Expression of biologically active recombinant porcine GM-CSF by baculovirus gene expression system.
The full length porcine granulocyte/macrophage colony stimulating factor (GM-CSF) cDNA, including secretion signal peptide coding region was recloned into baculovirus transfer vector pAcYM1. The vector was then transfected with Autographica californica nuclear polyhedrosis virus (AcNPV) DNA into SF21AE cells and the recombinant virus AcPGM was recovered. Recombinant porcine GM-CSF (rpGM-CSF) was obtained from the serum-free culture medium of Tn5 cells infected with the AcPGM virus, and was shown to be a glycosylated 21 kDa protein as confirmed by tunicamycin treatment and [3H]-glucosamine uptake. The biological activities of rpGM-CSF in AcPGM-infected cell culture supernatants were demonstrated by porcine bone marrow cell proliferation and haematopoietic cell colony formation assays. The use of rpGM-CSF enabled us to culture porcine monocytes/macrophage and dendritic-like cells, derived from either porcine bone marrow or peripheral blood, for up to 4 months.
Laboratory of Bioengineering, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
S
S
Inumaru
T
T
Kokuho
S
S
Denham
M S
MS
Denyer
E
E
Momotani
S
S
Kitamura
A
A
Corteyn
S
S
Brookes
R M
RM
Parkhouse
H
H
Takamatsu
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D003114:Colony-Forming Units Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000138:chemical synthesis*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000494:pharmacology; D013552:Swine
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor