Expression of a bioactive bovine interleukin-12 using baculovirus.
Recombinant baculoviruses that express recombinant bovine interleukin-12 (rboIL-12) subunits, p35 and p40 subunits were constructed. A recombinant virus containing the p40 subunit gene expressed the p40 subunit as a 40kDa monomer and an 80kDa disulfide-linked homodimer in the infected insect cells and in the culture supernatant. The p35 subunit was expressed in a 30kDa monomer in the infected cells but not in the supernatant. Superinfection of both recombinant viruses into the cells in a spinner flask resulted in the formation of a 70kDa disulfide-bonded heterodimer detected in the supernatant by immunoblotting using anti-p40 and anti-p35 subunits antibodies. The superinfected culture supernatant showed induction of IFNgamma mRNA synthesis and IFNgamma production in bovine peripheral blood mononuclear cells. Thus, the bioactive rboIL-12 was produced in large scale using a baculovirus expression system.
Takehara
K
K
Laboratory of Poultry Diseases, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, 034-8628, Aomori, Japan. takehara@vmas.kitasato-u.ac.jp
Nagata
T
T
Kikuma
R
R
Takanashi
T
T
Yoshiya
S
S
Yamaga
A
A
Yokomizo
Y
Y
Nakamura
M
M
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D015870:Gene Expression; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics; D018664:Interleukin-12 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D021122:Protein Subunits; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera
D017931:DNA Primers; D018076:DNA, Complementary; D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D018664:Interleukin-12; D007371:Interferon-gamma