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目的 :克隆表达大肠杆菌脱氢奎尼酸合成酶基因aroB ,并测定其生物学活性。方法 :根据大肠杆菌中aroB基因的序列设计了一对引物 ,利用PCR技术扩增得到aroB基因 ,将其克隆入pGEM Teasy载体中 ,以双脱氧法进行测序 ,然后再克隆入高效原核表达载体pBV2 2 0 ,对该重组子的SD序列进行调节后利用温度诱导表达 ,并测酶的生物活性。结果 :构建了aroB基因的克隆和表达重组子 ,经SD序列调节后 ,aroB基因获得高效表达 ,SDS PAGE图谱显示新增 38.8× 10 3 蛋白带 ,酶活性测定结果表明脱氢奎尼酸合成酶的相对酶活性为 5 .4。结论 :实现了脱氢奎尼酸合成酶基因在大肠杆菌中的高效表达 ,为构建产脱氢奎尼酸工程菌种奠定了基础。
OBJECTIVE: To clone and express aroB gene of E. coli dehydroquinate synthase and determine its biological activity. Methods: According to the sequence of aroB gene in Escherichia coli, a pair of primers was designed. The aroB gene was amplified by PCR and cloned into pGEM Teasy vector. The aroB gene was sequenced by dideoxy method, and then cloned into prokaryotic expression vector pBV2 20, the SD sequence of the recombinant after the regulation of the use of temperature-induced expression, and test the biological activity of the enzyme. Results: The aroB gene was cloned and expressed recombinant. The aroB gene was highly expressed after the regulation of SD sequence. SDS-PAGE showed that a new 38.8 × 10 3 protein band was obtained. The results of enzyme activity assay showed that the dehydroquinate synthetase The relative enzyme activity was 5.4. Conclusion: The high expression of dehydroquinate synthetase gene in Escherichia coli is achieved, which lays the foundation for the construction of dehydroquinate gene engineering strain.