Expression of Human Granulocyte-Macrophage Colony-Stimulating Factor in Stably-Transformed BmN and Sf-9 Cells and Silkworms by a Non-Transposon Vector

摘要

This study aimed to explore the possibility of non-transposon vector mediated foreign gene expression in cultured insect cells and transgenic silk worms. To this end, the human Granulocyte-Macrophage Colony-Stimulating Factor (hGM-CSF) gene was inserted into the insect cell expression vector pIZT-V5-His to generate the recombinant vector pIZT-hGM-CSF. After transfection of BmN and Sf-9 cells with the pIZT-hGM-CSF vector, stably-transformed cells expressing the hGM-CSF gene were selected using the antibiotic zeocin at a final concentration of 300-400 μg mL-1. Expression of a 22 kDa protein band representing hGM-CSF was detected in the transformed cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The expression levels of hGM-CSF in BmN and Sf-9 cells were determined by Enzyme-Linked Immunosorbent Assay (ELISA) to be about 0.7 and 0.3 ng/106 cells, respectively. The transgenic vector pIZT-hGM-CSF was transferred into silkworm eggs using sperm-mediated gene transfer. Transgenic silkworms were obtained after screening for the gfp gene and were verified by polymerase chain reaction, dot hybridization and Western blotting. The expression level of hGM-CSF determined by ELISA was about 4.7 ng g-1 of freeze-dried silk glands in the G5 generation. These results suggest that heterologous genes can be integrated into cultured BmN and Sf-9 cells and into the silkworm genome using a non-transposon vector and can be expressed successfully.