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
Expression of rice codon optimized cry2A* gene in Escherichia coli and purification of its expressed proteins
通过PCR方法从克隆载体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Nde I和BamH I双酶切定向插入到原核表达载体pET-28a(+)中，成功构建了在表达蛋白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0.05 mmol/L、诱导时间为3小时、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亲和柱纯化得到了高纯度目的蛋白，薄层扫描分析蛋白纯度达到95%。
he coding sequence of rice preferable codon optimized cry2A* 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 6His-tag 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 under conditions of isopropyl-β-D-thiogalactopyranoside(IPTG) at a final concentration of 0.05 mmol/L for 3h at 20℃. The recombinant protein was purified by Ni-NTA affinity chromatography, and the purity was up to 95% through thin layer scanning analysis.
林希瑾、贺晓云、秦伟、黄昆仑、李欣、罗云波、许文涛
生物工程学分子生物学生物化学
抗虫基因ry2A*转基因水稻表达纯化
insecticidal geneCry2A*transgenic riceexpressionpurification
林希瑾,贺晓云,秦伟,黄昆仑,李欣,罗云波,许文涛.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[EB/OL].(2007-06-05)[2025-08-11].http://www.paper.edu.cn/releasepaper/content/200706-74.点此复制
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