The Study Of Expression Of HGM-CSF In Silkworm Silk Gland Bioreactor Based On The Gene-targeted Vector And The Insect Expressing Vector

The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is an important kind of the glycoprotein that has the immunity adjustment function and has the important clinical practice value. It has become the focus of research of the gene engineering that how to obtain the high active, little side effect hGM-CSF efficiently. Currently, veriety of the recombinant hGM-CSF expression systems were introduced, and Insect expression system has its own advantage gradually come to the fore.The use of transgenic silkworm expression system for producing recombinant proteins is safer than baculovirus expression system (BEVS), because the viral protein would be synthesized and protein processing may vary as viral replication proceeds in BEVS. However it still has many disadvantages, including effect induction, expression of exogenous gene and steady heredity of transgenic individual. In our study, the gene-targeting vector with the two fragments from fibroin heavy chain gene (fib-H) as the homologous arms, pSK-HL-A3GFP-FLP-GMCSF-FLPA-HR including a gfp gene driven by A3 promoter and an hGM-CSF gene driven by the fibroin light chain (fib-L) promoter was transferred into the silkworm eggs using sperm-mediated gene transfer; to target the exogenous gene into the H-chain 1st intron region of the silkworm genome. The green fluorescent silkworms were obtained after being screened for GFP. These results of polymerase chain reaction (PCR), dotting hybridization and western blotting showed that the hGM-CSF and gfp genes were integrated into the H-chain locus through homologous recombination mechanism. According to ELISA technique, the expression level of hGM-CSF in the PSGs of the G4 generation transformation silkworms was approximately 1.26 ng per gram fresh posterior silk gland. In the same time, we constructed the insect expression vector PIZT-hGM-CSF, including the hGM-CSF. BmN cells and Sf9 cells were transfected with both transposon vector and helper plasmid, and screened with antibiotics Zeocin. Two stable expression transformed cells line was obtained. ELISA assay indicated that the expression level of hGM-CSF in transformed BmN cells and Sf9 cells line was about 0.7ng/106 cells and 0.3ng/106 cells, respectly. And then, the vector was transferred into the silkworm eggs using sperm-mediated g plZT/V5- His nic silkworm silk gland was 22 kD. The result of ELISA showed that the expression level of hGM-CSF in transgenic silkworm silk gland was 4.7 ng per gram of freeze-dried silk gland.