Basic studies on production of useful foreign gene products in the silkworm, Bombyx mori, using insect virus vectors

利用昆虫病毒载体生产家蚕有用外源基因产物的基础研究

基本信息

批准号：
58440014
负责人：
KAWAI Takashi
金额：
$ 16.83万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1983
资助国家：
日本
起止时间：
1983 至 1985
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-58440014/
关键词：
Insect virus protein silkworm vector gene expression polyhedrosis virus 多角体病ウイルス樹立細胞株

项目摘要

Many wild isolates of insect viruses and established cell lines were screened for the studies of insect viral replication. Extensive searching showed that Bombyx mori nuclear polyhedrosis virus (SlNPV) can replicate in BmN cells, and Spodoptera litura NPV can replicate in CLS79, SF21AE, and TN368.More than two hundred viral isolates of Spodoptera litura nuclear polyhedrosis virus were plaque purified on established cell lines. These viral clones were classified into four groups concerning in vitro host range. Viral DNAs, polypeptides, polyhedral proteins were purified and compared. Biochemical characters of the viruses between host range groups were rellativelly different, however, there were a few changes in biochemical characters within the same host range group. These results indicate that wild isolates of SlNPV are a mixture of different viral clones.DNA fragments containing polyhedrin gene in BmNPV were cloned into pUC19 plasmid using cDNA as a probe. The cDNA was made from mRNA isolated from infected fat bodies at the late stage of infection. Nucleotide sequences of polyhedral coding region and its 5' and 3' regions were determined by the dideoxy sequencing method described by Sanger.A complete gene library of BmNPV was made using pUC9, PUC19, pBR322 plasmids as vectors. A physical map of BmNPV was also constructed. Transfection of viral DNA purified from viral particles of BmNPV produced intact viral particles in the cell fluids, indicating that recombinant NPV can be produced by cotransfection of a recombinant plasmid and viral DNA.Heat stability and viral growth of a recombinant virus with insertion of an E2 protein of BPV-2 were not different from that of T3 wild isolate of BmNPV.

筛选了许多昆虫病毒的野生分离株和已建立的细胞系，用于昆虫病毒复制的研究。广泛检索表明，家蚕核型多角体病毒（SlNPV）可以在BmN细胞中复制，斜纹夜蛾NPV可以在CLS79、SF21AE和TN368中复制。在已建立的细胞上噬菌斑纯化了200多个斜纹夜蛾核型多角体病毒分离株线。根据体外宿主范围，这些病毒克隆被分为四组。纯化并比较病毒 DNA、多肽、多面体蛋白。不同宿主范围内的病毒生化特性存在较大差异，但同一宿主范围内的病毒生化特性也存在一定的变化。这些结果表明，SlNPV野生分离株是不同病毒克隆的混合物。以cDNA为探针，将BmNPV中含有多角体蛋白基因的DNA片段克隆到pUC19质粒中。该cDNA是由感染后期从受感染的脂肪体中分离出的mRNA制成的。采用Sanger描述的双脱氧测序方法测定多面体编码区及其5'和3'区的核苷酸序列。以pUC9、PUC19、pBR322质粒为载体构建BmNPV完整基因文库。还构建了 BmNPV 的物理图。从 BmNPV 病毒颗粒纯化的病毒 DNA 转染在细胞液中产生了完整的病毒颗粒，表明可以通过重组质粒和病毒 DNA 共转染来产生重组 NPV。插入 E2 的重组病毒的热稳定性和病毒生长BPV-2 的蛋白质与 BmNPV T3 野生分离株的蛋白质没有差异。