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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 ,neo基因在受染细胞内从早期到晚期均可发生转录
OBJECTIVE: To construct the promoter of baculovirus early gene promoter using the IE1 gene promoter of Alfalfa Sapindus nuclear polyhedrosis virus (Ac NPV). Methods: A fragment of Ac NPV p10 was used as a flanking sequence and a neomycin resistance gene (neo) was inserted into the downstream of IE1 promoter to obtain transfer vector p Ac PIneo. It was co-transfected with wild-type Ac NPV DNA into insect cell Sf9. Due to the neo gene expression, pure culture of the recombinant virus v Ac PIneo was obtained by the selection and enrichment of G418. Results: Confirmation of digestion and Southern blotting showed that the neo gene was integrated into the p10 gene of Ac NPV genome. Conclusion: The baculovirus early promoter vector was constructed successfully. The neo gene could be transcribed from early to late stage in infected cells