88 might be the key residues of CoTI1. In order to confirm the speculation, the three above residues were replaced as Asp by site-directed mutagenesis, respectively, and analysis the inhibitory activity of the mutants and CoT1 to trypsin and insects' digestive enzyme. Compared with CoT1, the inhibitory activity of the mutant CoTI1R86D to trypsin decreased most obviously, and the inhibitory effect of the original 93% was lost. CoTI1L84D lost 59% of the inhibitory effect; while the inhibitory activity of CoTI1T88D decreased by 64%. The average inhibitory activity decreased 88.7%, 57% and 60.7% to digestive enzymes of Helicoverpa armigera, Beet armyworm and Spodoptera litura, respectively. The result shows that Arg86, Leu84 and Thr88 are the key residues of CoT1, and it was useful for the molecular mechanism and anti-insects study of CoTI1.
Key words: Cassia obtusifolia    Trypsin inhibitor    Site-directed mutagenesis
收稿日期: 2016-04-26 出版日期: 2016-10-25
ZTFLH:  Q754  
基金资助:

国家自然科学基金（31371232，31500276）、成都市科技技术研发项目（2015-HM01-00051-SF）资助项目
通讯作者: 廖海,电子信箱:ddliaohai@home.swjtu.edu.cn;周嘉裕,电子信箱:spinezhou@home.swjtu.edu.cn     E-mail: ddliaohai@home.swjtu.edu.cn;spinezhou@home.swjtu.edu.cn
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引用本文:

向缅, 朱建全, 俞继华, 李洋洋, 李娟娟, 刘祖碧, 王万军, 廖海, 周嘉裕. 决明胰蛋白酶抑制剂1活性相关残基的定点突变与抑制活性分析[J]. 中国生物工程杂志, 2016, 36(10): 15-20.

XIANG Mian, ZHU Jian-quan, YU Ji-hua, LI Yang-yang, LI Juan-juan, LIU Zu-bi, WANG Wan-jun, LIAO Hai, ZHOU Jia-yu. The Site-directed Mutation of Key Residues and the Analysis about Inhibitory Activity of Cassia obtusifolia Trypsin Inhibitor. China Biotechnology, 2016, 36(10): 15-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20161003    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I10/15

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