摘要
质粒 pAcIEneo携带杆状病毒极早期基因IE1启动子驱动的新霉素抗性基因 (neo) ,经酶切回收后插入到质粒pAc34DZ1的SacI位点上 ,构建成多角体外膜蛋白基因 (pe)失活的转移载体 pAc34DZ2。我们曾构建了一个多角体完整 (ocu+ )的表达苏云金杆菌 (Bt)截短cryIAb基因的重组病毒 (1)vAcPhBtT。为了改进这一重组病毒的杀虫效率 ,将转移载体 pAc34DZ2与重组病毒 (1)vAcPhBtTDNA共转染Sf9细胞 ,进行第二次同源重组。由于neo基因的表达 ,用G4 18筛选得到重组病毒 (2 )vAcPhBtTPE-;Southernblot证明vAcPhBtTPE-的构建是正确的 ,经SDS PAGE分析 ,重组病毒 (2 )仍然能在昆虫细胞中表达 80kD的Bt截短毒蛋白 ,但不表达 34kD的多角体外膜蛋白。电镜观察重组病毒 (2 )无多角体外膜 ,碱解时病毒粒子释放的速度快于重组病毒 (1)。以重组病毒 (2 )感染甜菜夜蛾三龄幼虫 ,LC50 比野生型病毒小了接近 1倍 ,LT50 提前近 2d。
Plasmid pAcLEneo which bears neomycin gene drived by the baculovirus IE1 promotor was digested and the gene was harvested.A transfer vector pAc34DZ2 in which the polyhedrin envelope gene has been inactivated by insertion of expression cassette of P IE1/ neo was constructed by inserting the neo cassette into the Sac I site of plasmid pAc34DZ1.We have constructed the polyhedrin positive recombinant virus (Ⅰ) vAcPhBtT which was able to express Bt truncated endotoxin gene.In order to improve the insecticide efficiency of recombinant virus (Ⅰ),an anti\|neomycin recombinant virus (Ⅱ) vAcPhBtTPE - was obtained by second co\|transfection into the Sf9 cells with pAc34DZ2 and recombinant virus (Ⅰ) DNA.Southern blot and SDS\|PAGE analysis indicated that the recombinant virus (Ⅱ) was still able to express the 80kD Bt truncated δ endotoxin,but did not express the 34kD polyhedrin envelope protein.The recombinant virus (Ⅱ) without envelope released virion faster than recombinant virus (Ⅰ) after alkaline lysis.Bioassay was carried out using Spodoera exigua. LC 50 of the recombinant virus (Ⅱ) was about half of wild type virus and LT 50 reduced about two days.
出处
《生物工程学报》
CAS
CSCD
北大核心
2000年第4期451-456,共6页
Chinese Journal of Biotechnology
基金
国家九五攻关课题!( 96 0 0 1 0 2 0 4 0 3 )
关键词
重组杆状病毒
PE
生物防治
杀虫活性
Recombinant baculovirus,polyhedrin envelope gene,Bt truncated δ endotoxin gene,neomycin gene
