Secretory production of bioactive recombinant human granulocyte-macrophage colony-stimulating factor by a baculovirus...

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 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 10(6). U ml-1 of hGM-CSF in the medium was obtained on the third day after infection.