Expression of 6×his-hGM-CSF fusion gene in larvae of silkworm(Bombyx mori) using recombinant baculovirus

Six×his-hGM-CSF fusion gene was inserted in silkworm baculovirus transfer vector pBacPAK8 to construct the recombinant transfer vector pBacPAKHis-GM-CSF.The recombinant transfer vector DNA and linear virus Bm-BacPAK6 DNA were co-transfected into BmN cells.The homologous recombination occurred inside the cells, so that the recombinant virus vBacPAKHis-GM-CSF was obtained.The BmN cells and the fifth instar silkworm larvae were infected by the recombinant virus vBacPAKHis-GM-CSF and the fusion protein were expressed in silkworm larvae.The biological activity of the expressed products was determined by TF-1 cells.The highest detectable level of fusion protein in hemolymph of silkworm larvae yielded up to 15μg/mL at the 120 h.The fusion proteins expressed in silkworm larvae hemolymph were purified using the poly-his protein purification kit.SDS-PAGE and Western-blotting analysis indicated that the expression products are three glycosylated proteins with molecular weights of 18 kD,20 kD and 31 kD.