Wilson L E, Wilkinson N, Marlow S A, Possee R D, King L A
Oxford Brookes University, NERC Institute of Virology and Environmental Microbiology, England, UK.
Biotechniques. 1997 Apr;22(4):674-6, 678-81. doi: 10.2144/97224st02.
A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperda cells resulted in the synthesis of a 30-kDa protein, which was confirmed as GFP by Western blotting and by the emission of green fluorescence when illuminated with longwave UV light (495 or 365 nm). To use GFP as a marker for the selection of recombinant baculoviruses, we prepared a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of lacZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfection to prepare recombinant baculoviruses, the virus DNA was linearized with Bsu361 to improve the recovery of non-parental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with the non-parental phenotype. Plaques were rapidly identified by simply exposing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains.
本文描述了一种利用维多利亚多管水母绿色荧光蛋白（GFP）的自发荧光特性来快速制备和鉴定重组杆状病毒的方法。在草地贪夜蛾细胞中表达GFP cDNA（无信号肽序列）可合成一种30 kDa的蛋白质，通过蛋白质免疫印迹法以及在长波紫外光（495或365 nm）照射下发出绿色荧光，证实该蛋白质为GFP。为了将GFP用作选择重组杆状病毒的标记，我们制备了一种病毒BacGFP1，其中GFP cDNA替代了BacPAK6中的lacZ。在使用BacPAK6或BacGFP1进行共转染以制备重组杆状病毒之前，用Bsu361将病毒DNA线性化，以提高非亲本病毒斑的回收率。使用BacGFP1 DNA可使具有非亲本表型的病毒斑回收率达到79% - 91%。只需将病毒斑短暂暴露于长波紫外光（365 nm）下，无需外源底物或生物染色剂，即可快速鉴定病毒斑。
