人粒细胞集落刺激因子在家蚕细胞及幼虫中的高效表达

High level expression of human granulocyte colony stimulating factor ( hG-CSF ) in silkworm cells and larvae

含信号肽序列的ｈＧ－ＣＳＦｃＤＮＡ基因克隆于Ｍ１３ｍｐ１８中，测序表明其基因序列与高活性ｈＧ－ＣＳＦｃＤＮＡ一致。将ｈＧ－ＣＳＦ基因插入家蚕核型多角体病毒（ＢｍＮＰＶ）转移载体ｐＢＥ２８４，经与野生型病毒ＤＮＡ共转染家蚕细胞后，通过体内同源重组的方式构建了重组病毒ＢｍＮＰＶ－Ｇ－ＣＳＦ，Ｓｏｕｔｈｅｒｎ印迹杂交表明重组病毒ＤＮＡ中含Ｇ－ＣＳＦ基因片段。重组病毒感染单层ＢｍＮ细胞后第四天表达量达１． ２×１０６ＣＦＵ／ｍｌ培养液，Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析可见分子量为１９×１０３左右一条带。家蚕幼虫感染重组病毒后第四天表达量达１．４×１０７ＣＦＵ／ｍｌ血淋巴（ｈｅｍｏｌｙｍｐｈ）。

The cDNA of hG-CSF containing signal sequence was cloned into M13mp1 8 vector , and the sequence analysis showed that the cloned fragment was the high active form of hG-CSF. Insertion of the hG-CSF. cDNA into BmNPV transfer vector pBE284 , formed a recombinant transfer vector pBE284-G. BmN cell was cotransfected with wild type viral DNA and pBE284-G. The recombinant virus BmG-CSF was obtained by homologous recombination in vivo Southern-hybridization analysis suggests that the recombinant viral DNA contains hG-CSF cDNA fragrnent. The biological activity of G-GSF was 1 . 2 × 1 06CFU /ml at day 4 in the supernatant of BmN cell culture infected with BmG-CSF and was 1 . 4× 107CFU /ml at day 4 in the hemolymph of silkworm larvae infected with BmG-CSF.