Zhu F X, Qi Y P, Huang Y X, Bing Q
Department of Virology and Molecular Biology, College of Life Science, Wuhan University, Hubei, People's Republic of China.
J Virol Methods. 1996 Oct;62(1):71-9. doi: 10.1016/0166-0934(96)02112-x.
A transfer vector was constructed in which the neomycin resistance (neo) gene was under the control of a copy of Autographa californica nuclear polyhedrosis virus (AcMNPV) IE1 gene promoter at the p10 locus. After cotransfection of Spodoptera frugiperda (Sf9) cells with the transfer vector and infectious AcMNPV DNA, the recombinant virus-containing neo gene was selected by serial passage of the mixed progenies from cotransfection. This was done at low MOI in the presence of G418 in growth medium and was followed by limited dilution. RNA dot hybridization showed that the neo gene was transcribed from immediately early phase to very late phase, in infected Sf9 cells. These results demonstrate that a new baculovirus vector system had been constructed in infected cells. Furthermore, a new method for selection of positive recombinant baculovirus had been developed.
构建了一种转移载体，其中新霉素抗性（neo）基因受位于p10位点的苜蓿银纹夜蛾核多角体病毒（AcMNPV）IE1基因启动子的一个拷贝的控制。将转移载体与感染性AcMNPV DNA共转染草地贪夜蛾（Sf9）细胞后，通过共转染混合子代的连续传代来选择含有neo基因的重组病毒。这是在生长培养基中存在G418的低感染复数下进行的，随后进行有限稀释。RNA斑点杂交表明，在感染的Sf9细胞中，neo基因从极早期转录到极晚期。这些结果表明，已在感染细胞中构建了一种新的杆状病毒载体系统。此外，还开发了一种选择阳性重组杆状病毒的新方法。
