以neo作选择标记富集和筛选阳性重组杆状病毒

将苜蓿银纹夜蛾(AcNPV)极早期基因IEI启动子控制的neo基因表达盒插入p10型转载体pAcEP106中得到pAcPIneo,将它和野生型AcNPV DNA共转染Sf细胞得到neo^+重组病毒.因为neo基因的表达使得neo^+基因的重组病毒可以在G418的存在下复制,而野生型则不能.将共转染得到的上清液以很低的感染复数连续传代,通过G418的选择作用,使重组病毒得到富集,三代以后重组病毒比例可达90%以上,如此之高的重组比例使得重组病毒的筛选不须经空斑纯化只须有限稀释即可得到.利用杆状病毒早期基因启动子表达neo基因为用早期基因表达外源毒素基因作重组病毒杀虫剂打下了基础,同时也建立了一种简便有效的筛选、富集阳性重组病毒的方法.

AN EFFICIENT METHOD TO IDENTIFY POSITIVE RECOMBINANT BACULOVIRUS BY USING NEO AS SELECTION MARKER

A cassette of neomycin resistance gene under the AcNPV IE1 gene promotorwas inserted into a p10-based transfer vector pAcEP106. Recombinant plasmidpAcPIneo was selected and cotransfected Sf cells with wild type AcNPV DNA.Expression of the neo gene under IE1 promotor enable recombinant virus multiply in theSf cells treated with antibiotic G418 which inhibit the wild-type virus growth. Passagethe cotransfection suspend at low MOI, the proportion of neo-containing virusesincrease. After three passages, the recombinants accounted for over 90%. This work laya foundation for using baculovirus early gene promoter to drive foreign gene expressionand also established a simplified procedure for selection of positive recombinantbaculoviruses.