TY - JOUR
T1 - Secretory production of bioactive recombinant human granulocyte-macrophage colony-stimulating factor by a baculovirus expression system
AU - Au, Lo Chun
AU - Liu, Tsun Jen
AU - Shen, Horng Der
AU - Choo, Kong Bung
AU - Wang, Sheng Yuan
PY - 1996/11/1
Y1 - 1996/11/1
N2 - The proliferation and differentiation of hematopoietic cells are stimulated by a group of glycoproteins called colony stimulating factors (CSFs). Previously, we found that the human hepatoma cell line HA22T/VGH secreted a high level of human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The cDNA of hGM-CSF, including the signal peptide sequence, was amplified from the total RNA of HA22T/VGH by a reverse-transcription polymerase chain reaction and was cloned into the pUC18 vector. After confirming the nucleotide sequence, the cDNA was inserted into a pVL1393 baculovirus transfer vector. The recombinant baculovirus carrying hGM-CSF cDNA was generated by co-transfecting the hGM-CSF recombinant transfer vector and BaculoGold(TM) baculovirus DNA into the Sf9 insect cells. The expected hGM-CSF transcript was detected in the recombinant virus-infected Sf9 cells. The conditioned media of the infected cells were analyzed by a slot-blot immunoassay. The results indicate that the infected insect cells produced and secreted hGM-CSF. According to colony forming assay, a maximum titer of 2.1 x 106 U ml-1 of hGM-CSF in the medium was obtained on the third day after infection.
AB - The proliferation and differentiation of hematopoietic cells are stimulated by a group of glycoproteins called colony stimulating factors (CSFs). Previously, we found that the human hepatoma cell line HA22T/VGH secreted a high level of human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The cDNA of hGM-CSF, including the signal peptide sequence, was amplified from the total RNA of HA22T/VGH by a reverse-transcription polymerase chain reaction and was cloned into the pUC18 vector. After confirming the nucleotide sequence, the cDNA was inserted into a pVL1393 baculovirus transfer vector. The recombinant baculovirus carrying hGM-CSF cDNA was generated by co-transfecting the hGM-CSF recombinant transfer vector and BaculoGold(TM) baculovirus DNA into the Sf9 insect cells. The expected hGM-CSF transcript was detected in the recombinant virus-infected Sf9 cells. The conditioned media of the infected cells were analyzed by a slot-blot immunoassay. The results indicate that the infected insect cells produced and secreted hGM-CSF. According to colony forming assay, a maximum titer of 2.1 x 106 U ml-1 of hGM-CSF in the medium was obtained on the third day after infection.
KW - Baculovirus
KW - Recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF)
KW - Secretion
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U2 - 10.1016/0168-1656(96)01587-8
DO - 10.1016/0168-1656(96)01587-8
M3 - Article
C2 - 8987879
AN - SCOPUS:0030298309
VL - 51
SP - 107
EP - 113
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
IS - 2
ER -