关键词: 抗虫基因, cry2A*, 转基因水稻, 表达, 纯化

Abstract: 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.

Key words: insecticidal gene, cry2A*, transgenic rice, expression, purification