棉花半胱氨酸蛋白酶抑制剂基因的克隆、表达与活性研究  

Molecular cloning' expression and activity of gene encoding the cysteine proteinase inhibitor in cotton

作　　者：潘正[1] 李生伟[1,2] 陈敏[1,2] 蔡应繁[1] 江怀仲[1] 孙全[1] 何晓红[1] 高运玲[1] 杨建平[1] 江明锋[2] 

机构地区：[1]重庆邮电大学生物信息学院,重庆400065 [2]西南民族大学生命科学与技术学院,成都610041

出　　处：《四川大学学报（自然科学版）》2010年第6期1441-1446,共6页Journal of Sichuan University(Natural Science Edition)

基　　金：国家自然科学基金(30771311);重庆市自然科学基金

摘　　要：通过从已构建好的突变体湘棉-18的cDNA文库中筛选分离出一个半胱氨酸蛋白酶抑制剂基因的全长cDNA序列,经测序和序列分析,发现该片段包含一个完整的开放阅读框,长378个碱基,编码125个氨基酸,氨基酸序列中包含了一个半胱氨酸蛋白酶抑制剂家族的保守区段Gln-Val-Val-Ala-Gly;信号肽预测发现该序列包含一个N端信号肽,保守区分析该基因编码的氨基酸包含一个半胱氨酸蛋白酶抑制剂相似区域.半胱氨酸蛋白酶抑制剂在大肠杆菌中表达重组蛋白,在条件为30℃,IPTG 5mmol/L,时间24～36h时,CPI实现最优表达,Western Blotting分析表明表达的蛋白正确.通过木瓜蛋白酶活性抑制实验,结果表明该重组CPI具有一定的酶活抑制作用.A cDNA clone encoding a novel cystatin, Cysteine Proteinase Inhibitor （CPI）, was isolated from a normalized cDNA library constructed from upland cotton of a gland mutant Xiangmian-18. The cDNA insert comprises 378 bp and encodes 125 amino acid residues that contain the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily （Gln-Val-Val-Ala-Gly）. In accordance with the characteristics of CPI, this gene contains an N-terminal signal Peptide by using signal peptide prediction and the same region also contains code for amino acid of cysteine protease inhibitors. The cpi cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The conditions for CPI to achieve optimal expression were 30 ℃, Isopropyl β-D-1-Thiogalactopyranoside （IPTG） 5 mmol/L-1 ,and the time at 24-36 hours. Western Blotting analysis showed the correct expression of the protein. The result of experiment through inhibition of the enzyme activity of papain showed that the recombinant CPI had the activity of inhibition of enzyme.

关 键 词：陆地棉 半胱氨酸蛋白酶抑制剂 基因克隆 原核表达 免疫印迹 

分 类 号：Q78[生物学—分子生物学]