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目的构建AvTPS1的原核表达载体并进行蛋白表达,为该基因在原核表达系统中的功能鉴定奠定基础。方法用In-Fusion方法构建AvTPS1截掉转运肽的表达载体;用Gateway方法构建AvTPS1包含完整阅读框的表达载体;两种表达载体分别转化至大肠杆菌BL21(DE3)和BL21DE3plys中进行诱导表达,用变性聚丙烯酰氨凝胶电泳(SDS-PAGE)以及Western杂交检测蛋白的表达情况。结果构建了原核表达载体pET-32a(+)-(-tp)AvTPS1和pDEST17-AvTPS1;这两个表达载体在大肠杆菌BL21(DE3)和BL21DE3plys中均没有表达出明显的相应蛋白,但是这两个载体在大肠杆菌BL21(DE3)中大量表达后,用Western杂交检测有蛋白存在。结论成功构建了阳春砂单萜合酶基因AvTPS1的表达载体和工程菌。
Objective To construct prokaryotic expression vector of AvTPS1 and express its protein, which lays a foundation for the functional identification of AvTPS1 in prokaryotic expression system. Methods The expression vector of AvTPS1 was cloned by In-Fusion method. The expression vector of AvTPS1 was constructed by Gateway method. The two expression vectors were transformed into E. coli BL21 (DE3) and BL21DE3plys respectively, Denatured polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot detection of protein expression. Results The prokaryotic expression vectors pET-32a (+) - (- tp) AvTPS1 and pDEST17-AvTPS1 were constructed. The two expression vectors did not express any obvious corresponding proteins in E. coli BL21 (DE3) and BL21DE3plys, After a large amount of vector was expressed in E. coli BL21 (DE3), the presence of protein was detected by Western blotting. Conclusion The expression vector and engineered bacteria of Avian tundra synthase gene AvTPS1 were successfully constructed.