Construction of modular and versatile plasmid vectors for the high-level expression of single or multiple genes in insects and insect cell lines
Construction of modular and versatile plasmid vectors for the high-level expression of single or multiple genes in insects and insect cell lines
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DOI：
10.1006/jmbi.1999.2674
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发表时间：
1999-04-23
影响因子：
5.6
通讯作者：
Zieler, H
中科院分区：
文献类型：
--
作者：
Huynh, CQ;Zieler, H
我们构建了一系列外源基因在昆虫或昆虫细胞系中的表达质粒载体。我们将果蝇hsp70和actin 5C启动子以及hr5增强子驱动的杆状病毒ie1启动子整合到质粒中，从而方便地将外源基因克隆到多个克隆位点。我们将这些启动子与来自Heliothis virescens p63伴侣蛋白基因的短双聚腺苷化位点结合，或者将小t内含子与SV40的早期3'未翻译区和聚腺苷化位点融合。启动子和多聚腺苷化序列两侧独特的八个碱基切割酶切位点使得将整个转录单元转移到其他序列环境中成为可能，例如，转移到转座元件或转移到其他承载可选择标记基因的质粒中。在同一质粒上结合我们的两个转录单位也很方便，以便同时表达多个基因。为了验证载体驱动报告基因表达的能力，将表达质粒制成荧光素酶衍生物，并通过电穿孔法导入白纹伊蚊C6/36细胞，或通过生物粒子轰击法导入冈比亚按蚊胚胎。所有三种启动子都指导高水平的荧光素酶表达。然而，在两个实验系统中，它们的相对活性存在差异。在C6/36细胞中，actin 5C和hr5-ie1启动子的活性明显高于hsp70启动子。按蚊胚胎中，hsp70和actin 5C活性最大，hr5-ie1活性较弱。我们还发现含有SV40小t内含子和早期3'未翻译区序列的构建体比含有Heliothis多聚腺苷化序列的构建体表达水平更高。我们最活跃的结构结合了actin 5C启动子、SV40内含子和3'未翻译区序列。该载体还用于驱动可见标记物——增强型绿色荧光蛋白基因的表达，使C6/36细胞中容易表达可见绿色荧光蛋白。(C) 1999学术出版社。
We have constructed a series of plasmid vectors for the expression of foreign genes in insects or insect cell lines. We incorporated the Drosophila hsp70 and actin 5C promoters, as well as the hr5 enhancer-driven baculovirus ie1 promoter, into plasmids that allow convenient cloning of heterologous genes into multiple cloning sites. We combined these promoters with either a short, double poly-adenylation site derived from the Heliothis virescens p63 chaperonin gene, or with a fusion of the small t intron with the early 3' untranslated region and poly-adenylation sites of SV40. Unique eight base cutter restriction sites flanking the promoters and poly-adenylation sequences make it possible to transfer the entire transcription units into other sequence contexts, for example, into transposable elements or into other plasmids bearing selectable marker genes. It is also convenient to combine two of our transcription units on the same plasmid in order to express multiple genes simultaneously. To test the ability of our vectors to drive expression of reporter genes, luciferase derivatives were made of the expression plasmids and introduced into Aedes albopictus C6/36 cells by electroporation or into Anopheles gambiae embryos by biolistic particle bombardment. All three promoters directed high levels of luciferase expression. However, there were differences in their relative activities in the two experimental systems. In C6/36 cells, the actin 5C and hr5-ie1 promoters were significantly more active than the hsp70 promoter. In Anopheles embryos, hsp70 and actin 5C had maximal activities, while hr5-ie1 was weaker. We also found that the constructs containing the SV40 small t intron and early 3' untranslated region sequences had higher expression levels than their counterparts containing the Heliothis poly-adenylation sequence. Our most active construct combines the actin 5C promoter with the SV40 intron and 3' untranslated region sequences. This vector was also used to drive expression of a visible marker, the enhanced green fluorescent protein gene, resulting in readily visible green fluorescent protein expression in C6/36 cells. (C) 1999 Academic Press.