SpltNPV和SeNPV几丁质酶基因大肠杆菌表达产物对病毒的增效作用  

The synergistic activity of SpltNPV or SeNPV chitinase expressed in Escherichia coli with the baculovirus in infecting host insects

作　　者：刘艳荷[1] 王利华[1] 杨凤霞[1] 方继朝[1] 

机构地区：[1]江苏省农业科学院植物保护研究所

出　　处：《植物保护学报》2008年第3期209-214,共6页Journal of Plant Protection

基　　金：中国博士后科学基金项目(20060390946);江苏省博士后基金 ;江苏省农业科技创新项目(cx(07)606)

摘　　要：为明确杆状病毒几丁质酶对病毒杀虫剂的增效作用,采用PCR扩增获得斜纹夜蛾核型多角体病毒(SpltNPV)不含N-端信号肽序列的几丁质酶基因片段SpltNPVChiA/S-和甜菜夜蛾核型多角体病毒(SeNPV)几丁质酶基因编码区全序列SeNPVChiA,分别克隆入原核表达载体pET-28a,转化大肠杆菌BL21(DE3),获得高效表达,表达蛋白的分子量为62 kD和66 kD。将含有表达产物SpltNPVChiA/S-和SeNPVChiA的大肠杆菌菌液分别与SpltNPV或SeNPV一起加入人工饲料,饲喂3龄斜纹夜蛾或甜菜夜蛾幼虫,两种幼虫取食后致死时间均显著缩短,其中,SpltNPV+Splt-NPVChiA/S-和SpltNPV+SeNPVChiA的LT50比SpltNPV对照处理缩短0.90天和0.89天,LT90缩短1.90天和1.97天;SeNPV+SpltNPVChiA/S-和SeNPV+SeNPVChiA的LT50比SeNPV对照处理缩短0.47天和0.50天,LT90缩短0.57天和0.59天;而pET-28a/BL21对病毒的侵染无明显增效作用。两种病毒几丁质酶基因的大肠杆菌表达产物对病毒早期侵染均有明显的增效作用,但同源几丁质酶和异源几丁质酶对病毒的增效作用没有显著差异。In order to explore the synergistic activity of baculovirus chitinase with the baculovirus in infecting its host insects, the coding region of the complete chitinase of Spodoptera exigua nucleopolyhedrovirus （SeNPVChiA） and the matured chitinase gene of Spodoptera litura nucleopolyhedrovirus （ SpltNPVChiA/S- ） were amplified by PCR from the genomic DNA, and then chitinase genes were separately cloned into the prokaryotic expression vector pET-28a for highly efficient expression in Escherichia coli BL21 （DE3）. Molecular weight of SeNPVChiA and SpltNPVChiA/S- fusion proteins expressed in E. coli was 66 kD and 62 kD respectively. The total cellular proteins involving one of the chitinases ex- pressed in E. coli were mixed with SpltNPV or SeNPV, and fed to the third instar larvae of S. litura or S. exigua. The results showed that the lethal time of the third S. litura or S. exigua are all significantly shorter than that of controls. LT50 of S. litura fed with SpltNPV and one of the expressed chitinases was shorter 0.90 and 0.89 days, LT90 was shorter 1.90 and 1.97 days than that of larvae fed with SpltNPV alone.LT50 of S. exigua larvae fed with SeNPV and one of the expressed chitinases was shorter 0.47 and 0.50 days, LT90 was shorter 0.57 and 0.59 days than that of larvae fed with SeNPV alone, too. pET-28a/ BL21 had not any synergistic activity with the baculovirus. It＇ s revealed herein that the baculovirus chitinases expressed in E. coli showed an evident synergistic effect with the baculoviruses upon infecting the host insects, and the homologous synergism between the chitinases and the baculoviruses had not significantly different with heterologous synergism.

关 键 词：核型多角体病毒 几丁质酶基因 大肠杆菌表达产物 病毒 增效作用 

分 类 号：S476[农业科学—农业昆虫与害虫防治]