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%。
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.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2008年第7期267-271,共5页
Food Science
基金
国家863计划项目(2006AA10Z440)
农业部948项目(2005-Z32)
