DENSOVIRUS OF AEDES-AEGYPTI AS AN EXPRESSION VECTOR IN MOSQUITO CELLS
DENSOVIRUS OF AEDES-AEGYPTI AS AN EXPRESSION VECTOR IN MOSQUITO CELLS
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DOI：
10.1006/expr.1994.1095
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发表时间：
1994-11-01
影响因子：
2.1
通讯作者：
BEATY, BJ
中科院分区：
文献类型：
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作者：
AFANASIEV, BN;KOZLOV, YV;BEATY, BJ
我们在细菌质粒中构建了含有埃及伊蚊致密病毒（AeDNV）基因组的感染性DNA克隆。将该克隆转染到白纹伊蚊C6/36蚊子细胞中，AeDNV基因组从质粒中获救并复制为野生型病毒。为了研究克隆病毒作为表达载体，将编码β -半乳糖苷酶（β -gal）的报告基因插入到AeDNV基因组中观察到的四个大开放阅读框（ORF）中。将这些重组构建体转染到白纹伊蚊C6/36细胞中，β -gal在右侧ORF（编码衣壳蛋白，Vps）和中间ORF（编码推定的非结构蛋白2）中高效表达。左侧ORF（编码非结构蛋白1 NS1）表达水平较低，而在AeDNV基因组负链上的ORF未检测到表达。来自右、中、左orf的表达可被NS1反式激活。在AeDNV Vps的n端观察到的一个假定的核靶向序列被认为是嵌合β -半乳糖转运到细胞核的原因。AeDNV衣壳蛋白提供的重组AeDNV基因组（携带β -gal基因）可以包装成感染性转导颗粒。我们的研究结果表明，AeDNV基因组可以作为外源基因在蚊子细胞中传递和表达的载体，随后将产物靶向到所需的细胞区室。(C) 1994学术出版社，Inc.
We have constructed an infectious DNA clone containing the genome of Aedes aegypti densovirus (AeDNV) in a bacterial plasmid. When this clone was transfected into Aedes albopictus C6/36 mosquito cells, the AeDNV genome rescued from the plasmid and replicated as the wild-type virus. To investigate the cloned virus as an expression vector, the reporter gene encoding beta-galactosidase (beta-gal) was inserted into four large open reading frames (ORF) observed in the AeDNV genome. When these recombinant constructs were transfected into Aedes albopictus C6/36 cells, the beta-gal was expressed efficiently from the right ORF (encoding capsid proteins, Vps) and the mid ORF (encoding putative nonstructural protein 2). A low level of expression was found from the left ORF (encoding nonstructural protein 1, NS1), and no expression was detected from the ORF observed on the minus strand of the AeDNV genome. The expression from the right, mid, and left ORFs can be trans-activated with NS1. A putative nuclear targeting sequence observed in the N-terminus of the AeDNV Vps is presumed to be responsible for transport of the chimeric beta-gal into nucleus. The recombinant AeDNV genomes (carrying the beta-gal gene) supplied with the AeDNV capsid proteins can be packaged into infectious transducing particles. Our results indicate that the genome of AeDNV can serve as a vector for delivery and expression of foreign genes in mosquito cells with subsequent targeting of the product to the desired cell compartment. (C) 1994 Academic Press, Inc.