转宿主粘虫颗粒体病毒(PuGV-Ps)增效蛋白基因的克隆表达及活性  

Expression of Enhancin Gene from Pseudaletia unipuncta granulovirus-Ps in Escherichia coli and Bioassay of Its Activity

作　　者：徐健[1] 赵松[2] 刘琴[1] 杨青[3] 李传明[1] 

机构地区：[1]江苏里下河地区农业科学研究所,扬州225007 [2]江苏省血吸虫病防治研究所,无锡214064 [3]中国科学院动物研究所,北京100101

出　　处：《中国生物防治学报》2013年第3期389-394,共6页Chinese Journal of Biological Control

基　　金：国家自然科学基金(31071740;30901246);江苏省自然科学基金(BK2010295);公益性行业(农业)科研专项(201103021);江苏省农业三新工程(SX-2011-051)

摘　　要：以东方粘虫为转宿主增殖的颗粒体病毒(PuGV Ps)DNA为模板通过PCR反应扩增出一条2.7 kb的特异性基因片段,纯化的PCR产物克隆到载体质粒pET 15b中,构建重组质粒pET 15b En,重组质粒外源基因测序证明PCR扩增产物是粘虫颗粒体病毒转宿主病毒PuGV Ps增效蛋白的全长基因。与原始美洲粘虫颗粒体病毒(PuGV)增效蛋白基因的序列比较,两者同源性达99.59%。11个突变碱基中有7个集中在5′端下游500 bp,而3′端上游500 bp仅1个碱基发生突变。PuGV Ps增效蛋白基因重组质粒pET 15b En转化到大肠杆菌中构建重组菌,在IPTG诱导下表达了108 kD的表达产物,Ni2+柱纯化证明表达产物是目标增效蛋白。LB培养液中加入0.2%的葡萄糖后有利于增效蛋白基因的表达。生物测定结果表明,粗提的表达产物具有增效活性,可以提高Btδ内毒素对棉铃虫、甜菜夜蛾的敏感性。Using the DNA from PuGV-Ps （Pseudaletia unipuncta granulovirus propagated in the larvae of P. separata） as template, a 2.7 kb specific fragment was amplified by PCR reaction. Purified PCR product was cloned into plasmid pET-15b and a recombinant plasmid pET-15b-En was constructed. The PCR product was proved to be the full length gene encoding enhancin of PuGV-Ps by DNA sequencing. Sequence analysis revealed that enhancin gene from PuGV-Ps was tested to have 99.56% identity to that from PuGV, which was reported previously. Among the 11 mutation nucleotides, 7 of them concentrated in the 500 bp of 5＇ terminal half, while one was different within 500 bp in 3＇ terminal half. pET-15b-En vector was recombinanted into Escherichia eoli BL21（DE3）. When induced by IPTC~ the protein with molecular weight 108 kD was expressed successfully. The protein expressed by the inserted foreign gene proved to be the target protein by Ni1＋ column purification. It was promoted the target protein expression by adding 0.2 percent of glucose in the substrate. Bioassay showed that the extracted expressing protein had synergistic activity, enhanced the toxicity effect of delta-endotoxin of B. thruingiensis to larvae ofH. armigera and S. exigua.

关 键 词：转宿主粘虫颗粒体病毒 增效蛋白 克隆表达 生物测定 

分 类 号：S476.13[农业科学—农业昆虫与害虫防治] Q785[农业科学—植物保护]