新型真核表达质粒pcDNA6/myc-his-EGFP B 的构建及其在重组基因表达中的应用

增强型绿色荧光蛋白（EGFP, enhanced green fluorescent protein）、myc抗原和6×His已在众多真核表达载体中用作重组蛋白的表达标记，EGFP能发出的绿色荧光，myc抗原能用相应的抗体检测，6×His能被相应的树脂特异吸附。但目前为止，没有一个质粒表达载体能够同时整合三者的功能。本研究构建了一个能够同时整合EGFP、myc抗原和6×His功能的新型真核质粒表达载体，我们将其命名为pcDNA6/myc-his-EGFP B。值得注意的是，为确保目的基因与EGFP基因融合表达后，融合表达产物各组成部分能够保持原有的生物活性，我们运用LINKER程序在EGFP基因的5'端设计了一段编码八肽的连接DNA序列。将一段含有人白细胞介素2（IL-2, human interleukin 2）信号肽编码序列的基因亚克隆进pcDNA6/myc-his-EGFP B的多克隆位点中，使之与EGFP、myc抗原和6×His融合表达，构建成质粒pMHES。用pcDNA6/myc-his-EGFP B和pMHES转染2.2.15细胞，48 h后成功观察到绿色荧光；用pcDNA6/myc-his-EGFP B尾静脉注射Balb/c小鼠，8 h后在小鼠肝脏冰冻切片中同样观察到绿色荧光。用同源建模软件Modeller8V2模拟IL-2与EGFP、myc抗原和6×His融合表达产物的三维结构，结果表明：IL-2、EGFP、myc和6×His各部分互不干扰，连接八肽具有一定的柔性。以上结果表明pcDNA6/myc-his-EGFP B可望作为外源基因在哺乳动物细胞中表达研究和基因治疗的新型载体。

Construction of a Novel Eukaryotic Expression Plasmid pcDNA6/myc-his-EGFP B and Its Applications in Expression of Recombinant Genes

Enhanced green fluorescent protein(EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin. Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo-peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his-EGFP B in frame with EGFP, myc and 6×His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6×his did not interfere each other and octo-peptide linker owned certain flexibility. Our data suggest that pcDNA6/myc-his-EGFP B might be useful as a genetic tool for mammalian cells and a vector for gene therapy.