A gene coding for insulin-like growth factor II (IGF II) was constructed from 16 oligodeoxynucleotides synthesized chemically and cloned into RI-I 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 silkworm larvae as the host and 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.

Keyword(s): B. mori NPV , baculovirus and IGF II

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