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

筛选了许多昆虫病毒的野生分离物和已建立的细胞系，用于昆虫病毒复制的研究。广泛的研究表明，家蚕核型多角体病毒可在BMN细胞中复制，斜纹夜蛾核型多角体病毒可在CLS79、SF21AE和TN368中复制。根据病毒在体外的寄主范围，将这些克隆分为四组。对病毒DNA、多肽、多角体蛋白进行提纯和比较。不同寄主范围组间的病毒生化特性存在较大差异，而同一寄主范围组内的病毒生化特性变化不大。这些结果表明，SLNPV野毒株是不同病毒克隆的混合体。将BmNPV中含有多角体蛋白基因的DNA片段克隆到pUC19载体上。从感染后期感染的脂肪体中分离出mRNA，获得了该基因的c DNA。以pUC9、PUC19、pBR322为载体，采用双脱氧测序方法测定了BmNPV的多角体编码区及其5‘和3’区的核苷酸序列。构建了BmNPV的物理图谱。从BmNPV病毒颗粒中纯化的病毒DNA在细胞液中获得完整的病毒颗粒，表明重组质粒与病毒DNA共转染可产生重组NPV。插入BPV-2的E2蛋白的重组病毒的热稳定性和病毒生长与BmNPV的T3野毒株没有差异。

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.