Mori H, Nakazawa H, Shirai N, Shibata N, Sumida M, Matsubara F
Department of Applied Biology, Kyoto Institute of Technology, Japan.
J Gen Virol. 1992 Jul;73 ( Pt 7):1877-80. doi: 10.1099/0022-1317-73-7-1877.
A nuclear polyhedrosis virus (NPV) (Baculoviridae)-based gene expression system was improved by DNA recombination. The BmN cell line established from Bombyx mori and the Sf21 cell line established from Spodoptera frugiperda are non-permissive for Autographa californica multicapsid NPV (AcMNPV) and B. mori NPV (BmNPV) replication, respectively. After cotransfection of AcMNPV DNA and BamHI-digested BmNPV DNA into Sf21 cells, progeny viruses were isolated by plaque purification on BmN cell monolayers and the host specificity of one viral isolate was analysed. The virus had a wider host range, and replicated and produced polyhedra in Sf21 cells, BmN cells and larvae of the silkworm, B. mori. DNA restriction endonuclease analysis showed that the isolate was a hybrid of AcMNPV and BmNPV. Using the AcMNPV transfer vector pAcYM1 a portion of the polyhedrin gene of the hybrid virus was replaced with the coding region of the firefly luciferase gene, producing a recombinant virus. The latter expressed firefly luciferase in both cell lines and in silkworm larvae under the control of the polyhedrin promoter.
一种基于核型多角体病毒（NPV，杆状病毒科）的基因表达系统通过DNA重组得到了改进。从家蚕建立的BmN细胞系和从草地贪夜蛾建立的Sf21细胞系分别对苜蓿银纹夜蛾多粒包埋核型多角体病毒（AcMNPV）和家蚕核型多角体病毒（BmNPV）的复制不敏感。将AcMNPV DNA和经BamHI酶切的BmNPV DNA共转染到Sf21细胞中后，通过在BmN细胞单层上进行蚀斑纯化分离出子代病毒，并分析了一种病毒分离株的宿主特异性。该病毒具有更广泛的宿主范围，能在Sf21细胞、BmN细胞以及家蚕幼虫中复制并产生多角体。DNA限制性内切酶分析表明该分离株是AcMNPV和BmNPV的杂交种。利用AcMNPV转移载体pAcYM1，将杂交病毒多角体蛋白基因的一部分替换为萤火虫荧光素酶基因的编码区，产生了一种重组病毒。后者在多角体蛋白启动子的控制下，在两种细胞系和家蚕幼虫中均表达萤火虫荧光素酶。
