@article{mbs:/content/journal/jgv/10.1099/0022-1317-68-10-2599, author = "Marumoto, Yasumasa and Sato, Yoshinari and Fujiwara, Hiroyuki and Sakano, Katsuichi and Saeki, Yoshiyuki and Agata, Mitsuko and Furusawa, Mitsuru and Maeda, Susumu", title = "Hyperproduction of Polyhedrin-IGF II Fusion Protein in Silkworm Larvae Infected with Recombinant Bombyx mori Nuclear Polyhedrosis Virus", journal= "Journal of General Virology", year = "1987", volume = "68", number = "10", pages = "2599-2606", doi = "https://doi.org/10.1099/0022-1317-68-10-2599", url = "https://www.microbiologyresearch.org/content/journal/jgv/10.1099/0022-1317-68-10-2599", publisher = "Microbiology Society", issn = "1465-2099", type = "Journal Article", keywords = "IGF II", keywords = "B. mori NPV", keywords = "baculovirus", abstract = "SUMMARY A gene coding for insulin-like growth factor II (IGF II) was constructed from 16 oligodeoxynucleotides synthesized chemically and cloned into EcoRI-SalI sites of pBR322. In this gene an ATG codon for methionine was introduced for cleavage by CNBr at the beginning of mature IGF II. For expressing foreign genes, a new host- vector system, with Bombyx mori silkworm larvae as the host and B. mori nuclear polyhedrosis virus (BmNPV) as the vector, has been developed. BmNPV genomic DNA codes polyhedrin which is a major protein of inclusion bodies and is mass- produced in infected silkworm larvae. We employed this polyhedrin production system to obtain a large yield of a foreign gene product. The coding region of the carboxy- terminal half of polyhedrin was removed and the remainder was ligated with the IGF II gene in phase to create a fusion protein gene consisting of the coding region of the amino-terminal half of polyhedrin and the IGF II gene. This fusion protein gene was combined in a plasmid with the promoter and 5′ and 3′ flanking regions of the polyhedrin gene. The resulting plasmid and the wild-type BmNPV genomic DNA were cotransfected into BM-N cells, and a recombinant virus was isolated by the limiting dilution method. The silkworm larvae infected with the recombinant virus produced 3·6 mg of the fusion protein per larva and the infected BM-N cells produced 0·3 mg per ml of culture. IGF II was released from the fusion protein produced by BM-N cells infected with the recombinant virus by CNBr treatment, purified by extraction with guanidine-HCl, column chromatography and HPLC and the correct amino-terminal amino acid sequence confirmed.", }