BmNPV几丁质酶基因的表达及其产物对Bt杀虫剂的增效作用  

Expression of BmNPV Chitinase Gene in Escherichia coli and Insect Cells and Synergistic Effect of the Expressed Products with Bt on Killing Silkworm

作　　者：顾奇伟[1] 林建国[1] 王芳[1] 张传溪[1] 

机构地区：[1]浙江大学应用昆虫研究所分子生物学实验室,杭州310029

出　　处：《农业生物技术学报》2005年第1期96-101,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30070032);高等学校全国优秀博士学位论文作者专项资金(No.200055)资助

摘　　要：用PCR方法分别扩增出BmNPV几丁质酶基因编码区全片段和去信号肽片段。将全片段克隆入原核表达载体pET28a,在大肠杆菌(Escherichiacoli)BL21(DE3)进行融合表达。将去信号肽片段克隆入pFastBacb转移载体,重组到杆状病毒Bac-to-Bac表达系统,在粉纹夜蛾(Trichoplusiani)细胞系(Tn-5B1-4)中进行融合表达。SDS-PAGE分析和薄层扫描显示,2片段均得到高效表达,表达产物分别占细胞总蛋白的22%和12%。将以上2种表达产物加入Bt菌液中喂食2龄家蚕(Bombyxmori),观察并记录不同处理下家蚕的死亡时间及体重。结果表明,2种表达产物对Bt杀虫剂均有明显的增效作用。取食添加以上2种表达产物的家蚕与对照处理相比,LT50分别提前24.5和24.6h,体重增量约是对照组的1/4。The coding regions of the complete chitinase and matured chitinase genes were amplified by PCR from Bombyx mori nucleopolyhedrovirus (BmNPV) genome. The fragment of the complete coding region was cloned into the prokaryotic expression vector pET28a for highly efficient expression, while the fragment encoding for chitinase without signal peptide was cloned into the pFastBacb transfer vector. The transfer vector was then recombined into a baculovirus expression vector and expressed in Trichoplusia ni cells. The expressed target proteins accumulated up to about 22% and 12% of the total cellular proteins in the bacteria and insect cells, respectively. The two chitianse products expressed in Escherichia coli and Tn cells were mixed with Bt and fed to the larvae of second instar of the silkworm (B. mori ). The results indicated that when compared with their corresponding controls, the lethal time of the silkworm larvae fed with Bt and two products of chitianse gene expressed in E.coli and Tn cells were shortened to 24.5 and 24.6 hours respectively, and the weights augmented to only a quarter of the controls.

关 键 词：表达产物 融合表达 对照组 处理 薄层扫描 ET 体重 BT杀虫剂 家蚕 增效作用 

分 类 号：S482[农业科学—农药学] S436.3[农业科学—植物保护]