融合6个组氨酸的人粒细胞-巨噬细胞集落刺激因子在家蚕幼虫中的表达

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

将融合6个组氨酸(6×his)序列的hGM-CSF基因插入杆状病毒转移载体pBacPAK8中得到杆状病毒重组转移载体pBacPAKHis-GM-CSF,pBacPAKHis-GM-CSF DNA与线性化病毒Bm-BacPAK6 DNA共转染BmN细胞,获得了表达rhGM-CSF融合蛋白的重组病毒vBacPAKHis-GM-CSF.用重组病毒感染家蚕五龄起蚕,分别在24、48、72、961、20 h和144 h剪腹足取蚕血淋巴,ELISA法测得rhGM-CSF融合蛋白在120 h的蚕血淋巴中表达量最高,约为15μg/mL蚕血淋巴.融合蛋白通过Poly-His Protein Purification Kit纯化.SDS-PAGE和Western blotting分析表明,表达产物是3种糖基化程度不同的,分子量分别约为18、203、1 kD的蛋白质.

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.