利用杆状病毒在昆虫细胞内表达  

EXPRESSION OF A Bacillus thuringiensis var israelensisδ -ENDOTOXIN GENE IN INSECT CELLS BYRECOMBINANT BACULOVIRUSES

作　　者：黎路林[1] Poss.,RD 

机构地区：[1]华中师范大学昆虫学研究所

出　　处：《病毒学报》1995年第4期357-364,共8页Chinese Journal of Virology

基　　金：中英奖学金

摘　　要：苏云金杆菌以色列亚种（Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓｖａｒｉｓｒａｅｌｅｎｓｉｓ）的分子量为２７ｋＤ的δ－内毒素蛋白基因，与一个拷贝杆状病毒ｇｐ６７信号肽基因连接后，被插入苜蓿银纹夜蛾核型多角体病毒（ＡｃＭＮＰＶ）基因组。在筛选出的３种不同的重组病毒株ＡｃＢＴＩ５－１、ＡｃＢＴＩ５－３和ＡｃＢＴＩ６－３中，前两种表现为多角体阳性，后者为多角体阴性，ＡｃＢＴＩ５－１和ＡｃＢＴＩ６－３在Ｓｆ细胞中表达产生分子量为２６～３０．５ｋＤ的４种与２７ｋＤδ－内毒素蛋白有关的多肽。在ＡｃＢＴＩ５－３感染的细胞中未检测出类似的多肽。粉纹夜蛾在吃了涂有ＡｃＢＴＩ５－１感染的细胞收集物的饲料后，表现典型的苏云金杆菌δ－内毒素中毒症状。被ＡｃＢＴＩ５－１感染的粉纹夜蛾幼虫血淋巴与２７ｋＤδ－内毒素蛋白抗血清呈阳性反应。he gene encoding the 27kD δ-endotoxin, a cytolysin, from Bacillus thuringiensis varisraelensis was expressed in insect cells by a baculovirus expression vector. The 27kD crystalprotein gene was conjugated with a copy of gp67 signal gene of Autographa californicanuclear polyhedrosis virus (AcMNPV) then inserted into a transfer vector containingpolyhedrin gene and flanking sequence and a copy of p10 promoter of AcMNPV. The foreigngene was placed under control of the p10 promoter that is upstream to the polyhedrinpromoter in opposite orientation. The recombinant transfer vector was used to co-transfectIPLB-Sf-21(Sf)cells with DNA of AcPAK6,a polyhedrin -negative mutant of AcMNPV.There stiains of the recombinant viruses produced in the cells, designated AcBTI5-1, AcBTI5-3and AcBTI6-3, were characterized.In Sf cells, AcBTI5-1, a polyhedrin-positive recombinant,produced four polypeptides,that reacted with antisera specific for the 27kD crystal protein.AcBTI6-3, with polyhedrin-negative phenotype, produced the same type of polypeptidesas AcBTI5-1.No polypeptide related to the 27kD crystal protein eas found in the cellsinfected by AcBTI5-3, although the gene of the crystal protein exsists in its genome, asshown by the result of southern hybridization.The products of the 27kD crustal protein genewas also detected in hemolymph of the larvae of Trichoplusia ni infected with AcBTI5-1.When extracts from the cells infected with AcBTI5-1 were fed to second instar larvae of T.ni, feeding by the insects was inhibited, the insects were paralysed and died whthin 7 days. Thepossibility using the toxin gene to improve baculovirus insecticide in disscussed.

关 键 词：杆状病毒 表达载体 苏云金杆菌 以色列亚种 

分 类 号：Q939.403[生物学—微生物学]