Hyperproduction of polyhedrin-IGF II fusion protein in silkworm larvae infected with recombinant Bombyx mori nuclear polyhedrosis virus.
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 at 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.
Marumoto
Y
Y
Research Institute, Daiichi Seiyaku Co. Ltd., Tokyo, Japan.
Sato
Y
Y
Fujiwara
H
H
Sakano
K
K
Saeki
Y
Y
Agata
M
M
Furusawa
M
M
Maeda
S
S
D000818:Animals; D012831:Bombyx / Q000235:genetics; D002460:Cell Line; D004274:DNA, Recombinant; D005814:Genes, Viral; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000077183:Occlusion Body Matrix Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013002:Somatomedins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*; D015678:Viral Structural Proteins
D004274:DNA, Recombinant; D000077183:Occlusion Body Matrix Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013002:Somatomedins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D007335:Insulin-Like Growth Factor II