Coxpression Of BmNPV/hGM-CSF In Bombyx Mori Cells And Larvae Of Silkworms

Human granulocyte-macrophage colony stimulating factor(hGM-CSF) is a glycoprotein that stimulates the production of granulocftes,macrophages,and white blood cell. Expression of hGM-CSF in Bombyx mori cells and Larvae of Silkworms has been successful, but it's quantity of expression is much less than BmNPV. In order to enhance it's quantity of expression in Bombyx mori cells and Larvae of Silkworms, we co-express BmNPV and hGM-CSF in Bombyx mori cells and Larvae of Silkworms, and investigate it's outcome.Fusion gene by PCR was inserted into Bombyx mori baculovirus transfer vector pBacPAK.8 and contransfected with lineared DNA of Bm-BacPAK6 virus into BmN cells. The homologous recombination occurred inside the cells,and the recombinant virus BacPAK-6aa-hGM-CSF was expressed, as identified by PCR and Southern Hybridization. The BmN cells and the fifth instars were infected by the recombinant virus BacPAK-6aa-hGM-CSF. The bioactivity of the protein product was determined by TF-1 growth assay in vitro. 6aa-hGM-CSF showed significantly enhancemental effect on TF-1. The expression activity reached 31.98 mg/L in BmN cells, 210.9 mg/L in larvae, 37.56 mg/L in silkworm. ELISA assay showed that coexpression product had immunoreacivity of hGM-CSF. Western blotting assay showed that product expression in larvae of silkworm was three proteins, and weights were 17kD, 20kD,and 29kD.Because quantity of expression wasn't enhanced, the co-expression of methology in this study is not desirable.