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当前位置: 首页 > 文章 > 水稻密码子优化的cry2A*基因在大肠杆菌中的表达及其表达产物的纯化 食品科学 2008,29 (7) 267-271
Position: Home > Articles > Expression of Rice Codon Optimized cry2A* Gene in Escherichia coli and Purification of Its Expressed Proteins FOOD SCIENCE 2008,29 (7) 267-271

水稻密码子优化的cry2A*基因在大肠杆菌中的表达及其表达产物的纯化

作  者：
秦伟;黄昆仑;贺晓云;李欣;许文涛;林希瑾;罗云波
单  位：
中国农业大学食品科学与营养工程学院食品生物技术实验室
关键词：
抗虫基因;cry2A*;转基因水稻;表达;纯化
摘  要：
通过PCR从克隆载体pUC18-3Z/Cry2A*上扩增水稻偏爱型密码子优化的抗虫基因cry2A*,经限制性内切酶NdeI和BamHI双酶切定向插入到原核表达载体pET-28a(+),成功构建了在表达蛋白的N端只带有6个组氨酸标签的融合蛋白表达载体pET-28a(+)/Cry2A*,并转入大肠杆菌BL21(DE3)中。通过对其表达条件进行优化,发现在IPTG浓度为0.05mmol/L、诱导时间为3h、诱导温度为20℃的表达条件下目的蛋白大部分以可溶形式进行表达。采用Ni-NTA亲和柱纯化得到高纯度目的蛋白,薄层扫描分析蛋白纯度达到95%。
译  名：
Expression of Rice Codon Optimized cry2A* Gene in Escherichia coli and Purification of Its Expressed Proteins
作  者：
QIN Wei,HUANG Kun-lun,HE Xiao-yun,LI Xin,XU Wen-tao,LIN Xi-jin,LUO Yun-bo* (Laboratory of Food Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China)
关键词：
insecticidal gene;cry2A*;transgenic rice;expression;purification
摘  要：
The coding sequence of rice preferable codon optimized cry2A* gene was amplified via polymerase chain reaction (PCR) from recombinant plasmid pUC18-3Z/Cry2A*. Then the PCR products of cry2A* gene were inserted into expression vector pET-28a(+) using restriction endonucleases Nde I and BamH I, resulting in the recombinant expression plasmid pET-28a(+)/ Cry2A* expressing Cry2A* proteins with only 6 His-tags attached to its N-terminus. Subsequently, the expression vector pET-28a(+)/Cry2A* was introduced into E. coli BL21 (DE3). The Cry2A* protein was expressed mainly in soluble form in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG) with final concentration 0.05 mmol/L by inducing for 3 h at 20 ℃. The recombinant protein was purified by Ni-NTA affinity chromatography, and the purity is up to 95% according to thin layer scanning analysis.

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