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Expression of biologically active recombinant porcine GM‐CSF by baculovirus gene expression system
Author(s) -
Inumaru S,
Kokuho T,
Denham S,
Denyer MS,
Momotani E,
Kitamura S,
Corteyn A,
Brookes S,
Parkhouse Rme,
Takamatsu H
Publication year - 1998
Publication title -
immunology and cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.999
H-Index - 104
eISSN - 1440-1711
pISSN - 0818-9641
DOI - 10.1046/j.1440-1711.1998.00734.x
Subject(s) - recombinant dna , microbiology and biotechnology , tunicamycin , biology , complementary dna , virus , transfection , granulocyte macrophage colony stimulating factor , cell culture , virology , haematopoiesis , bone marrow , genetic enhancement , gene , cytokine , immunology , stem cell , biochemistry , genetics , unfolded protein response
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 [ 3 H]‐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.