Zuidema D, Schouten A, Usmany M, Maule A J, Belsham G J, Roosien J, Klinge-Roode E C, van Lent J W, Vlak J M
Department of Virology, Agricultural University, Wageningen, The Netherlands.
J Gen Virol. 1990 Oct;71 ( Pt 10):2201-9. doi: 10.1099/0022-1317-71-10-2201.
An improved polyhedrin-based baculovirus expression vector was constructed to expedite distinguishing infections by putative baculovirus recombinants from infections by wild-type (wt) baculovirus. The vector utilizes the Escherichia coli beta-galactosidase gene (lacZ) as a genetic marker for positive recombination between wt Autographa californica nuclear polyhedrosis virus and the baculovirus transfer vector. The marker gene/expression cassette was constructed so that lacZ and the deleted polyhedrin gene were transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator. In the constructed vector, lacZ is transcribed from the Drosophila melanogaster heat-shock promoter (hsp 70), which is constitutively expressed in baculovirus-infected Spodoptera frugiperda (Sf) cells, thereby making the site of the deleted polyhedrin gene available for the insertion and expression of foreign genes under the control of the polyhedrin promoter. Recombinant baculoviruses are readily selected in plaque assays by the development of a blue colour upon the addition of X-Gal. The colour selection renders the retrieval of recombinants less dependent on a high frequency of recombination between the transfer vector and wt baculovirus DNA. The usefulness of this new vector was illustrated by expressing gene I of cauliflower mosaic virus, which encodes a protein of Mr 46,000. Expression of gene I was at the same level as in cells infected with a conventional polyhedrin-based expression vector. Gene I protein formed large hollow fibre-like structures in the cytoplasm of infected Sf cells. This is the first plant virus protein to be expressed in insect cells by a recombinant baculovirus.
构建了一种改良的基于多角体蛋白的杆状病毒表达载体，以加快区分假定的杆状病毒重组体感染与野生型（wt）杆状病毒感染。该载体利用大肠杆菌β-半乳糖苷酶基因（lacZ）作为wt苜蓿银纹夜蛾核型多角体病毒与杆状病毒转移载体之间正向重组的遗传标记。标记基因/表达盒的构建方式使得lacZ和缺失的多角体蛋白基因以相反方向转录，两者均在作为双向终止子的猿猴病毒40 DNA片段处终止。在构建的载体中，lacZ由果蝇热休克启动子（hsp 70）转录，该启动子在杆状病毒感染的草地贪夜蛾（Sf）细胞中组成型表达，从而使缺失的多角体蛋白基因位点可用于在多角体蛋白启动子控制下插入和表达外源基因。通过在添加X-Gal后出现蓝色，可在噬斑测定中轻松筛选重组杆状病毒。颜色筛选使得重组体的检索较少依赖于转移载体与wt杆状病毒DNA之间的高频率重组。通过表达花椰菜花叶病毒的基因I（其编码分子量为46,000的蛋白质）说明了这种新载体的实用性。基因I的表达水平与用传统的基于多角体蛋白的表达载体感染的细胞中的表达水平相同。基因I蛋白在受感染的Sf细胞细胞质中形成大的中空纤维状结构。这是第一个通过重组杆状病毒在昆虫细胞中表达的植物病毒蛋白。
