杆状病毒早期启动子载体的构建及报道基因的表达

Expression of report gene in insect cells by a new tranfer vector with baculovirus early promoter

目的 :用苜蓿尺蠖核多角体病毒 (Ac NPV)的 IE1基因启动子构建杆状病毒早期基因启动子载体。 方法 :以 Ac NPV p10基因为侧翼序列 ,并将新霉素抗性基因 (neo)插入 IE1基因启动子下游 ,得到转移载体 p Ac PIneo。将它和野生型 Ac NPV DNA共转染昆虫细胞 Sf9,由于 neo基因的表达 ,通过 G418的选择和富集作用 ,得到重组病毒 v Ac PIneo的纯培养。 结果 :用酶切和 Southern印迹杂交证明 ,neo基因整合于 Ac NPV基因组的 p10基因位相。 结论 :成功地构建了杆状病毒早期启动子载体 。

Objectives:Using IE 1 gene promoter of Autograph californica nuclear polyhedrosis virus(AcNPV),a transfer vector with an immediately early gene promoter was constructed. Methods:Transfer vector pAcPIneo which contains neomycin resistance gene(neo)coding sequence downstream of IE 1 promoter was constructed and cotransfected with the wild type of AcNPV DNA into Sf9 insect cells.Recombinant virus was selected by G418 resistance since the neo gene can be expressed in Sf9 cells. Results:Northern blot hybridzation with 32 P labeled neo gene fragement as probe showed that the neogene was integrated in plogene of AcNPV genome. Conclusions:Transfer vector with an immediately early gene promoter of baculovirus was constructed successfully,the neo gene was transcribed from the immediately early phase to the very late phase in infected cells.