豇豆胰蛋白酶抑制剂cDNA在大肠杆菌中的克隆与表达  

cDNA Cloning and Expression of Cowpea Trypsin Inhibitor in Escherichia coli

作　　者：刘春明[1] 朱祯[1] 周兆斓 孙宝林 李向辉[1] 

机构地区：[1]中国科学院遗传研究所

出　　处：《生物工程学报》1993年第2期152-157,共6页Chinese Journal of Biotechnology

摘　　要：从即将成熟的豇豆子叶中分离出总RNA,逆转录合成cDNA第一条链。参照已知的几种Bowman-Birk型胰蛋白酶抑制剂基因序列,设计并合成了两段寡核苷酸引物,以单链cDNA为模板,进行PCR扩增,得到320bp的均一扩增产物,克隆到pBluescrip SK(+)的EcoRV位点上并转化大肠杆菌JM101。酶切图谱及DNA序列分析表明克隆到的片段含有编码完整80个aa的豇豆胰蛋白酶抑制剂结构基因和一段编码27aa的前导序列。利用限制酶NcoI对其前导序列进行缺失,只保留成熟蛋白结构基因上游第一个ATG密码子并克隆到大肠杆菌表达载体pKK233-2中进行表达研究,从转化细菌的提取物中检测到了外源CpTI基因表达产物对胰蛋白酶的抑制活力。Total RNA was isolated from cowpea cotyledons as they were approaching maturity. Single stranded cDNA was synthesized by the reverse transcriptase. A 320bp fragment was amplified and cloned by polymerase chains reaction. DNA sequence analysis indicated that the cloned fragment containing the entire coding sequence for 80aa CpTI mature protein and a 5' coding sequence for 27aa leader. The leader sequence was partially deleted and the remaining CpTI cDNA fragment was cloned into E.coli expression vector pKK233-2 to study its expression. The trypsin inhibitor activity was detected in the extracts of transformed bacteria.

关 键 词：豇豆 胰蛋白酶 抑制剂 

分 类 号：S188[农业科学—农业基础科学]