pQE30-bgln原核表达质粒的构建和鉴定

Construction and Identification of the Recombinant Prokaryotic Expression Plasmid pQE30-bgln

构建重组原核表达质粒pQE30-bgln使之可以在大肠杆菌中表达以获得β-葡糖苷酶,用于提高从植物中提取白藜芦醇的得率.以克隆质粒pUCP67为模板,用降落PCR的方法扩增β-葡糖苷酶基因片段(bgln).利用原核表达载体pQE30构建pQE30-bgln重组质粒,用酶切电泳验证重组结果的正确性,测序检测质粒重组后序列情况.PCR结果显示扩增片段2.2 kb,与预期相同.重组质粒酶切后显示其大小约5.6kb,大小及酶切图谱与预期相同.经测序发现插入片段读码框正确.可用于原核表达的pQE30-bgln质粒构建成功.

A recombinant plasmid pQE30-bgln was constructed for obtaining the BGLN protein expressed in E. coil so as to develop the efficiency of extracting resveratrol from plants. Touch-down PCR was used to amplify the bgln gene from plasmid pUCP67 in which bgln was cloned. A new plasmid pQE30-bgln, was then constructed by inserting the amplified bgln gene into pQE30, a prokaryotic expression vector. Restriction analysis and sequencing were used to confirm the structure of pQE30-bgln. Results showed that a DNA fragment in size of 2.2 kb was amplified. Furthermore, restriction analysis showed that the amplified gene was inserted into pQE30 correctly and the size of recombinant plasmid is abont 5.6 kb. The forepart interface between the vector and the insert was sequenced by TaKaRa company, and it was confirmed that the insert was not out of frame. In conclusion, the plasmid pQE30-bgln is constructed successfully.