阳春砂单萜合酶基因AvTPS1载体构建和原核表达
发布时间：2018-11-19 19:11
【摘要】：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。
[Abstract]:Objective to construct the prokaryotic expression vector of AvTPS1 and express the protein in order to lay a foundation for the functional identification of the gene in the prokaryotic expression system. Methods the expression vector of AvTPS1 truncated peptide was constructed by In-Fusion method, and the expression vector containing complete reading frame of AvTPS1 was constructed by Gateway method. The two expression vectors were transformed into Escherichia coli BL21 (DE3) and BL21DE3plys to induce expression, and the protein expression was detected by denatured polyacrylamide gel electrophoresis (SDS-PAGE) and Western hybridization. Results the prokaryotic expression vectors pET-32a ()-(- tp) AvTPS1 and pDEST17-AvTPS1; were constructed. The two expression vectors did not express the corresponding proteins in E. coli BL21 (DE3) and BL21DE3plys, but after the two vectors were expressed in E. coli BL21 (DE3), the proteins were detected by Western hybridization. Conclusion the expression vector and engineering strain of AvTPS1 were successfully constructed.
【作者单位】： 广州中医药大学中药资源科学与工程研究中心岭南中药资源教育部重点实验室(广州中医药大学)国家中成药工程技术研究中心南药研发实验室;广州中医药大学中药学院;
【基金】：国家自然科学基金青年科学基金(81303163) 广东省高等学校优秀青年教师培养计划项目(Yq2013042) 广州中医药大学青年英才培养项目(AAC414124A08)
【分类号】：S567.239
本文编号：2343181
[Abstract]:Objective to construct the prokaryotic expression vector of AvTPS1 and express the protein in order to lay a foundation for the functional identification of the gene in the prokaryotic expression system. Methods the expression vector of AvTPS1 truncated peptide was constructed by In-Fusion method, and the expression vector containing complete reading frame of AvTPS1 was constructed by Gateway method. The two expression vectors were transformed into Escherichia coli BL21 (DE3) and BL21DE3plys to induce expression, and the protein expression was detected by denatured polyacrylamide gel electrophoresis (SDS-PAGE) and Western hybridization. Results the prokaryotic expression vectors pET-32a ()-(- tp) AvTPS1 and pDEST17-AvTPS1; were constructed. The two expression vectors did not express the corresponding proteins in E. coli BL21 (DE3) and BL21DE3plys, but after the two vectors were expressed in E. coli BL21 (DE3), the proteins were detected by Western hybridization. Conclusion the expression vector and engineering strain of AvTPS1 were successfully constructed.
【作者单位】： 广州中医药大学中药资源科学与工程研究中心岭南中药资源教育部重点实验室(广州中医药大学)国家中成药工程技术研究中心南药研发实验室;广州中医药大学中药学院;
【基金】：国家自然科学基金青年科学基金(81303163) 广东省高等学校优秀青年教师培养计划项目(Yq2013042) 广州中医药大学青年英才培养项目(AAC414124A08)
【分类号】：S567.239
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