NeuroBactrus, a Novel, Highly Effective, and Environmentally Friendly Recombinant Baculovirus Insecticide
NeuroBactrus, a Novel, Highly Effective, and Environmentally Friendly Recombinant Baculovirus Insecticide
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DOI：
10.1128/aem.02781-12
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发表时间：
2012-10
影响因子：
4.4
通讯作者：
H. Shim;J. Choi;Yong Wang;X. Tao;Qin Liu;J. Roh;Jae Su Kim;W. Kim;S. Woo;B. Jin;Y. Je
中科院分区：
文献类型：
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作者：
H. Shim;J. Choi;Yong Wang;X. Tao;Qin Liu;J. Roh;Jae Su Kim;W. Kim;S. Woo;B. Jin;Y. Je
摘要构建了一种新的重组杆状病毒，NeuroBactrus，以开发一种改进的杆状病毒杀虫剂，与野生型杆状病毒相比，具有额外的有益特性，例如更高的杀虫活性和改善的回收率。为了构建Neurobactrus，通过在多角体蛋白启动子的控制下融合多角体蛋白-cry 1 -5-多角体蛋白基因，将苏云金芽孢杆菌晶体蛋白基因（这里称为cry 1 -5）引入苜蓿银纹夜蛾核型多角体病毒（AcMNPV）基因组中。在相反的方向，一个昆虫特异性的神经毒素基因，AaIT，从Androctonus australis的控制下的早期启动子从菜蛾绒茧蜂杆状病毒的orf 603的部分片段的融合。由NeuroBactrus表达的多角体蛋白-Cry 1 -5-多角体蛋白融合蛋白不仅被封闭在多角体中，而且还被胰蛋白酶处理激活，产生约65-kDa的活性毒素。此外，定量PCR显示，神经毒素的表达从感染的早期阶段。NeuroBactrus对小菜蛾幼虫表现出高水平的杀虫活性，与野生型AcMNPV相比，对甜菜夜蛾幼虫的半数致死时间显著降低。通过体外连续传代产生来自NeuroBactrus的AaIT和/或cry 1 -5缺失的再重组突变体。外源蛋白的表达（B.苏云金杆菌毒素和AaIT）在神经杆菌的连续传代过程中持续减少。此外，从S.用连续传代的Neurobactrus感染的甜菜幼虫显示出与野生型AcMNPV相似的杀虫活性。这些结果表明，NeuroBactrus可以通过连续传代恢复到野生型AcMNPV。
ABSTRACT A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin–cry1-5–polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin–Cry1-5–polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an ∼65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.