Expression Of HGM-CSF In Silkworm Silk Gland Bioreactor

The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is an important kind of the glycoprotein that has the immunity adjustment function, it plays the vital role in hemopoiesis regulation and the immunity adjustment, and has the important clinical practice value. Currently, Veriety of the recombinant hGM-CSF expression systems were introduced, and Insect expression system has its own advantage gradually come to the fore.Bombyx mori is an important traditional economical insect, keeping a long history in our country, and has a clear genetic background. The larva of silkworm has a pair of highly developed silk glands with the ability to synthesize fibroin. At present, researchers have made it become bioreactor and expressed many kinds of proteins. However it still has many disadvantages, including effect induction, expression of exogenous gene and steady heredity of transgenic individual. Transposon expression vector based on piggyBac was constructed successfully in this thesis. The transgenic vector contains a hGM-CSF gene controlled by fibroin H-chain promoter and a neomycin resistance gene controlled by baculovirus early promoter. BmN cells were transfected with both transposon vector and helper plasmid, and screened with antibiotics G418. A stable expression transformed cell line was obtained. ELISA assay indicated that the expression level of hGM-CSF in cell line was about 1534.55 pg/106 cells. In order to introduce the transposon vector into Bombyx mori, several transgenic methods such as sperm-mediated gene transfer, particle delivery system transfer, egg pricking and gonadal injection were used. And screened with GFP and antibiotics G418, combined identification of PCR and Southern blot, transgenic silkworms were obtained. The result of western blot displayed that the molecular weight of hGM-CSF expressed by transgenic silkworm silk gland was 22 kD. The result of ELISA showed that the expression level of hGM-CSF in transgenic silkworm silk gland was 5.15 ng per gram of freeze-dried silk gland. Developed a new pathway of hGM-CSF expression and provided a new method of using silkworm.